Effects of Voluntary Exercise on the Central Melanocortin Receptor Knockout Mice

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Title:
Effects of Voluntary Exercise on the Central Melanocortin Receptor Knockout Mice
Physical Description:
1 online resource (520 p.)
Language:
english
Creator:
Schaub,Jay
Publisher:
University of Florida
Place of Publication:
Gainesville, Fla.
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Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences, Pharmacodynamics
Committee Chair:
Haskell-Luevano, Carrie
Committee Members:
Keller-Wood, Maureen
Millard, William J
Katovich, Michael J
Rowland, Neil E

Subjects

Subjects / Keywords:
exercise -- hypothalamus -- knockout -- liver -- mc3r -- mc4r -- melanocortin -- mouse -- muscle
Pharmacodynamics -- Dissertations, Academic -- UF
Genre:
Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract:
Human obesity, defined as possessing a body mass index value of greater than 30, is reaching critical levels world wide. Obese individuals are at greater risk for other diseases including cardiovascular disease, type 2 diabetes, and certain types of cancer. In addition to environmental and lifestyle causes, certain genetic mutations have been shown to result in obesity. Genetic deletion of the melanocortin-3 and -4 receptors (MC3R and MC4R respectively) have been shown to result in altered metabolisms and phenotypes in mice. Voluntary exercise has been shown by the Haskell-Luevano laboratory to delay the onset of the obesity of melanocortin-4 receptor knockout (KO) mice, preventing early onset weight gain and increases in fat mass. Running wheel exercise was also found to change hypothalamic expression levels of genes involved in energy homeostasis. This dissertation investigates the effects of voluntary exercise on the phenotypes and gene expression profiles of male mice lacking the MC3R, the MC4R, or both the MC3 and MC4 receptors (DKO mice). Additionally the effects of MC4R deletion and voluntary exercise were investigated in male and female mice to determine what effects, if any, gender had on the phenomenon seen in male MC4R KO mice allowed to exercise. Genotype and exercise both had significant effects on both phenotype and gene expression in male mice lacking the one or both of the central melanocortin receptors. Voluntary exercise resulted in a significant decrease in body weight in MC4R KO and DKO mice (P<0.001) and total fat mass in exercising MC3R KO, MC4R KO, and DKO mice (P<0.05) compared to the same genotypes in conventional cages. Furthermore, changes in expression levels of genes involved in liver fatty acid metabolism seen in sedentary MC4R KO mice were prevented by voluntary exercise. Significant differences in phenotype and hypothalamic gene expression were seen between male and female MC4R KO mice. The obese phenotype was diminished in female mice, in part due to the lack of hyperphagia generally associated with MC4R KO dysfunction. Overall, voluntary exercise had a beneficial effect on central melanocortin receptor KO mice, delaying the onset of the associated phenotypes and preventing changes in gene expression.
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility:
by Jay Schaub.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
Local:
Adviser: Haskell-Luevano, Carrie.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

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UFRGP
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Applicable rights reserved.
Classification:
lcc - LD1780 2011
System ID:
UFE0043130:00001


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1 EFFECTS OF VOLUNTARY EXERCISE ON THE CENTRAL MELANOCORTIN RECEPTOR KNOCKOUT MICE By JAY SCHAUB A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Jay Schaub

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3 For Jim Harshman, Spike Black, Mike Stewart, and Doug Gilliland

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4 ACKNOWLEDGMENTS I would like to thank my adviser, Dr. Carrie Haskell Luevano for her su pport for the past six years. I would like to thank the members of my committee, Dr. Neil Rowland, Dr. William Millard, Dr. Maureen Keller Wood, and Dr. Michael Katovich for their role in my graduate education. I would like to acknowledge the many people who have contributed in some form Zhimin Xiang, Erin Bruce, Kim Haskell, Sarah Carey, Yunyang Wang, Dr. Hua Yao, Dr. Francois Rouzaud, Dr. Boman Irani, Marcus Moore, Dr. Sally Litherland, Dr. Glenn Walter, Dr. Ronald J. Mandel, Dr. Henry Baker, Dr. William Millard, and Dr. Carrie Haskell Luevano. I would also like to thank all of the technicians and supervisors at the University of Florida Animal Care Services who have gr aciously worked with us and accommodated our research. I would like to thank my friends Brandon and Krista Wilson who took me into their home while I was looking for my own and have supported me through my graduate career. Thank you to Dr. Anamika Singh f or always being available to listen when my project was not working at all and for letting me misbehave with her three year old during Post Bac dinners. I also want to thank Erica Haslach for being such a good friend and colleague helping me get through so me very interesting times in the lab and no fewer than four lab managers. I want to state that I was not born with a love of science, but it was cultured in me by my excellent high school science teachers to whom this dissertation is dedicated. I also want to thank the Mouse Prayer Team of Sarasota for their love, support, and prayers during my graduate study. The members include: Dr. Katherine Keeley, Dr.

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5 Susan DeWyngaert, Elizabeth Marshall, Ann Moore, Linda Hollen, Lisa Herring, Karen Turner, Kathy Seide r, Sarah Hudson, Karen Eastmoore, Jacki Boedecker, Karen Peterson, Mary Jane Hartenstine, Karen McGaharan, and Leigh Ann Webb. I want to thank my family, John, Valerie, Megan and Tyler Schaub for their love and support my entire life. I also want to thank my beautiful and patient wife Martha Pennock Schaub who was brave (or foolish) enough to marry a graduate student part way through his Ph.D. program. I would also like to thank Martha for proof reading my entire dissertation and being supportive of my stud ies. I want to acknowledge that I was financially supported by an Alumni Fellowship from the University of Florida for the four years of my graduate work. I was also supported in my final year by a fellowship from the American Foundation for Pharmaceutica l Education. The animal research in Dr. Haskell supported by an American Diabetes Association research grant and by the National Institutes of Health.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 11 LIST OF FIGURES ................................ ................................ ................................ ........ 13 LIST OF ABBREVIATIONS ................................ ................................ ........................... 29 ABSTRACT ................................ ................................ ................................ ................... 32 CHAPTER 1 OBESITY, EXERCSE, AND THE MELANOCORTIN SYSTEM .............................. 34 Obesity ................................ ................................ ................................ .................... 34 Definition ................................ ................................ ................................ .......... 34 Causes ................................ ................................ ................................ ............. 35 Melanocortins ................................ ................................ ................................ ......... 35 Receptors ................................ ................................ ................................ ......... 35 Ligands ................................ ................................ ................................ ............. 36 Agonists ................................ ................................ ................................ ..... 36 Antagonists ................................ ................................ ................................ 37 Knockout mice ................................ ................................ ................................ .. 37 Phenotypes ................................ ................................ ................................ 37 Link to clinical obesity ................................ ................................ ................ 38 Exercise ................................ ................................ ................................ .................. 39 Treatment for Diseases ................................ ................................ .................... 39 Animal Models of Exercise ................................ ................................ ............... 39 Gene Expression ................................ ................................ ................................ .... 41 General Background ................................ ................................ ........................ 41 Genes Involved in Energy Balance Pathways ................................ .................. 45 Central Gene Expression ................................ ................................ ................. 49 Peripheral Gene Expression ................................ ................................ ............. 50 Liver ................................ ................................ ................................ ........... 50 Pancreas ................................ ................................ ................................ .... 50 Muscle ................................ ................................ ................................ ........ 50 Overview and Objectives ................................ ................................ ........................ 51 2 METHODS AND MATERIALS ................................ ................................ ................ 58 Experimental Animals ................................ ................................ ............................. 58 Selection Criteria for Mice ................................ ................................ ................ 58 Breeding ................................ ................................ ................................ ........... 59

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7 Genotyping ................................ ................................ ................................ ....... 60 Housing ................................ ................................ ................................ ............ 61 Food ................................ ................................ ................................ ................. 62 Experimental Design ................................ ................................ ............................... 62 Weights and Lengths ................................ ................................ ........................ 63 Blood Draws ................................ ................................ ................................ ..... 63 Plasma Hormones ................................ ................................ ............................ 64 Bo dy Composition ................................ ................................ ............................ 64 Running Wheel Activity ................................ ................................ ..................... 65 Sacrifice and Dissection ................................ ................................ ................... 65 Gene Expression ................................ ................................ ................................ .... 65 RNA Extraction ................................ ................................ ................................ 65 cDNA Synthesis ................................ ................................ ............................... 67 R T PCR Studies ................................ ................................ ............................... 68 Data Analysis ................................ ................................ ................................ .......... 68 RT PCR Data ................................ ................................ ................................ ... 68 Running Wheel D ata ................................ ................................ ........................ 69 Statistical Analysis ................................ ................................ ............................ 70 3 EFFECT OF VOLUNTARY EXERCISE ON MALE, CENTRAL MELANOCORTIN RECEPTOR KNOCKOUT MICE ................................ ............... 77 Introductory Remarks ................................ ................................ .............................. 77 Methods and Results ................................ ................................ .............................. 78 Brief Overview of Experime nts ................................ ................................ ......... 78 Animal Selection ................................ ................................ ............................... 79 Statistical Analysis ................................ ................................ ............................ 80 Body Size and Composition ................................ ................................ ............. 81 Weight ................................ ................................ ................................ ........ 81 Fat mass ................................ ................................ ................................ .... 86 Lean mass ................................ ................................ ................................ 92 Body length ................................ ................................ ................................ 96 Food Intake ................................ ................................ ................................ .... 100 Effects of genotypes within housing ................................ ......................... 101 Summary ................................ ................................ ................................ .. 103 Effect of housing within genotypes ................................ ........................... 104 Summary ................................ ................................ ................................ .. 104 Whole Blood Glucose ................................ ................................ ..................... 105 Plasma Hormone Concentrations ................................ ................................ ... 105 Insulin ................................ ................................ ................................ ...... 107 Leptin ................................ ................................ ................................ ....... 109 Running Wheel Activity ................................ ................................ ................... 112 Minimum Amount of Exercis e Needed to Prevent Obesity ............................. 113 Correlation of exercise and body weight ................................ .................. 114 Correlation of exercise and fat mass ................................ ........................ 114 Additional MC3R Data and Statistics ................................ .............................. 114 MC3R body length ................................ ................................ ................... 115

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8 MC3R fat mass ................................ ................................ ........................ 116 MC3R lean mass ................................ ................................ ..................... 118 Discussion ................................ ................................ ................................ ............ 119 Discussion of Results ................................ ................................ ..................... 1 20 Body weight ................................ ................................ ............................. 120 Fat mass ................................ ................................ ................................ .. 121 Lean mass ................................ ................................ ............................... 122 Body length ................................ ................................ .............................. 123 Food intake ................................ ................................ .............................. 125 Plasma hormone concentrations ................................ .............................. 126 Running wheel activity ................................ ................................ ............. 129 Correlations ................................ ................................ .............................. 131 Strain Differences Between MC3R and MC4 R Breeding Colony Mice ........... 133 Method By Which Exercise Causes Changes ................................ ................ 135 Concluding Remarks ................................ ................................ ............................. 135 4 EFFECTS OF VOLUNTARY EXERCISE ON GENE EXPRESSION IN ORGANS INVOLVED IN ENERGY HOMEOSTASIS ................................ ........... 330 Introductory Remarks ................................ ................................ ............................ 330 Methods and Results ................................ ................................ ............................ 332 RNA Extraction ................................ ................................ ............................... 332 Hypothalamus, liver, and muscle ................................ ............................. 332 Pancreas ................................ ................................ ................................ .. 333 Selection of Housekeeping Genes ................................ ................................ 333 Relative Expression ................................ ................................ ........................ 333 Hypothalamus ................................ ................................ ................................ 334 Agouti related protein (AGRP) ................................ ................................ 334 5' AMP activated protein kinas e (PRKAA1) ................................ ............. 334 Cocaine and amphetamine regulated transcript (CART) ......................... 335 Carnitine palmitoyltransferase 2 (CPT2) ................................ .................. 335 Glucokinase (GCK) ................................ ................................ .................. 335 Hypocretin (HCRT) ................................ ................................ .................. 335 Insulin receptor (INSR) ................................ ................................ ............. 335 Leptin receptor (LEPR) ................................ ................................ ............ 336 Melanocortin 3 receptor (MC3R) ................................ .............................. 336 Melanoc ortin 4 receptor (MC4R) ................................ .............................. 336 Proopiomelanocortin (POMC) ................................ ................................ .. 336 Neuropeptide Y (NPY) ................................ ................................ ............. 337 Neuropeptide Y receptor type 1 (NPY1R) ................................ ................ 337 Suppressor of cytokine signaling 3 (SOCS3) ................................ ........... 337 Uncoupling protein 2 (UCP2) ................................ ................................ ... 338 Relative expression ................................ ................................ .................. 338 Liver ................................ ................................ ................................ ................ 339 5' AMP activated protein kinase (PRKAA1) ................................ ............. 339 Carnitine palmitoyltransferase 1a (CPT1A) ................................ .............. 339 Carnitine palmitoyltransferase 2 (CPT2) ................................ .................. 340

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9 Diglyceride acyltransferase 1 (DGAT1) ................................ .................... 340 Diglyceride acyltransferase 2 (DGAT2) ................................ .................... 340 Fatty acid synthase (FASN) ................................ ................................ ..... 341 Fructose 1,6 biphosphatase (FBP1) ................................ ........................ 341 Glucokinase (GCK) ................................ ................................ .................. 342 Glucose 6 phosphatase (G6PC3) ................................ ............................ 342 Glucose transporter 2 (SLC2A2) ................................ .............................. 343 Glycogen phosphorylas e (PYGL) ................................ ............................. 343 Glycogen synthase 2 (GYS2) ................................ ................................ ... 344 Hormone sensitive lipase (LIPE) ................................ .............................. 344 Insulin receptor (INSR) ................................ ................................ ............. 345 Leptin receptor (LEPR) ................................ ................................ ............ 345 Phosphofructokinase (PFKL) ................................ ................................ ... 345 Melanocortin 3 receptor and melanocortin 4 receptor (MC3R and MC4R) ................................ ................................ ................................ .. 346 Relative expression ................................ ................................ .................. 346 Skeletal Muscle (Gastrocnemius of the Leg) ................................ .................. 351 5' AMP activated protein kinase (PRKAA1) ................................ ............. 351 Carnitine palmitoyltransferase 1b (CPT1B) ................................ .............. 351 Carnitine palmitoyltransferase 2 (CPT2) ................................ .................. 351 Glucokinase (GCK) ................................ ................................ .................. 351 Glucose transporter 4 (SLC2A4) ................................ .............................. 352 Glycogen phosphorylase (PYGM) ................................ ............................ 352 Glycogen synthase 1 (GYS2) ................................ ................................ ... 352 Interleukin 6 (IL 6) ................................ ................................ .................... 352 Phosphofructokinase (PFKM) ................................ ................................ .. 352 Uncoupling protein 2 (UCP2) ................................ ................................ ... 353 Uncoupling protein 3 (UCP3) ................................ ................................ ... 353 Relative expression ................................ ................................ .................. 353 Discussion ................................ ................................ ................................ ............ 355 Discussion of Results ................................ ................................ ..................... 355 Pancreas ................................ ................................ ................................ .. 355 Hypo thalamus ................................ ................................ .......................... 355 Liver ................................ ................................ ................................ ......... 360 Muscle ................................ ................................ ................................ ...... 364 Relative Expression ................................ ................................ ........................ 366 Hypothalamus ................................ ................................ .......................... 366 Liver ................................ ................................ ................................ ......... 367 Muscle ................................ ................................ ................................ ...... 369 Shifts in Metabolic Pathways ................................ ................................ .......... 370 Strain Differences Between MC3R and MC4R Breeding Colony Mice ........... 373 Concluding Remarks ................................ ................................ ............................. 374 5 EFFECT OF GENDER ON THE MITIGATING EFFECTS OF VOLUNTARY EXERCISE ON PREVENTION OF THE OBESE PHENOTYPE OF THE MELANOCORTIN 4 RECEPTOR MICE ................................ ............................... 415

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10 Introductory Remarks ................................ ................................ ............................ 415 Methods and Results ................................ ................................ ............................ 416 Brief Overview of Experiment ................................ ................................ ......... 41 6 Statistical Analysis ................................ ................................ .......................... 417 Body Size and Composition ................................ ................................ ........... 418 Body weights ................................ ................................ ............................ 418 Fat masses ................................ ................................ .............................. 420 Lean masses ................................ ................................ ............................ 421 Body lengths ................................ ................................ ............................ 422 Food Intake ................................ ................................ ................................ .... 423 Insulin ................................ ................................ ................................ ............. 424 Leptin ................................ ................................ ................................ .............. 425 Hypothalamic Melanocortin Gene Expression ................................ ................ 426 Running Wheel Activity ................................ ................................ ................... 429 Discussion ................................ ................................ ................................ ............ 430 6 CONCLUSION ................................ ................................ ................................ ...... 447 APPENDIX: ADDITIONAL INFORMATION ABOUT EXPERIMENTAL MICE ............. 471 LIST OF REFERENCES ................................ ................................ ............................. 505 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 520

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11 LIST OF TABLES Table page 1 1 Body m ass index and corresponding body type classifications set by the World Health Organization. ................................ ................................ ................. 53 2 1 Mice used to study the physiological effects of exercise on male MC3R, MC4R, and DKO mice. St udies presented in Chapters 3 and 4. ........................ 71 2 2 Mice used to study the physiological effects of exercise on male and female MC4R KO mice. Study presented in Chapter 5. ................................ ................. 71 2 3 Proteinase K tail digest buffer for DNA extraction for making 1 L of solution. ..... 72 2 4 Primers used for genotyping melanocortin receptor KO mice. ............................ 72 2 5 Genotyping PCR mastermix solution for DNA amplification per reaction. .......... 73 2 6 Polymerase chain reaction mastercycler program f or MC3R genotyping. .......... 73 2 7 Polymerase chain reaction mastercycler program for MC4R genotyping. .......... 73 2 8 Volume of RNAlater used fo r storage of fresh tissue until RNA extraction. ........ 73 2 9 cDNA synthesis mastermix solution recipe provided with High Capacity cDNA Archive Kit for cDNA synthesis per reaction. ................................ ............ 74 2 10 Polymerase chain reaction mastercycler program for cDNA synthesis. ............. 74 2 11 Gene name, gene symbol, and Applied Biosystems catalog number o f TaqMan probes and associated genes. ................................ .............................. 75 2 12 RT PCR mastermix solution for TaqMan Gene Expression Assay per reaction well. ................................ ................................ ................................ ....... 76 2 13 Real time polymerase chain reaction cycler program for evaluating gene expression by TaqMan Gene Expression Assay. ................................ ............... 76 A 1 Details for male mice that were part of the experiments presented in Chapters 3 and 4. ................................ ................................ ............................. 472 A 2 Details for male MC3R mice that were part of the expanded statistical analysis presented in Chapter 3. ................................ ................................ ...... 475 A 3 Details for male and female MC4R mice from RW 11 presented in Chapter 5. 477 A 4 Body weights (in grams) of male mice presented in Chapters 3 and 4. ............ 480

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12 A 5 Fat masses (in grams) of male mice presented in Chapters 3 and 4. ............... 483 A 6 Lean masses (in grams) of male mice presented in Chapters 3 and 4. ............ 486 A 7 Body lengths (in millimeters) of male mice presented in Chapters 3 and 4. ..... 489 A 8 Average daily food intake (in grams) of male mic e presented in Chapters 3 and 4. ................................ ................................ ................................ ............... 492 A 9 Average daily number of running wheel rotations during the dark cycle of male mice presented in Chapters 3 and 4. ................................ ....................... 495 A 10 Average weekly plasma insulin concentration (pM) for male MC3R mice presented in Chapters 3 and 4. ................................ ................................ ........ 497 A 11 Average weekly plasma leptin concentration (pM) for male MC3R mice presented in Chapters 3 and 4. ................................ ................................ ........ 498 A 12 Hypothalamic gene expression presented as group averages of fold change (from conventionally housed WT group). ................................ .......................... 499 A 13 Liver gene expression presented as group averages of fold change (from conventionally housed WT group). ................................ ................................ ... 501 A 14 Skeletal muscle (gastrocnemi us) gene expression presented as group averages of fold change (from conventionally housed WT group). ................... 503

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13 LIST OF FIGURES Figure page 1 1 Graphical representation of the effects of shifting energy input and output on body weight. ................................ ................................ ................................ ....... 53 1 2 Graphical representation of functions of the melanocortin receptors and ligands. ................................ ................................ ................................ ............... 54 1 3 Photo of melanocortin receptor WT and KO mice with corresponding body weights and fat masses. ................................ ................................ ..................... 55 1 4 Metabolic pathways of the body to be inves tigated in metabolically active organs in the body. ................................ ................................ ............................. 56 1 5 Organs and genes to be investigated to elucidate the effects of genetic obesity caused by melanocortin receptor dysfunction and volun tary exercise by running wheel. ................................ ................................ ............................... 57 2 1 Breeding schemes used to generate experimental mice. ................................ ... 72 2 2 Example of a good and poor RN A quality visualized on 1% agarose gels. ........ 74 3 1 Body weights of male mice during running wheel experiments. ....................... 137 3 2 Body weights at five weeks of age. ................................ ................................ ... 138 3 3 Body weights at six weeks of age. ................................ ................................ .... 139 3 4 Body weights at seven weeks of age. ................................ ............................... 140 3 5 Body weights at eight weeks of age. ................................ ................................ 141 3 6 Body weights at nine weeks of age. ................................ ................................ 142 3 7 Body weights at ten weeks of age. ................................ ................................ ... 143 3 8 Body weights at 11 weeks of age. ................................ ................................ .... 144 3 9 Body weights at 12 weeks of age ................................ ................................ ..... 145 3 10 Body weights at 13 weeks of age ................................ ................................ ..... 146 3 11 Body weights at five weeks of age ................................ ................................ .... 147 3 12 Body weights at six weeks of age ................................ ................................ ..... 148 3 13 Body weights at seven weeks of age ................................ ................................ 149

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14 3 14 Body weights a t eight weeks of age ................................ ................................ 150 3 15 Body weights at nine weeks of age ................................ ................................ .. 151 3 16 Body weights at ten weeks of age. ................................ ................................ ... 152 3 17 Body weights at 11 weeks of age. ................................ ................................ .... 153 3 18 Body weights at 12 weeks of age ................................ ................................ ..... 154 3 19 Body weights at 13 weeks of age ................................ ................................ ..... 155 3 20 Fat masses of male mice during running wheel experiments ........................... 156 3 21 Fat masses at 5 weeks of age ................................ ................................ .......... 157 3 22 Fat masses at 6 weeks of age ................................ ................................ .......... 158 3 23 Fat masses at 7 weeks of age ................................ ................................ .......... 159 3 24 Fat masses at 8 weeks of age ................................ ................................ .......... 160 3 25 Fat masses at 9 weeks of age ................................ ................................ .......... 161 3 26 Fat masses at 10 weeks of age ................................ ................................ ........ 162 3 27 Fat masses at 11 weeks of age ................................ ................................ ........ 163 3 28 Fat masses at 12 weeks of age ................................ ................................ ........ 164 3 29 Fat masses at 13 weeks of age ................................ ................................ ........ 165 3 30 Fat masses at 5 weeks of age. ................................ ................................ ......... 166 3 31 Fat masses at 6 weeks of age ................................ ................................ .......... 167 3 32 Fat masses at 7 weeks of age ................................ ................................ .......... 168 3 33 Fat masses at 8 weeks of age ................................ ................................ .......... 169 3 34 Fat masses at 9 weeks of age ................................ ................................ .......... 170 3 35 Fat masses at 10 weeks of age ................................ ................................ ........ 171 3 36 Fat masses at 11 weeks of age ................................ ................................ ........ 172 3 37 Fat masses at 12 weeks of age ................................ ................................ ........ 173 3 38 Fat masses at 13 weeks of age ................................ ................................ ........ 174

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15 3 39 Lean masses of male mice during running wheel experiments. ....................... 175 3 40 Lean masses at 5 weeks of age. ................................ ................................ ..... 176 3 41 Lean masses at 6 weeks of age. ................................ ................................ ..... 177 3 42 Lean masses at 7 weeks of age ................................ ................................ ...... 178 3 43 Lean masses at 8 weeks of age ................................ ................................ ...... 179 3 44 Lean masses at 9 weeks of age ................................ ................................ ...... 180 3 45 Lean masses at 10 weeks of age ................................ ................................ .... 181 3 46 Lean masses at 11 weeks of age ................................ ................................ .... 182 3 47 Lean masses at 12 weeks of age ................................ ................................ .... 183 3 48 Lean masses at 13 weeks of age ................................ ................................ .... 184 3 49 Lean masses at 5 weeks of age. ................................ ................................ ..... 185 3 50 Lean masses at 6 weeks of age ................................ ................................ ...... 186 3 51 Lean masses at 7 weeks of age ................................ ................................ ...... 187 3 52 Lean masses at 8 weeks of age ................................ ................................ ...... 188 3 53 Lean masses at 9 weeks of age ................................ ................................ ...... 189 3 54 Lean masses at 10 weeks of age ................................ ................................ .... 190 3 55 Lean masses at 11 weeks of age ................................ ................................ .... 191 3 56 Lean masses at 12 weeks of age ................................ ................................ .... 192 3 57 Lean masses at 13 weeks of age ................................ ................................ .... 193 3 58 Body Lengths of male mice during running wheel experiments. ....................... 194 3 59 Body Lengths at 5 weeks of age. ................................ ................................ ...... 195 3 60 Body Lengths at 6 weeks of age. ................................ ................................ ...... 196 3 61 Body Lengths at 7 weeks of age ................................ ................................ ....... 197 3 62 Body Lengths at 8 weeks of age ................................ ................................ ....... 198 3 63 Body Lengths at 9 weeks of age ................................ ................................ ....... 199

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16 3 64 Body Lengths at 10 weeks of age. ................................ ................................ .... 200 3 65 B ody Lengths at 11 weeks of age ................................ ................................ ..... 201 3 66 Body Lengths at 12 weeks of age ................................ ................................ ..... 202 3 67 Body Lengths at 13 weeks of age ................................ ................................ ..... 203 3 68 Body Lengths at 5 weeks of age ................................ ................................ ....... 204 3 69 Body Lengths at 6 weeks of age ................................ ................................ ....... 205 3 70 Body Lengths at 7 weeks of age ................................ ................................ ....... 206 3 71 Body Lengths at 8 weeks of age ................................ ................................ ....... 207 3 72 Body Lengths at 9 weeks of age ................................ ................................ ....... 208 3 73 Body Lengths at 10 weeks of age. ................................ ................................ .... 209 3 74 Body Lengths at 11 weeks of age. ................................ ................................ .... 210 3 75 Body Lengths at 12 weeks of age ................................ ................................ ..... 211 3 76 Body Lengths at 13 weeks of age ................................ ................................ ..... 212 3 77 Food intake of male mice during running wheel experiments. .......................... 213 3 78 Average daily food intake for 5 weeks of age. ................................ .................. 214 3 79 Average daily food intake for 6 weeks of age ................................ ................... 215 3 80 Average daily food intake for 7 weeks of age ................................ ................... 216 3 81 Average daily food intake for 8 weeks of age ................................ ................... 217 3 82 Average daily food intake for 9 weeks of age ................................ ................... 218 3 83 Average daily food intake for 10 weeks of age. ................................ ................ 219 3 84 Average daily food intake for 11 weeks of age ................................ ................. 220 3 85 Average daily food intake for 12 weeks of age ................................ ................. 221 3 86 Average daily food intake for 13 weeks of age ................................ ................. 222 3 87 Average daily food intake for 5 weeks of age. ................................ .................. 223 3 88 Average daily food intake for 6 weeks of age ................................ ................... 224

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17 3 89 Average daily food intake for 7 weeks of age ................................ ................... 225 3 90 Avera ge daily food intake for 8 weeks of age ................................ ................... 226 3 91 Average daily food intake for 9 weeks of age ................................ ................... 227 3 92 Average daily food intake for 10 weeks of age ................................ ................. 228 3 93 Average daily food intake for 11 weeks of age ................................ ................. 229 3 94 Average daily food intake for 12 weeks of a ge ................................ ................. 230 3 95 Average daily food intake for 13 weeks of age ................................ ................. 231 3 96 Average plasma insulin concentration male MC3R and DKO mice. ................. 232 3 97 Plasma insulin concentration for 5 week old male DKO mice in conventional and running wheel cages. ................................ ................................ ................. 233 3 98 Plasma insu lin concentration for 6 week old male DKO mice in conventional and running wheel cages. ................................ ................................ ................. 234 3 99 Plasma insulin concentration for 7 week old male DKO mice in conventional and running wheel cag es ................................ ................................ .................. 235 3 100 Plasma insulin concentration for 8 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 236 3 101 Plasma insulin concentration for 9 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 237 3 102 Plasma insulin concentration for 10 week old male DKO mice in conventional and running whee l cages ................................ ................................ .................. 238 3 103 Plasma insulin concentration for 11 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 239 3 104 P lasma insulin concentration for 12 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 240 3 105 Plasma insulin concentration for 13 week old male DKO mice in conventional and runni ng wheel cages ................................ ................................ .................. 241 3 106 Plasma insulin of 13 week old male MC3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. ................................ ................................ ................................ ... 242

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18 3 107 Plasma insulin of 13 week old male MC3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. ................................ ................................ ................................ ... 243 3 108 Plasma insulin of 13 week old male MC3R WT and MC3R KO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. ................................ ................................ ................................ .................. 244 3 109 Plasma insuli n of 13 week old male MC3R WT and MC3R KO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. ................................ ................................ ................................ .................. 245 3 110 Average plasma leptin concentration for male MC3R and DKO mice in running wheel experiments. ................................ ................................ .............. 246 3 111 Plasma leptin concentration for 5 week old male mice from MC3R strain in conventional and running wheel cages. ................................ ............................ 247 3 112 Plasma leptin concentration for 6 week old male mice from MC3R strain in conventional and running wheel cages. ................................ ............................ 248 3 113 Plasma leptin concentrati on for 7 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 249 3 114 Plasma leptin concentration for 8 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 250 3 115 Plasma leptin concentration for 9 week old male mice from MC3R strain in conventional and running wheel cages. ................................ ............................ 251 3 116 Plasma leptin concentration for 10 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 252 3 117 Plasma leptin concentration for 11 week old male mice f rom MC3R strain in conventional and running wheel cages ................................ ............................. 253 3 118 Plasma leptin concentration for 12 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 254 3 119 Plasma leptin concentration for 13 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 255 3 120 Plasma lepti n concentration for 5 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 256 3 121 Plasma leptin concentration for 6 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 257

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19 3 122 Plasma leptin concentration for 7 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 258 3 123 Plasma lept in concentration for 8 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 259 3 124 Plasma leptin concentration for 9 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 260 3 125 Plasma leptin concentration for 10 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 261 3 126 Plasma le ptin concentration for 11 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 262 3 127 Plasma leptin concentration for 12 week old male DKO mice in conventional and running wheel c ages ................................ ................................ .................. 2 63 3 128 Plasma leptin concentration for 13 week old male DKO mice in conventional and running wheel cages ................................ ................................ .................. 264 3 129 Plasm a leptin concentration for 5 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 265 3 130 Plasma leptin concentration for 6 week old male mice from MC3R strain in conv entional and running wheel cages ................................ ............................. 266 3 131 Plasma leptin concentration for 7 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 267 3 132 Plasma leptin concentration for 8 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 268 3 133 Plasma leptin concentration for 9 wee k old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 269 3 134 Plasma leptin concentration for 10 week old male mice from MC3R strain in conventional and running wheel cage s. ................................ ............................ 270 3 135 Plasma leptin concentration for 11 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 271 3 136 Plasma leptin concentration for 12 week old male mice from MC3R strain in conventional and running wheel cages ................................ ............................. 272 3 137 Plasma leptin concentration for 13 week old male mice from MC3R s train in conventional and running wheel cages ................................ ............................. 273

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20 3 138 Plasma leptin of 13 week old male MC3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Lu evano et al. ................................ ................................ ................................ ... 274 3 139 Plasma leptin of 13 week old male MC3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al ................................ ................................ ................................ .... 275 3 140 Plasma leptin of 13 week old male MC3R WT and MC3R KO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. ................................ ................................ ................................ .................. 276 3 141 Average number of running wheel turns of male mice during the dark cycle of running wheel experiments. ................................ ................................ .............. 277 3 142 Average number turns of running wheel during dark cycle for 6 wee k old mice.. ................................ ................................ ................................ ................ 278 3 143 Average number turns of running wheel during dark cycle for 7 week old mice.. ................................ ................................ ................................ ................ 279 3 144 Average number tu rns of running wheel during dark cycle for 8 week old mice. ................................ ................................ ................................ ................. 280 3 145 Average number turns of running wheel during dark cycle for 9 week old mice. ................................ ................................ ................................ ................. 281 3 146 Average number turns of running wheel during dark cycle for 10 week old mice. ................................ ................................ ................................ ................. 282 3 147 Average number turns of running wheel during dark cycle for 11 week old mic e.. ................................ ................................ ................................ ................ 283 3 148 Average number turns of running wheel during dark cycle for 12 week old mice.. ................................ ................................ ................................ ................ 284 3 149 Average number turns of r unning wheel during dark cycle for 13 week old mice. ................................ ................................ ................................ ................. 285 3 150 Linear regression plots of final body weights versus total amount of exercise performed by male mice in running wheel cages.. ................................ ............ 286 3 151 Linear regression plots of final masses versus total amount of exercise performed by male mice in running wheel cages ................................ .............. 287 3 152 Body weights of male MC3R WT and KO mice in conventional and running wheel cages. ................................ ................................ ................................ ..... 288

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21 3 153 Average daily food intake of male MC3R WT and KO mice in conventional and running wheel cages ................................ ................................ .................. 289 3 154 Dark cycle activity as measured by turns of a running wheel for male MC3R WT and KO mice in running wheel cages ................................ ......................... 290 3 155 Body lengths of male MC3R WT and KO mice in conventional and running wheel cages. ................................ ................................ ................................ ..... 291 3 156 Body lengths of 5 week old male mice from a MC3R background. ................... 292 3 157 Body lengths of 6 week old male mice from a MC3R background. ................... 293 3 158 Body lengths of 7 week old male mice from a MC3R background .................... 294 3 159 Body lengths of 8 week old male mice from a MC3R background .................... 295 3 160 Body lengths of 9 week old male mice from a MC3R background .................... 296 3 161 Body lengths of 10 week old male mice from a MC3R background .................. 297 3 162 Body lengths of 11 week old male mice from a MC3R backgr ound .................. 298 3 163 Body lengths of 12 week old male mice from a MC3R background .................. 299 3 164 Body lengths of 13 week old male mice from a MC3R background .................. 300 3 165 Fat masses of male MC3R WT and KO mice in conventional and running wheel cages ................................ ................................ ................................ ...... 301 3 166 Fat Masses of 5 week old male mice from a MC3R background. ..................... 302 3 167 Fat Masses of 6 week old male mice from a MC3R background. ..................... 303 3 168 Fat Masses of 7 week old male mice from a MC3R background. ..................... 304 3 169 Fat Masses of 8 week old male mice from a MC3R background ...................... 305 3 170 Fat Masses of 9 week old male mice from a MC3R background ...................... 306 3 171 Fat Masses of 10 week old male mice from a MC3R background. ................... 307 3 172 Fat Masses of 11 week old male mice from a MC3R background. ................... 308 3 173 Fat Masses of 12 week old male mice from a MC3R background .................... 309 3 174 Fat Masses of 13 week old male mice from a MC3R background .................... 310 3 175 Fat Masses of 5 week old male mice from a MC3R background. ..................... 311

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22 3 176 Fat Masses of 6 week old male mice from a MC3R background. ..................... 312 3 177 Fat Masses of 7 week old male mice from a MC3R background ...................... 313 3 178 Fat Masses of 8 week old male mice from a MC3R background. ..................... 314 3 179 Fat Masses of 9 week old male mice from a MC3R background. ..................... 315 3 180 Fat Masses of 10 week old male mice from a MC3R background .................... 316 3 181 Fat Masses of 11 week old male mice from a MC3R background. ................... 317 3 182 Fat Masses of 12 week old male mice from a MC3R background .................... 318 3 183 Fat Masses of 13 week old male mice from a MC3R backg round .................... 319 3 184 Lean masses of male MC3R WT and KO mice in conventional and running wheel cages. ................................ ................................ ................................ ..... 320 3 185 Lean mass of 5 week o f male MC3R KO and WT mice in conventional and running wheel cages. ................................ ................................ ........................ 321 3 186 Lean mass of 6 week of male MC3R KO and WT mice in conventional and running wheel cages ................................ ................................ ......................... 322 3 187 Lean mass of 7 week of male MC3R KO and WT mice in conventional and running wheel cages. ................................ ................................ ........................ 323 3 188 Lean mass of 8 week of male MC3R KO and WT mi ce in conventional and running wheel cages. ................................ ................................ ........................ 324 3 189 Lean mass of 9 week of male MC3R KO and WT mice in conventional and running wheel cages ................................ ................................ ......................... 325 3 190 Lean mass of 10 week of male MC3R KO and WT mice in conventional and running wheel cages ................................ ................................ ......................... 326 3 191 Lean mass of 11 week of male MC3R KO and WT mice in conventional and running wheel cages ................................ ................................ ......................... 327 3 192 Lean mass of 12 week of male MC3R KO and WT mice in conventional and running wheel cages ................................ ................................ ......................... 328 3 193 Lean mass of 13 week of male MC3R KO and WT mice in conventional and running wheel cages ................................ ................................ ......................... 329 4 1 Potential gene expression results in experiments for the identification of potential drug targ ets. ................................ ................................ ....................... 376

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23 4 2 Hypothalamic expression of the AGRP gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 377 4 3 Hypothalamic expression of t he PRKAA1 (AMPK) gene in sedentary or exercising male mice. ................................ ................................ ....................... 377 4 4 Effect of voluntary exercise on the hypothalamic expression of the PRKAA1 (AMPK) gene.. ................................ ................................ ................................ .. 378 4 5 Hypothalamic expression of the CART gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 378 4 6 Hypothalamic expression of the CPT2 gene in sedentary or exercising m ale mice. ................................ ................................ ................................ ................. 379 4 7 Hypothalamic expression of the GCK gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 379 4 8 Hypothalamic expression of the HCRT gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 380 4 9 Effect of voluntary exercise on the hypothalamic expression of the HCRT1 gene. ................................ ................................ ................................ ................ 380 4 10 Hypothalamic expression of the INSR gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 381 4 11 Hypothalamic expression of the LEPR gene in sedentary or exercising male mice ................................ ................................ ................................ .................. 381 4 12 Hypothalamic expression of the MC3R gene in sedentary or exercising male mice ................................ ................................ ................................ .................. 382 4 13 Hypothalamic expression of the MC3R gene in sedentary or exercising male mice excluding mice lacking the MC3R. ................................ ........................... 382 4 14 Hypothalamic expression of the MC4R gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 383 4 15 Hypothalamic expression of the MC4R gene in sedentary or exercising male mice excluding mice lacking the MC4R ................................ ............................ 383 4 16 Hypothalamic expression of t he POMC gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 384 4 17 Hypothalamic expression of the NPY gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 384

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24 4 18 Hypothalamic expression of the NPY1R gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 385 4 19 Hypothalamic expression of the SOCS3 gene in sedentary or exercising male mice ................................ ................................ ................................ ......... 38 5 4 20 Effect of voluntary exercise on the hypothalamic expression of the SOCS3 gene. ................................ ................................ ................................ ................ 386 4 21 Hypothalamic expression of the UCP2 gene in sedentary or exercising male mice. ................................ ................................ ................................ ................. 386 4 22 Relative expression of the MC3R gene to the MC4R gene in the hypothalamus. ................................ ................................ ................................ .. 387 4 23 Relative expression of the AGRP gene to the POMC gene in the hypothalamus ................................ ................................ ................................ ... 387 4 24 Liver expression of the PRKAA1 (AMPK) gene in sedentary and exercising male mice. ................................ ................................ ................................ ........ 388 4 25 Liver expression of the CPT1A gene in sedentary and exercising male mice. 388 4 26 Effect of voluntary exercise on the expression of the C PT1A gene in male mice. ................................ ................................ ................................ ................. 389 4 27 Liver expression of the CPT2 gene in sedentary and exercising male mice. .... 389 4 28 Effect of vol untary exercise on the expression of the CPT2 gene in male mice. ................................ ................................ ................................ ................. 390 4 29 Liver expression of the DGAT1 gene in sedentary and exercising male mice. 390 4 30 Liver expression of the DGAT2 gene in sedentary and exercising male mice. 391 4 31 Effect of voluntary exercise on the expression of the DGAT2 gene in male mice ................................ ................................ ................................ .................. 391 4 32 Liver expression of the FASN gene in sedentary and exercising male mice. ... 392 4 33 Liver expression of the FASN gene in se dentary and exercising male mice excluding the DKO genotype ................................ ................................ ............ 392 4 34 Effect of voluntary exercise on the expression of the FASN gene in male mice excluding the DKO genotype. ................................ ................................ .. 393 4 35 Liver expression of the FBP1 gene in sedentary and exercising male mice. .... 393 4 36 Liver expression of the GCK gene in sedentary and exerc ising male mice.. .... 394

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25 4 37 Effect of voluntary exercise on the expression of the GCK gene in male mice. 394 4 38 Liver expressio n of the G6PC3 gene in sedentary and exercising male mice. 395 4 39 Liver expression of the SLC2A2 (GLUT2) gene in sedentary and exercising ... 395 4 40 Effect of voluntary exercise on the expression of the SLC2A2 (GLUT2) gene in male mice. ................................ ................................ ................................ .... 396 4 41 Liver expression of the PYGL gene in sedentary and exercising male mice. ... 396 4 42 Liver expression of the GYS2 gene in sedentary and exercising male mice. ... 397 4 43 Liver expression of the LIPE gene in sedentary and exercising male mice. .... 397 4 44 Effect of voluntary exercise on the expression of the LIPE gene in male mice. 398 4 45 Liver expression of the INSR gene in sedentary and exercising male mice. .... 398 4 46 Liver expression of the LEPR gene in sedentary and exercising male mice.. ... 399 4 47 Effect of voluntary exercise on the expression of the LEPR gene in male mice. ................................ ................................ ................................ ................. 399 4 48 Liver expression of the PFKL gene in sedentary and exercising ma le mice. .... 400 4 49 Relative expression of the GCK gene to the G6PC3 gene in the livers of male 400 4 50 Relative expression of th e GYS2 gene to the PYGL gene in the livers of male mice. ................................ ................................ ................................ ................. 401 4 51 Effect of voluntary exercise on the relative expression of the GYS2 gene to the PYGL gene in the livers of male mice. ................................ ........................ 401 4 52 Relative expression of the FBP1 gene to the PFKL gene in the livers of male mice ................................ ................................ ................................ .................. 402 4 53 Effect of voluntary exercise on the r elative expression of the FBP1 gene to the PFKL gene in the livers of male mice. ................................ ........................ 402 4 54 Relative expression of the DGAT1 gene to the DGAT2 gene in the livers of male mice. ................................ ................................ ................................ ........ 403 4 55 Effect of voluntary exercise on the relative expression of the DGAT1 gene to the DGAT2 gene in the livers of male mice. ................................ ..................... 403 4 56 Relative expression of the LIPE gene to the DGAT1 gene in the livers of male mice ................................ ................................ ................................ ......... 404

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26 4 57 Effect of voluntary exercise on the relative expression of the LIPE gene to the DGAT1 gene in the livers of m ale mice. ................................ ........................... 404 4 58 Relative expression of the LIPE gene to the DGAT2 gene in the livers of male mice.. ................................ ................................ ................................ ....... 405 4 59 Relative express ion of the FASN gene to the CPT1A gene in the livers of male mice.. ................................ ................................ ................................ ....... 405 4 60 Relative expression of the FASN gene to the CPT2 gene in the livers of male mice. ................................ ................................ ................................ ................. 406 4 61 Skeletal muscle expression of the PRKAA1 (AMPK) gene in male mice in conventional and running wheel cages. ................................ ............................ 406 4 62 Skeletal muscle expression of the CP T1B gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 407 4 63 Skeletal muscle expression of the CPT2 gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 407 4 64 Skeletal muscle expression of the GCK gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 408 4 65 Skeletal muscle expression of the G CK gene in male mice in conventional and running wheel cages ................................ ................................ .................. 408 4 66 Skeletal muscle expression of the SLC2A4 (GLUT4) gene in male mice in conventional and running wheel cages. ................................ ............................ 409 4 67 Skeletal muscle expression of the PYGM gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 409 4 68 Skeletal muscle expression of the GYS1 gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 410 4 69 Skeletal muscle expression of the IL 6 gene in male mice in conventional and running wheel cages. ................................ ................................ ........................ 410 4 70 Skeletal muscle expression of the PFKM gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 411 4 71 Skeletal muscle expressio n of the UCP2 gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 411 4 72 Skeletal muscle expression of the UCP2 gene in male mice in conventional and running wheel cages. ................................ ................................ ................. 412

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27 4 73 Skeletal muscle expression of the UCP3 gene in male mice in conventional and running wheel cages ................................ ................................ .................. 412 4 74 Skeletal muscle expressio n of the UCP3 gene in male mice in conventional and running wheel cages ................................ ................................ .................. 413 4 75 Relative expression of the UCP2 gene to the UCP3 gene in the skeletal muscle tissue of male mice ................................ ................................ ............... 413 4 76 Relative expression of the GYS1 gene to the PYGM gene in the skeletal muscle tissue of male mice ................................ ................................ ............... 414 4 77 Relative expression of the GYS1 gene to the PYGM gene in the skeletal muscle tissue of male mice ................................ ................................ ............... 414 5 1 Weekly average body weights for male (A) and female (B) MC4R mice in conventional and running wheel cages. ................................ ............................ 436 5 2 Fat mass was measured by quantitative MRI for male (A) and female (B) MC4R mice in conventional and running wheel cages. ................................ .... 437 5 3 Lean Mass measured by quantitative MRI for male (A) and female (B) MC4R mice ................................ ................................ ................................ .................. 438 5 4 Linear growth was measured weekly by monitoring nasal anal length for male and female MC4R mice in sedent ary and exercise treatment groups.. .... 439 5 5 Female MC4R mouse food intake in conventional and running wheel cages. .. 440 5 6 Mean plasma insulin concentration for male (A) and female (B) MC4R mice in conventional and running wheel cages. ................................ ........................ 441 5 7 Average circulating leptin concentration for male (A) and female (B) MC4R mi ce in conventional and running wheel cages ................................ ................ 442 5 8 Hypothalamic gene expression of male and female MC4R mice in conventional cages normalized to male WT mouse group. .............................. 443 5 9 Hypothalamic gene expression for male and female MC4R mice. Hypothalamic gene expression for male mice (A) was normalized to male WT Conv group gene expression levels. ................................ ................................ 444 5 10 Average number of running wheel turns per dark cycle for male and female MC4R mice housed in running wheel cages ................................ ..................... 445 5 11 Correlations of body weights and fat ma sses to total amount of exercise performed ................................ ................................ ................................ ......... 446

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28 6 1 Figure summarizing the effects of genotype and exercise on MC3R KO mice compared at 13 weeks of age (after 8 weeks of exercise) to MC3R WT mi ce. 459 6 2 Figure summarizing the effects of genotype and exercise on MC4R KO mice compared at 13 weeks of age (after 8 weeks of exercise) to MC4R WT mice. 460 6 3 Figure summarizing the effects of genotype and exercise on DKO mice compared at 13 weeks of age (after 8 weeks of exercise) to either MC3R WT or MC4R WT mice. ................................ ................................ ........................... 461 6 4 Figure summarizing the effects of genotype and exercise on hypothalamic gene expression in male MC3R KO mice. ................................ ........................ 462 6 5 Figure summarizing the effects of genotype and exercise on hyp othalamic gene expression in male MC4R KO mice. ................................ ........................ 463 6 6 Figure summarizing the effects of genotype and exercise on hypothalamic gene expression in male DKO mice. ................................ ................................ 464 6 7 Figure summarizing the effects of genotype and exercise on hepatic gene expression in male MC3R KO mice. ................................ ................................ 465 6 8 Figure summarizing the effects of gen otype and exercise on hepatic gene expression in male MC4R KO mice. ................................ ................................ 466 6 9 Figure summarizing the effects of genotype and exercise on hepatic gene expression in male DKO mice. ................................ ................................ ......... 467 6 10 Figure summarizing skeletal muscle gene expression in male MC3R KO mice. ................................ ................................ ................................ ................. 468 6 11 Figure summarizing skeletal muscle gene expression i n male MC4R KO mice. ................................ ................................ ................................ ................. 469 6 12 Figure summarizing skeletal muscle gene expression in male DKO mice. ....... 470

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29 LIST OF ABBREVIATION S ACTH Adrenocorticotropic hormone A DP Adenosine diphosphate AGRP Agouti related protein AMP Adenosine monophosphate AMPK 5' adenosine monophosphate activated protein kinase ARC Arcuate nucleus ATP Adenosine triphosphate A y Lethal yellow agouti BBB Blood brain barrier BDNF Brain derived neu rotrophic factor BMI Body mass index bp Base pair cAMP Cyclic adenosine monophosphate CART Cocaine and amphetamine regulated transcript cDNA Complementary strand deoxyribonucleic acid Conv Conventional CNS Central nervous system CPT1 Carnitine palmitoyltr ansferase 1 CPT2 Carnitine palmitoyltransferase 2 db/db Diabetic gene mutation ddH 2 O Distilled deionized water DGAT1 Diglyceride acyltransferase 1 DGAT2 Diglyceride acyltransferase 1 DIO Diet induced obesity

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30 DKO Double knockout DMN Dorsomedial nucleus DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid FASN Fatty acid synthase FBP1 Fructose 1,6 biphosphatase GCK Glucokinase GDP Guanosine diphosphate GLUT2 Glucose transporter 2 GLUT4 Glucose transporter 4 GTP Guanosine triphosphate GYS1 Glycogen synthase 1 GYS2 Glycogen synthase 2 HCRT Hypocretin (Orexin) HPRT1 Hypoxanthine guanine phosphoribosyl transferase 1 LIPE Hormone sensitive lipase i.c.v. Intracerebroventricular IL 6 Interleukin 6 KO Knockout MTA Material transfer agreement MCR Melanocorti n receptor MC1R Melanocortin 1 receptor MC2R Melanocortin 2 receptor MC3R Melanocortin 3 receptor MC4R Melanocortin 4 receptor

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31 MC5R Melanocortin 5 receptor MRI Magnetic resonance imaging mRNA Messenger ribonucleic acid MSH Melanocyte stimulating hormone N aCl Sodium chloride NPY Neuropeptide Y NPY1R Neuropeptide Y receptor type 1 ob/ob Obese gene mutation PFK Phosphofructokinase POMC Proopiomelanocortin PPIA Peptidylprolyl isomerase A PTEN Phosphatase and tensin homolog PYG Glycogen phosphorylase RNA Ribonu cleic acid RW Running Wheel SDS Sodium dodecyl sulfate SOCS3 Suppressor of cytokine signaling 3 TAE Tris Acetic acid EDTA Tris Tris(hydroxymethyl)amino methane UCP2 Uncoupling protein 2 UCP3 Uncoupling protein 3 UV Ultraviolet WT Wild type

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32 Abstract of Dis sertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF VOLUNTARY EXERCISE ON THE CENTRAL MELANOCORTIN RECEPTOR KNOCKOUT MICE By J ay Schaub August 2011 Chair: Carrie Haskell Luevano Major: Pharmaceutical Sciences Human obesity, defined as possessing a body mass index value of greater than 30, is reaching critical levels world wide. Obese individuals are at greater risk for other d iseases including cardiovascular disease, type 2 diabetes, and certain types of cancer. In addition to environmental and lifestyle causes, certain genetic mutations have been shown to result in obesity. Genetic deletion of the melanocortin 3 and 4 recepto rs (MC3R and MC4R respectively) have been shown to result in altered metabolisms and phenotypes in mice. Voluntary exercise has been shown by the Haskell Luevano laboratory to delay the onset of the obesity of melanocortin 4 receptor knockout (KO) mice, pr eventing early onset weight gain and increases in fat mass. Running wheel exercise was also found to change hypothalamic expression levels of genes involved in energy homeostasis. This dissertation investigates the effects of voluntary exercise on the phen otypes and gene expression profiles of male mice lacking the MC3R, the MC4R, or both the MC3 and MC4 receptors (DKO mice). Additionally the effects of MC4R deletion and voluntary exercise were investigated in male and female mice to determine what effects,

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33 if any, gender had on the phenomenon seen in male MC4R KO mice allowed to exercise. Genotype and exercise both had significant effects on both phenotype and gene expression in male mice lacking the one or both of the central melanocortin receptors. Vol untary exercise resulted in a significant decrease in body weight in MC4R KO and DKO mice (P<0.001) and total fat mass in exercising MC3R KO, MC4R KO, and DKO mice (P<0.05) compared to the same genotypes in conventional cages. Furthermore, changes in expre ssion levels of genes involved in liver fatty acid metabolism seen in sedentary MC4R KO mice were prevented by voluntary exercise. Significant differences in phenotype and hypothalamic gene expression were seen between male and female MC4R KO mice. The obe se phenotype was diminished in female mice, in part due to the lack of hyperphagia generally associated with MC4R KO dysfunction. Overall, voluntary exercise had a beneficial effect on central melanocortin receptor KO mice, delaying the onset of the asso ciated phenotypes and preventing changes in gene expression.

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34 CHAPTER 1 OBESITY, EXERCSE, AN D THE MELANOCORTIN S YSTEM Obesity Obesity is an increasing health concern worldwide. Approximately one third of adult Americans are obese, with another third bein g classified as overweight, leaving only one in three adult Americans classified as normal weight. 1 Definition Human obesity is measured on the body mass index (BMI) scale which is calculated by dividing the weight of a person in kilograms by the square of their height in meters. This value can then be compared to scales de termined by health organizations to determine the weight class of the person (Table1 1). People with a BMI less than 18.49 are considered to be underweight. Patients with a body mass index between 18.5 and 24.99 are considered to be of normal weight. Perso ns with a BMI greater than 25 are considered to be overweight, while those with a BMI over 30 are classified as obese. BMI has also been shown to correlate with percentage of body fat in human patients, validating this method as a tool for estimating adipo sity. 2 Obesity is a significant risk factor for many other diseases including cardiovascular disease, type 2 diabetes, hypertension, stroke, asthma and certain types of cancer. 3 Health care costs associated with obesity were estimated to be as high as $147 billion in 200 6 in the United States alone. 4 When this cost was spread out to all obese patients it is estimated that they have approximately $1,400 more in healthcare costs per year compared to normal weight patients. 4 Both the link of obesity to other debilitating diseases and the large economic cost have made obesity a key area of research in the field of health sciences.

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35 Causes Weight change is caused by an imbalance in energy intake and expenditure. Weight gain occurs when energy intake is greater than energy expenditure, whereas weight loss occurs when the opposite occurs as illustrated in Figure 1 1. Humans can shift their energy balance to affect weight loss through caloric restriction (dieting) or by increas ing energy expenditure through exercise. Alternatively, over consumption of food and an inactive lifestyle can lead to weight gain. In addition to lifestyle, obesity has been linked to genetic defects that result in hyperphagia or changes in metabolism. Mo use models resembling human obese genetic defects include the db, the fat, the tub, and the ob gene mutations. 5 7 Also, mutations in the genes of the melanocortin system have resulted in mice with altered metabolisms. 8 1 2 Melanocortins Receptors The melanocortin system consists of five G protein coupled receptors (MC1 5R) and their endogenous peptide ligands (Figure 1 2). The melanocortin receptors are distributed throughout the body and are involved in cellular signalin g pathways for a wide variety of physiological processes. The melanocortin 1 receptor (MC1R) is involved in the control of skin, hair, and fur pigmentation. 13 The melanocortin 2 receptor (MC2R), involved in steroidogenesis and the stress response pathw ay, is expressed in cells in the islets of the pancreas. 13 15 The MC2R is unique among the melanocortin receptors in that it only stimulated by the endogenous A CTH agonist and not the others. 13 The melanocortin 3 receptor (MC3R) is expressed in the central nervous system, the pancreas, the heart, and the

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36 gastrointestinal tract. 16 18 Expression of the MC3R in these tissues and the altered metabolism of the MC3R KO mouse, has led to the hypothesis that the MC3R is involved in energy homeostasis. 8,9 The melanocortin 4 receptor (MC4R) is expressed in the central nervous system and has been demonstrated to be directly involved in food intake and energy homeostasis. 19 23 The involvement of the MC3R and MC4R in food intake and energy homeostasis have made them popular targets for the discovery of new compounds for the treatment of obesity and metabolic syndrome. The melanocortin 5 receptor (MC5R), is ubiquitously expressed throughout the body. 24 While the role of the MC5R is not completely understood, it is hypothesized to be involved in exocrine gland function. 24 Ligands Agonists The proopiomelanocortin (POMC) gene is post translationally cleaved by converting en melanocyte 1 2 MSH. ACTH. 25 MSH peptides all possess agonist activity at all of the melanocortin receptors, with the important exception of the MC2R which is only stimulated by ACTH. 13,26 The melanocortin agonists all possess a common tetrapeptide core sequence of His Phe Arg Trp that has been postul ated is required for receptor recognition and activation of the receptors (Figure 1 2). Further study of the chemistry and structure of the endogenous melanocortin agonists led to the development of highly potent synthetic agonists Nle 4 DPhe 7 MSH (NDP M SH) (Ac Ser Tyr Ser Nle Glu His DPhe Arg Trp Gly Lys Pro Val NH 2 ) and melanotan II (MTII) (Ac Nle c[Asp His DPhe

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37 Arg Trp Lys] NH 2 ). 27,28 Central adm inistration of synthetic melanocortin agonists results in a dose dependant decrease in food intake in rodents. 23,29 Antagonists The melanocortin system is unique among GPCRs in that it has the endogenous antagonists agouti signaling protein (ASP) and agouti related protein (AGRP). 12,30,31 Agouti is normally expressed in the skin two to seven days after birth and is directly involved in modulation of skin, hair, and fur pigmentation by antagonizing the MC1R. 32,33 The peptide ho rmone AGRP is expressed in the central nervous system and is an antagonist of the MC3R and MC4R. 31 Central administration of melanocortin antagonists increases food intake in rodent models. 23,29,34 Further development of synthetic melanocort in receptor ligands is anticipated to lead to drugs for the treatment of both overeating and wasting diseases (e.g. cachexia). Knockout mice Phenotypes Melanocortin 3 receptor knockout mouse. The melanocortin 3 receptor KO mice possess similar body weights to WT littermates, but have a significant increase in adipose tissue mass (Figure 1 3). 8,9 The effect of the MC3R on physical activity is unclear with varied reports of decreased activity compared to WT littermates while others report no difference in activity levels between WT and MC3R KO mice. 8,9,35 Further studies with MC3R KO mice have found evidence that the increased fat mass seen in MC3R KO mice may be due in part to decreased fatty acid oxidation. 36 Additional work has impli cated the MC3R for a role in anticipatory behavior in food restriction studies. 35

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38 Melanocortin 4 receptor knockout mouse. Generation of the melanocortin 4 receptor KO mouse was first reported in 1997 by Huszar et al. 10 The MC4R null mice present with increased body weight compared to their wildtype littermates with mice heterozygous for the MC4R deletion possessing an intermediate phenotype. 10 Haskell Lue vano et al. later demonstrated that the increase in body weight of male MC4R KO mice was largely due in part to an increase in adipose tissue (Figure 1 3) that was easily seen by imaging MRI scans. 37 The obesity of the MC4R KO mice has been determined to be caused by the hyperphagic behavior as opposed to hypometab olism. 38 Female MC4R KO mice have been reported to have changes in energy expenditure and fatty acid oxidation after being on a hig h fat diet. 39 Since their generation, the MC4R KO mice have been a valuable control and have been shown to be key in other signaling pathways including the leptin and serotonin pathways. 40 43 Double knockout mouse. Mice lacking both the MC3R and MC4R (herein referred to as double knocko ut or DKO mice) possess even greater body weights than either MC3R KO or MC4R KO mice. 9 The extent of the obesity of the DKO mice is greater than the additive obesity of the MC3R KO and MC4R KO mice combined, su ggesting a synergistic or compensating effect by the MC3R and MC4R when one or the other is deleted or dysfunctional. Link to clinical obesity Populations of obese individuals have been identified with mutations in melanocortin signaling pathway. Patients with mutations resulting in POMC gene product insufficiency suffer from early onset obesity, adrenal insufficiency, and have red pigmented hair. 44 Mutations of the central melanocort in 4 receptor have been

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39 documented in clinically obese patients. 45 48 Mutations of the MC3R gene have also been identified in obese human patients, with changes in pharmacology seen in some mutations. 49 54 The mechanism, dominance, and the contribution of these mutations to the obese phenotype remains controversial due to conflicting findings. 47,49 55 The same hyperphagic, overweight phenotype seen in human patients is also seen in the MC4R KO mouse. 10 Because of t he translational aspect of their phenotype, MC4R KO mice are a unique tool for the investigation for monogenetic onset obesity in an experimental setting. Exercise Treatment for Diseases Exercise is a well accepted method of controlling body weight by inc reasing the energy expenditure of the body. Exercise has been demonstrated to have a positive effect on general health in both humans and animals, including the added benefit of weight loss. 56 61 Much like restricted food intake, by shifting the energy balance of the body, weight l oss is achieved by the body burning energy stores resulting in loss of body weight (Figure 1 1). Exercise, along with dietary modification, is the only universally prescribed treatment for obesity, type 2 diabetes, and other metabolic disorders. 62 The effects of exercise are broad with exercise having been shown to positively affect thermoregulation, circulatory efficiency, metabolism, physical endurance, appetite, sleep patterns, stress pathways, mental health, and chronic diseases including obesity and type II diabetes. 37,56 72 Animal Models of Exercise Th ere are multiple experimental methods for studying the effects of exercise in animal models. Treadmill equipment is available for both mice and rats to study the

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40 effects of exercise. 73 75 Generally, treadmills are equipped with a shock grid, forced air, or cold water jets to mot ivate animals to exercise by providing a unpleasant stimulus if animals stop running and fall off the end of the treadmill. 76 Treadmills are a useful tool for studying exercise in rodents since the exercise program can be modified by varying the treadmill incline, speed, and duration as well as pairing the treadmill with calorimetric equipment to monitor the 76 Treadmills are an effective method for determining maximum endurance of an animal b y measuring how long an animal can run until it reaches a point of exhaustion (generally measured by animals submitting to a repeated number of stimuli without restarting exercise). Drawbacks to treadmill training include needing to train the animals to us e the treadmill, the cost of purchase and maintaining the treadmill equipment, and the fact that only one animal can be exercised at a time. measuring length of time before animal reaches a point of exhaustion and stops swimming. Disadvantages to swimming include that it is difficult to quantify the intensity of the exercise, that there is a risk of drowning of the experimental animals, and that swimming is a stressful activity use d in studies involving stress response. 76,77 Running wheels are one method that can be used to quantitatively me asure physical activity in the home cage. 8,35,37,63,64,74,78 80 One be nefit of using voluntary exercise equipment in the home cages is reducing the need to handle the experimental animals. This can decrease the amount of stress the mice experience which is particularly important for mice without full melanocortin receptor ac tivity. 81 The clear disadvantage of voluntary exercise in experimental settings is that the animals choose

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41 the time, length, and intensity of their bouts of exercise making it difficult to compare betwe en animals or experimental groups. One thing that is clear based on the literature is that the results obtained from different types of exercise are not directly comparable or indicative of results obtained using a different type of exercise. 74,82 Gene Expression General Background Deoxynucleic acid (DNA) is composed of a phosphate and sugar (deoxyribose) backbone with purine or pyrimidine bases linked to the sugar molecule. The purine bases are adenine and guanine while cytosine and thymi ne are the pyrimidine bases. The bases are grouped in units of three, called codons, which correspond to specific amino acids during protein synthesis. In order for the gene sequences that are found in the DNA to be converted to functional proteins, the DN A sequence must first be transcribed into messenger ribonucleic acid (mRNA) by RNA polymerase. Messenger RNA is chemically similar to DNA with the exception that ribose is used instead of deoxyribose for the sugar phosphate backbone, that the pyrimidine ba se uracil is used instead of thymine, and that mRNA is single stranded. Genes in DNA are composed of exons and introns, coding and non coding sections respectively. In mature mRNA, the introns sections have been removed by the RNA splicing process. Messeng er RNA is translated into chains of amino acids (peptides or proteins) by ribosomes using transfer RNA (tRNA) to supply the single amino acids. Proteins can be further modified after translation to modulate function and activity, often by the addition of f unctional groups such as phosphate, carbohydrates, and lipids as well as structural changes such as folding and the formation of disulfide bridges.

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42 While phenotype is ultimately determined by protein quantities, it can be influenced at the DNA level, the mRNA level, the protein level, or a combination of the three. Presence or lack of a gene in the genome has obvious influence over whether or not a protein can be expressed since without the DNA template, proteins will not be synthesized. An example of this are knockout or transgenic mice where a gene has been artificially removed or inserted respectively, resulting in an altered phenotype. 8 10,12 Depending on the presence of up or down regulatory transcription factors, mRNA levels can be up or down regulated resulting in changes in final protein quantity. Phenotype can also be affected by improper protein folding and localization due to changes in protein sequence or improper intra cellular trafficking. Finally, phenotype can be influenced at the protein level by changes in protein synthesis or degradation pathways resulting in different concentrations of protein or by chemical changes to the protein (commonly phosphorylation). Becau se the genomes of the MC3R KO, MC4R KO, and DKO mice are intact other than the melanocortin receptors, it would not be anticipated that studying the sequence of genomic DNA of the different strains would result in significant differences among them. Techni ques for evaluating and quantitatively measuring differences in mRNA, protein, and modified proteins are all well established. Messenger RNA can be measured by northern blot which separates the mRNA fragments based on size and then can probe specific sequ ences with labeled oligonucleotides sequences which can be measured by densitometry techniques. Alternatively, advances in technology allow for the selective amplification of targeted sequences of interest, with increases in copy number being measured in real time. This

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43 technique is called real time polymerase chain reaction (RT PCR). The technique used in the experiments described in Chapters 4 and 5 is called the TaqMan assay and is commercially available from Applied Biosystems. The TaqMan assay uses a two stranded complementary DNA (cDNA) molecule as a template that is synthesized from mRNA. The cDNA is amplified using a solution that includes a forward primer, a reverse primer, and primer that is linked to a fluorescent tag and quencher molecule. When the the fluorescent tag and quencher from the target primer. When the fluorescent tag is no longer in close proximity to the quencher molecule, it can be read by the ABI7 300 RT PCR machine. As the target gene sequence is amplified during each sequential cycle, the amount of free fluorescent tag in solution increases until it reaches a point where it can be measured by the RT PCR machine. The point at which the signal from the fluorescent molecule reaches a point ten times greater than the background signal is the threshold cycle (Ct). 37 The Ct value for the gene of interest is compared to a gene that has consistent expression for all the experimental groups to determine relative quantitative expression. RT PCR is widely used and is considered a valid method of studying gene expression. 35 37,39 One limitation of the RT PCR assay is that presence of mRNA does not guarantee translation of the gene or modification of protein to the active form. Proteins and modified proteins levels can be m easured by western blot which separates the proteins based on size and then uses antibodies to probe for specific sequences, structures, or modifications within the protein for identification. Antibodies linked with enzymes or fluorescent tags can be used to determine protein amount by

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44 densitometry. More advanced techniques for measuring protein concentrations involve the use of two dimensional gels (separation by charge and size) and mass spectrometry. Use of antibodies can also indicate activity status su ch as folding or post translational modification which cannot be determined by RT PCR. Limitations of measuring protein levels include the cost and complexity of experiments that use antibodies. Because each antibody is different, many require different i ncubation times and wash procedures, requiring optimization for each protein before the experiment. This contrasts with assays like the TaqMan RT PCR procedure where each probe is optimized by the commercial vendor for a standard procedure. Additionally, c oncerns exist over the effectiveness of primary antibodies for complex proteins imbedded in cellular membranes (such as the melanocortin receptors) cannot be guaranteed since the antigens used for antibody generation are sometime just fragments of the extr acellular domain. As a result, these antibodies would require verification of selectivity for functional receptors before use. Ultimately, mRNA and protein quantification experiments are complementary techniques since a change in protein levels can be aff ected at the transcriptional, the translational, the post translational, or all three levels. Simply put, an increase in mRNA expression does not directly translate to an increase in protein level, while a decrease in protein level does not necessarily ind icate a decrease in mRNA levels. In order to definitively know at which step protein levels are affected, both mRNA and protein levels for a specific gene must be measured. However, due to constraints involving time, equipment, and cost laboratories genera lly focus on one method or the other. The Haskell Luevano laboratory has successfully utilized the TaqMan RT PCR assay in the

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45 past, so that is the method that was used in the experiments described within this dissertation. 37 Genes Involved in Energy Balance Pathways Energy balance can be simplified to energy intake (calories from food and drink consumed) and energy output (energy needed for metabolic processes and physical activity). A shift in the energy balance resulting in a surplus of calories results in weight gain, often in the form of fatty adipose tissue, wh ile a deficit in energy results in weight loss (Figure 1 1). In the current obesity epidemic, the goal of many treatments is to shift the energy balance of the body towards a deficit to promote weight loss by restriction of caloric intake or by increasing the level of physical activity. Exercise increases the metabolic demand of the body by maintained muscle contractions and afterwards to replace depleted energy stores. Since exercise is rarely isolated to one part of the body, it can directly influence mul tiple homeostatic systems. Reports have shown exercise to positively effect thermoregulation, circulatory efficiency, endurance, metabolism, appetite, sleep patterns, stress pathways, and chronic diseases including obesity and type II diabetes. 56 61,65 While the benefits o f exercise are well documented, the physiological and cellular mechanisms by which exercise propagates its effects are still under investigation. There are numerous pathways in the body involved in energy homeostasis, including several major processes that control nutrient use, transport, storage, and availability. Glucose and fatty acids are two of the substrates the body commonly uses as energy sources for the generation of adenosine triphosphate (ATP). 83 The body is capable of storing both glucose and fatty acids in more complex molecules (glycogen

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46 and trigl ycerides respectively) and breaking them down in times of nutrient deficiency (e.g. during a fast). 83 Glucose transport into and out of cells is facilitated by glucose transporters (GLUT) which are members of the solute carrier 2 family which preferentially allow glucose to cross the cell membrane along its c oncentration gradient. 84 GLUT2 is expressed in the pancreas, liver, intestines, and kidneys and has a low aff inity for glucose with a high rate of turn over which is believed to facilitate glucose release from the liver. 85,86 Translocation of GLUT4, expressed in adipose tissue and in muscle cells, to the cell surface is induced by increased insulin concentration. 87 89 The first step of the intracellular glucose metabolism pathway is the phosphorylation of glucose by glucokinase (GCK) to form glucose 6 phosphate. 83 Glucose 6 phosphate can then enter either the glycolysis pathway to be further metabolized for the generation of ATP or the glycogen esis pathway where uridine diphosphate glucose can be added on to a growing chain of glycogen by glycogen synthase (GYS). 83 Conversely, glycogen can be broken down into glucose 1 phosphate subunits by glycogen phosphorylase (PYG) which can be converted to glucose 6 phosphate and either enter the glycolysis pat hway, or be hydrolyzed back into glucose and released into the blood stream by glucose 6 phosphatase (G6PC3). 83 Once in the glycolysis pathway, the rate limiting enzyme in the transition of glucose 6 phosphate to molecules of pyruvate is phosphofructokinase (PFK). Additionally, glucose 6 phosphate can be synth esized from derivatives such as pyruvate or lactic acid via the gluconeogenesis pathway. 83 Fructose 1,6 biphophatase (FBP1), the rate limiting enzyme of the gluconeogenic pathway, catalyzes the conversion of fructose 1,6

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47 biphosphate to fructose 6 phosphate, and is independently regulated by insulin and cyclic adenosine monophosphate (cAMP). 90,91 In addition to glucose, fatty acids are an important energy source, and similar to glucose can either be used immediately, or stored for future use. In order for fatty acids to be metabolized for energy production, they must first be transported into the mitochondrion. The enzymes carnitine palmitoyltransferase 1 and 2 (CPT1 and CPT2) are responsible for associating fatty acids with carnitine so that they can cross the outer and inner membrane s of the mitochondrion and be subjected to oxidation to generate acetyl Co A. 92 95 Fatty acids can be synthesized intracellularly from shorter carbon chains by fatty acid synthase (FASN) in a repetitive manner to form palmiti c acid. 96,97 Long chain fatty acids can be stored and transported throughout the bo dy as triglycerides, molecules consisting of one molecule of glycerol and three fatty acids. Addition of fatty acids to the final alcohol group of diglycerides is catalyzed by diglyceride acyltransferase (DGAT) via an acyl CoA intermediate of the fatty aci d. 98 Fatty acids are released from trig lycerides by hormone sensitive lipase (LIPE), levels of which are modulated by circulating hormone factors including the melanocortin agonist ACTH, epinephrine, glucagon, norepinephrine, growth hormone, thyroxine, and thyroid stimulating hormone. 99 Substrate o xidation for the formation of ATP is an important process to ensure there is enough ATP for the cell to properly complete the multitude of functions that require it. Hydrolysis of ATP, to provide energy for cellular functions, yields ADP, which can then do nate one of its remaining phosphates to another ADP forming an ATP and AMP. 83 The ratio of the concentration of AMP to ATP is monitored by the protein 5

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48 AMP activated protein kinase (AMPK) which is emerging as an important control mechanism in directing cellular energy homeostasis (Reviewed in 100 ). The AMPK enzyme undergoes a conformational change under high levels of intracellular AMP, l eading to its activation. 101 In the experiments presented in Chapter 4, gene expression of the alpha 1 catalytic subunit (PRKAA1) of the AMPK protein are studied. Generation of ATP in the mit ochondria is driven by the proton gradient that exists between the inner and outer mitochondrial membranes. 83 The uncoupling proteins are part of the mitochondrial anion carrier protein family, and act to dissipate the proton gradient before it can be used by ATP synthase for ATP production, thereby reducing A TP synthesis. 102 There are many organs involved with the regulation and control of energy homeostasis and energy stores in the body including the brain, the liver, the pancreas, as well as adipose and muscle tiss ues. In order for the body to manage its energy stores it is essential that the body be able to communicate with itself to monitor the size of energy stores available. Leptin and insulin are two endocrine hormones that are released into the blood stream th at carry information about the bod cells of the pancreas and induces glucose uptake into cells via the insulin inducible solute carrier GLUT4, essentially lowering circulating glucose concentrations. 87 89 Insulin is secreted in response to increasing blood glucose concentrations and is further influenced by a class of molecules secreted by the gastrointestinal tract called incretins. 103,104 Leptin is a polypeptide signaling molecule secreted primarily by the adipocytes of white adipose tissue, with leptin concentration correla ting closely to white adipose tissue mass. 105 107 Leptin signals through neurons in the hypothalamus to cause feelings of satiety which in

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49 turn reduces food intake. 43 Leptin must pass through the blood brain barrier (BBB) by a saturable transport mechanism independent of leptin production or leptin receptors in order to exert its anorexigenic effects on the brain. 108,109 It has been proposed that the saturable transport mechanism is why obese individuals with increased levels of circulating leptin do not red uce their food intake accordingly. This hypothesis has been supported by showing that centrally injected leptin is still able to cause reduction in food intake in animals with elevated circulating leptin. 110 Insulin and leptin signal through the insulin recep tor (INSR) and leptin receptor (LEPR) respectively. Suppressor of cytokine signaling 3 (SOCS3) is a protein that modifies the signaling potency of various cytokines, including decreasing the leptin sensitivity of POMC neurons. 111 Interleukin 6 (IL 6) acts as both a pro and anti inflammatory cytokine, which is released, in part, by contracting muscle tissue resulting in elevated circulating concentrations of IL 6. 112 It has been demonstrated that blocking the effects of IL 6 p revents the increase of central insulin and leptin sensitivity gained by exercise. 59 Figure 1 4 graphically summarizes the general metabolic pathways that were investigated in the experiments presented within this work with the gene of interest from each step of the pathway identified. Central Gene Expression The influence of the melanocortin ligands and receptors on the central nervous studies. 23,29,34 The POMC, AGRP, MC3R, MC4R, cocaine and amphetamine regulated transcript (CART), hypocretin (HCRT), neu ropeptide Y (NPY), and neuropeptide Y 1 receptor (NPY1R) genes have been previously studied for their involvement in energy homeostasis and metabolism. 37 Other genes involved in both brain tissue metabolism

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50 and signaling were identified for analysis to determine what effect, if any, inactivation of the central mela nocortin receptors and voluntarily exercise by running wheel have on their expression. These genes are PRKAA1 (AMPK), CPT2, GCK, INSR, LEPR, SOCS3, and UCP2 (Figure 1 5). In order to remain consistent with previously published data, gene expression was stu died in the hypothalamus of the brain. 37 Peripheral Gene Exp ression Liver The liver is an extremely important metabolic organ that is involved in processes such as maintaining proper blood glucose levels and storing excess energy in the form of glycogen and lipids. To evaluate the effect of inactivation of one or m ore of the centrally expressed melanocortin receptors and voluntarily exercise treatment on metabolic function of the liver, the expression levels of the PRKAA1 (AMPK), CPT1A, CPT2, DGAT1, DGAT2, FASN, FBP1, GCK, G6PC3, GLUT2, PYGL, GYS2, LIPE, INSR, LEPR, and PFKL genes were measured by RT PCR (Figure 1 5). Pancreas The pancreas is an organ central to metabolic homeostasis through the release of insulin and glucagon to control blood glucose levels. To study the effect of central melanocortin receptor inact ivation and the effects of voluntary exercise, the TaqMan RT PCR assay was used to measure GCK, GLUT2, MC2R, MC3R, and UCP2 gene expression in the pancreas (Figure 1 5). Muscle Skeletal muscle is clearly an important tissue to examine when considering the effects of voluntary exercise. Skeletal muscle would logically contribute a large amount to the extra energy expended during exercise. PRKAA1 (AMPK), CPT1B, CPT2, GCK,

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51 GLUT4, PYGM, GYS1, PFKM, UCP2, and UCP3 gene expression were all measured in muscle tiss ue taken from the leg to determine what effect, if any, MCR inactivation and running wheel exercise had on skeletal muscle gene expression (Figure 1 5). Additionally, IL 6 release by muscle tissue has been linked to some of the positive effects of exercise so IL 6 gene expression was measured to see if it increased with access to running wheel equipment. 59,112 Overview and Objectives Obesity is a disease that affects an overwhelming number of people worldwide including one in three adults in the United States of America. 1 In addition to environmental and lifestyle causes, certain gene mutations have been shown to result in obesity. 8 10,36,37,39,44,48,64,71,80,113 The MC4R KO mouse has emerged as an experimental animal model for obesity that mirrors the phenotype seen in human patients with dysfunctional melanocortin signaling. Voluntary exercise by running wheel has been shown to delay the obese phenotype and alter hypothalamic gene expression in male MC4R KO mi ce. 37,63,64 The objective of this dissertation is to test the hypothesis that voluntary exercise is able to prevent the metabolic dysfunction seen in mic e lacking the central melanocortin receptors. This hypothesis will be tested by: providing an in depth characterization of phenotypes of male MC3R KO, MC4R KO, and DKO mice in conventional and running wheel cages measuring the effects of central MCR inacti vation and running wheel exercise on both central and peripheral gene expression involved in energy homeostasis (Figure 1 5) investigating the effects of gender on the MC4R KO obese phenotype and its alleviation by voluntary exercise.

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52 This will generate n ovel data using established models of genetic obesity to contribute to the body of knowledge on the effects of obesity and exercise on the energy homeostasis of the body.

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53 Table 1 1. Body mass index and corresponding body type classifications set by the W orld Health Organization. 114 Body Mass Index Classification <18.49 Underweight 18.50 24.99 Normal Weight 25.00 29.99 Overweight > 30 Obese Figure 1 1. Graphical representation of the effects of shifting energy input and output on body weight.

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54 Figure 1 2. Graphical representation of functions of the melanocortin rece ptors and ligands.

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55 Figure 1 3. Photo of melanocortin receptor WT and KO mice with corresponding body weights and fat masses.

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56 Figure 1 4. Metabolic pathways of the body to be investigated in metabolically active organs in the body.

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57 Figure 1 5. Org ans and genes to be investigated to elucidate the effects of genetic obesity caused by melanocortin receptor dysfunction and voluntary exercise by running wheel.

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58 CHAPTER 2 METHODS AND MATERIAL S Breeding and genotyping of the experimental mice was done by Amy M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, and Erin B. Bruce. Mice cages were changed and cleaned by Amy M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, and Erin B. Bruce. Measurements and blood draws were done by Amy M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, Erin B. Bruce, and Jay Schaub. Running wheel activity data was compiled by Sarah B. Carey and Jay Schaub. All plasma hormone assays were run by Jay Schaub. Sacrifice and dissection of organs was performed by Dr. Zhim in Xiang, Dr. Sally Litherland, and Jay Schaub. Data analysis was performed by Jay Schaub. Experimental Animals All studies performed were conducted in accord with accepted standards of humane animal care and were approved by the Institutional Animal Care and Use Committee at the University of Florida. The MC3R KO, MC4R KO, DKO, and WT mice were generated using breeding colonies maintained at the University of Florida. The MC3R KO mice were provided by Merck and the MC4R KO mice were provided by Dennis Husz ar of Millennium Pharmaceutical by material transfer agreement (MTA). Permission to generate DKO mice was granted by both Merck (MC3R KO) and Genelogic (MC4R KO) by MTA. Knockout (MC4R / ) and wild type (MC4R +/+ ) are of mixed genetic backgrounds from the C57BL/6J and 129/Sv inbred mice strains. Selection Criteria for Mice Male mice were selected from multiple previous experiments (MC3RKO RW 1 [6/29/2009 8/30/2009], MC4RKO RW 11 [10/24/07 12/17/2007], MC4RKO RW 12

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59 [1/29/2008 4/1/2008], MC4RKO RW 13 [6/28/2 008 8/4/2008], and DKO RW 2 [5/13/2008 7/12/2008]) that are included in the peripheral gene expression study (Table 2 1). The MC4R KO MC4R WT and DKO mice were selected for use based on total body weight. The MC3R WT and MC3R KO mice were selected base d on total fat mass as determined by magnetic resonance imaging (MRI). Data for selected mice were compiled from previous studies into a single analysis file to allow for statistical analysis of measured parameters of the mice. Mice were age matched for an alysis and comparison. The total number of mice per group for the groups presented in Table 2 1 were limited by number of samples that could be simultaneously run in duplicate on a 96 well RT PCR plate. In order to evaluate if gender had a significant effe ct on the mitigating effects of exercise on the obese phenotype of MC4R KO mice, male and female MC4R KO and WT mice were studied in a single parallel experiment (MC4RKO RW 11 [10/24/07 12/17/2007]) (Table 2 2). The total number of mice per group for the t he groups outlined in Table 2 2 were limited by the number of male and female mice of the appropriate genotype generated from the heterozygous breeding scheme. Breeding The MC3R and MC4R KO mice and WT controls were generated using a standard heterozygous breeding scheme in conventional cages while DKO mice were generated using a breeding scheme using 3RHet/4RHet mice as breeders in running wheel cages (Figure 2 1). All experimental mice were generated using timed breeding to ensure similarly aged experime ntal mice. After an approximate 21 day incubation period, pups were born and left with their parents until postnatal day 21 at which point they were weaned into separate cages based on gender and were genotyped at 28 days of age.

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60 Genotyping Two millimeters blade under isoflurane anesthesia and placed in a clean 1.5 mL Eppendorf tube. The pup was then weighed and were fitted with a numbered ear tag in the right ear for future identification. T he tails were digested overnight using a proteinase K (Fisher Cat# BO1700 100) digest buffer (Table 2 3) in a 55C shaking water bath. The next day, the Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) solution (Invitrogen Cat#15593 031) was added to the tubes. The chloroform mixture was vortexed and then the tubes were spun for 10 minutes on high speed. The top layer was removed by pipet and placed in a clean 1.5 mL tube. DNA was precipitated using 1 mL of cold 100% Ethanol (Decon Labs, Inc. Cat #2701) and tubes were inverted several times to help DNA form pellet. DNA pellet was removed by J bend (flame sealed hematocrit tube) and placed in 500 L of ddH 2 O. The DNA was allowed to dissolve and sit overnight before proceeding to PCR amplification. Mouse genomic DNA was amplified using primers specified for the MC3R, the MC4R, or the gene inserts used to replace the respective receptors (Table 2 4) on an Eppendorf Mastercycler Gradient instrument. A solution containing polymerase, buffers, and primers was mixed using the recipes presented in Table 2 5. PCR reactions were run in 0.2 mL tubes (Eppendorf Cat#951010006). Mice from the MC3R colony were ge notyped using the primers MC3R F, MC3R R and MC3R NEO in Table 2 4, while mice from the MC4R colony were genotyped using the primers MC4R F, MC4R R and MC4R PGK in Table 2 4. Mice bred from the DKO colony were genotyped using both sets of primers in separa te reactions. The DNA being examined using the MC3R set of

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61 primers was amplified using the Mastercycler programs shown Table 2 6. The DNA being analyzed using the MC4R set of primers was amplified using the Mastercycler programs shown in Table 2 7. One gr am of 027) was dissolved in 100 mL of TAE buffer (BioRad Cat#161 0743), heated in a microwave on high for 2 minutes, or until the agarose had completely dissolved. Four microliters of 10mg/mL ethidium bromide (Invitrogen Cat#15585 011) was added to the solution, and then it was poured into a gel mold. The amplification product was loaded on the 1% agarose gel along with 1 KB ladder (Invitrogen Cat#15615 016). The samples were run at a voltage of ~150V for approximately an hour, or until the loading dye neared the end of the gel. Gels were visualized under UV light on a Gel Doc 2000 (BioRad) using Quantity One software (version 4.6.9) and an image of the gel was printed on a Mitsubishi P93 printer. For samples amplified usi ng the MC3R genotyping protocol, bands were expected at 514 bp if the mouse was a WT, 294 bp if the mouse was a MC3R KO, and at both 294 bp and 514 bp if the animal was heterozygous for the MC3R gene. For samples amplified using the MC4R genotyping protoco l, bands were expected at 313 bp if the mouse was a WT, 405 bp if the mouse was a MC4R KO, and at both 313 bp and 405 bp if the animal was heterozygous for the MC4R gene. Housing Mice were housed in standard polycarbonate conventional cages provided, clea ned, and maintained by University of Florida Animal Care Services until the start of experiments. At the beginning of the experimental treatment mice assigned to the exercise group were placed in clean cages equipped with Mini Mitter (Bend, Oregon) running wheel while mice in the control groups were placed in a new, clean

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62 conventional cage. DKO mice were housed in running wheel cages until start of the experimental measurements to try and delay the onset of the obese phenotype. Mice were housed in a room wi th a reverse 12 h light/dark cycle (11:00 to 23:00). All animals had ad libitum access to food and water for the duration of experiments. Mice in conventional cages were housed singly or group housed in pairs (n=2). All mice in running wheel cages were hou sed singly. Food Irradiated rodent chow (Harlan Teklad LM 485 mouse/rat sterilizable diet 7912) was provided for use by UF Animal Care Services. This diet contained a minimum of 19.0% and 5.0% of crude protein and crude fat, respectively, with a maximum o f 5.0% crude fiber for a digestible energy of 3.75 Kcal/g. Experimental Design All WT and single KO mice were housed in conventional cages for the fifth week of age to collect baseline readings. The DKO mice were housed in cages equipped with running wheel s to delay the onset of the obese phenotype until the start of the experiment. At six weeks of age, the mice in the treatment groups were moved to running wheel cages. Body weights, lengths, and food intake were measured twice a week. Blood was drawn two ( MC3RKO RW 1) or three (RW 11, RW 12, RW 13, DKO RW 2) times a week depending on individual experimental design. Body composition was measured once a week by MRI. Activity was monitored throughout the experiment for the mice in the cages equipped with runni ng wheels. At the end of each experiment, the mice were sacrificed and organs were collected for gene expression studies.

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63 Weights and Lengths Total body mass was measured twice a week using a Denver Instrument (model APX 1502) top loading balance. Food in take was determined by measuring the weight of food remaining in the cage top wire feeder and subtracting that value from the amount of food added twice weekly. Bedding was visually inspected for any intact food pellets to reduce the error associated with determining food intake. Average daily food intake was calculated by dividing weekly food intake by seven. For animals group housed in conventional cages, food intake values were divided by 2 to estimate average food consumption. 37,64 Body length (nose to anus, measured in mm) was measured by lightly anesthetizing the mice with isoflurane and extending the mouse to its full length. The nose of the mouse was aligned with a line on a piece of paper and a mark was made where the base of the tail meets the body, the distance between the two mar ks was measured with a ruler. 37,64 Blood Draws Blood was drawn two (MC3RKO RW 1) or three times a week (RW 11, RW 12, RW 13, DKO RW 2), depending on individual experimental design. Blood was drawn from the anterior facial vein using a 22 gauge needle and collected in a K 2 EDTA coated tube (BD Medical, Cat. #367861). Blood was transferred to 1.5 mL microcentrifuge tubes and spun at 10,000 rpm for 10 minutes. Plasma was removed by pipet, transferred to a clean microcentrifuge tube, frozen, and stored at negative 20C. At the end of each week, the plasma samples were thawed, pooled, aliquoted, and refrozen at minus 20C until ready to use for plasma hormone quantification. 37

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64 Plasma Hormones Plasma hormone concentrations of insulin and leptin were determined using a commercially available mouse endocrine panel kits (Linco Research, Millipore) on a Luminex 200 platform (Luminex, Austin, TX). Ten microliters of pooled plasma 37 Insulin and leptin were measured using a multiplex system using bead immobilized antibodies, a biotinylated secondary detection antibody, and a streptavidin phycoerythrin fluorescent label. Each well was sampled and 50 beads were read for each analyte, a minimum of 30 beads per analyte were required to use the well results. Hormone concentrations were determined using a known standard curve, the slope of which was calculated it function in the Luminex analysis software. The concentrations of the standards used to determine the standard curve were 0 (blank sample), 6.2 pM, 18.5 pM, 55.6 pM, 166.7 pM, 500 pM, 1500 pM, and 4500 pM. The minimal detection concentration for insulin using this assay was 55.6 pM and 6.2 pM for leptin. For samples where concentrations determined by the computer were given as <6.2 pM or >4500 pM, values of 6.2 pM and 4500 pM respectively were used for statistical analysis instead of omitting values. Acco rding to the manufacturer, there is an intra assay precision of 3.8 10.2% and an inter assay precision of 4.8 20.7%. Body Composition Once a week the body composition of the mice was measured in terms of fat and lean mass (measured in grams) using an Ec hoMRI 100 (Echo Medical Systems LLC, Houston, TX). 37 Conscious mice were placed in an ac rylic tube to reduce movement, inserted into the MRI machine horizontally, and scanned twice. If values for

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65 either fat or lean mass varied by more than 1 gram between readings, a third scan was done. Values for fat mass and lean mass were averaged for furt her analysis. Running Wheel Activity Nalgene cages were equipped with a running wheel (Mini Mitter, Bend, Oregon). Wheel revolutions were counted by recording the magnetic switch closures of a magnet attached to the wheel and recorded once a minute by a c omputer using VitalView software version 4.1 (Mini Mitter) as described previously. 37,64,80 The running wheel computer was stopped and activity was collected any time the mice were removed from their cages. Sacrifice and Dissection Mice were sacri ficed by decapitation and trunk blood was collected in EDTA coated tube (BD Medical, Cat. #367861). Brains were dissected out first, and followed by pancreas, heart, liver, kidneys, adipose tissue, skeletal muscle from the leg (gastrocnemius). Depending on the experimental design, tissues were either fresh frozen on dry ice and subsequently stored in a 80C freezer or placed in an appropriate volume of RNAlater (Ambion Cat#AM7021), allowed to sit at 4C for 24 hours and then moved to a 20C freezer until ready for use. Fresh tissue was stored in direction (volumes used for tissues examined in this dissertation shown in Table 2 8). Gene Expression RNA Extraction Total RNA was instructions. Briefly, tissue was homogenized in Trizol (Invitrogen Cat# 15596 018) solution using a 7mm PRO Scientific Inc. (Oxford, Connecticut) generator. One mL of

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66 Trizol was used for total hypo thalamus. Muscle and liver were homogenized in approximately 1 mL of Trizol per 100 mg of tissue (3 mL for muscle, 4 mL for liver). The homogenizer was rinsed in distilled deionized water (ddH 2 O), 1% sodium dodecylsulfate (SDS) solution, 1 M NaOH, 10% Blea ch, and finally in ddH 2 O again to prevent carry over between samples. For muscle and liver where more than 1 mL of Trizol was used for homogenization, 1 mL of homogenate was transferred to a new 1.5 mL microcentrifuge tube and remaining sample was stored at 20C until verification of RNA integrity after electrophoresis on agarose gel. To the homogenate, 0.2 mL of chloroform (Sigma Cat#C2432 500mL) was added and the samples vortexed (Fisher Cat#120812) for a full minute. Samples were spun at 12,000 x g for 15 minutes at 4C in an Eppendorf 5417R centrifuge. After centrifugation the sample separated by phase and the top (aqueous) phase was transferred by pipet to a new 1.5 mL microcentrifuge tube. Next, 0.5 mL of cold isopropanol (Sigma Cat#I9516 500mL) was added and the sample was briefly vortexed and then allowed to incubate at room temperature for 10 minutes. The samples were spun 12,000 g for 10 minutes at 4C to cause the precipitated RNA to form a pellet at the bottom of the tube. After centrifugation, the liquid was decanted off of the pellet and 1 mL of 75% ethanol (37.5 mL 200 proof ethanol [Decon Labs, Inc. Cat #2701] mixed with 12.5 mL of ddH 2 O) was added. The samples were inverted several times by hand and allowed to sit at room temperature for at least 5 minutes. The samples were spun a final time at 12,000 x g for 10 minutes at 4C. The ethanol was poured off and the sample tubes were inverted on a clean Kimwipe (Fisher Cat#34133) and allowed to dry for between 45 minutes and an hour, or until dro ps of ethanol were not obviously present. Sixty microliters of DEPC (Fisher Cat#BP561 1) treated water

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67 was added to the pellet and was allowed to dissolve for two to three hours and then frozen at 80C. Two microliters of the RNA solution were mixed with loading dye (Ambion Cat#AM8551) and loaded on a 1% agarose gel and run for approximately one hour at 150V. The gels were visualized under UV light and the integrity of the RNA was determined based on the intensity and the definition of the 18s and 28s bands. Examples of good and poor quality RNA as visualized after being run on a 1% agarose gel are shown in Figure 2 2 A and B respectively. The RNA Scientific). Samples were read in duplicate and values were averaged to determine concentration. The 260/280 ratio was checked for indications of protein contamination, with a ratio of 2.0 assumed to be little to no protein contamination. Samples were used for cDNA syn thesis if 260/280 ratio values were between 1.9 and 2.1 with clear bands seen after being run on a 1% agarose gel. If samples did not meet these criteria, RNA extraction was repeated from tissue homogenate until an acceplevel of purity was reached. cDNA S ynthesis Complementary strand DNA (cDNA) was synthesized using a High Capacity cDNA Archive Kit (Applied Biosystems Cat# 4322171) according to manufacturer instructions. Based on the average concentration determined by the NanoDrop spectrophotometer, enoug volume of the appropriate mastermix (Table 2 9). The reaction was carried out in a mastercycler w ith the program outlined in Table 2 10. After the completion of the

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68 reaction, cDNA was diluted with nuclease free water (Ambion Cat#AM9932) to a final RT PCR Studies A mastermix solution was made for each gene consisting of Gene Expression Mastermix (ABI Cat#4370074), the appropriate TaqMan gene probe (Table 2 11), and ddH 2 O in the quantities specified in Table 2 12. Each well in a 96 well plate (ABI was added for a total of 50 ng of cDNA in each well. Each sample was run in duplicate. The TaqMan Gene Expression probes used for the quantification of genes ordered from Applied Biosystems are shown in Table 2 11 Applied Biosystems considers the sequence of the probes proprietary, however the catalog number and gene accession number are provided in Table 2 11. After plating, they were run in the ABI 7300 Thermocycler with the program shown in Table 2 13. After completion of the run, the data were further analyzed using Sequence Detection Software version 1.4 (2006). The threshold cycle (Ct) was designated as the PCR cycle where fluorescent signal associated with the gene copy number exceeds the threshold (10x noise level). 37 The Ct was determined by the Auto analysis function of the ABI7300 software to eliminate error that could be associate d with manual determination of Ct over the many plates that were run for this dissertation. Data Analysis RT PCR Data Data from the RT PCR studies done on the ABI 7300 thermocycler were further compared to control group and a control gene. 115 The fold difference between

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69 experimental groups was calculated as 2 Ct Ct(Housekeeping 115 The genes HPRT1 and PPIA were both run as potential housekeeping genes and the gene with the least amount of variance in each tissue was used to determine fold chan ge differences for that tissue. Running Wheel Data In order to determine the total number of wheel turns in an entire dark cycle, raw data files of running wheel activity collected from the running wheel computer were further analyzed using VitalView and Microsoft Excel software. Using the VitalView software, data files were opened using the Data Load & Analysis function. Data from only one dark cycle at a time was opened using the Special Span Function and only specifying the times at which the lights in the room were not on (generally from 11:00 until 23:00). To simplify the data into one time point giving the total number of rotations of the wheel using the Analysis Menu to Filter Data. The filters for Invalid Points, Decimate, Scale, and Clipping were turned on and the values for Decimate, Scale, and Clipping were set to 720, 720, and 0 to 35, respectively, and the operations were applied. The number 720 was used because that is how many minutes there are in a 12 hour period. Values other than 720 were used for Decimate and Scale if data was not collected for the entire dark period. These values correspond to the number of minutes that data was collected. The data files were then saved as ASCII files and opened in Excel as both Tab and Comma delimited fi les. The number of rotations was pasted into another Excel file where the values were used to determine the average number of running wheel rotations per dark cycle during each week of the experiment.

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70 Statistical Analysis Prism 4.0 (GraphPad Software, Inc .) was used to perform one way and two way analysis of variance (ANOVA) tests and to graphically represent data. The PASW Statistics software (Version 18.0.0, SPSS Inc.) was used to perform two way repeated measure ANOVA tests. Data was analyzed using repe ated measures analysis of variance (ANOVA) followed by post hoc tests as appropriate (SPSS). To determine if there was significant interaction between factors, a generalized linear model was used with P<0.05 signifying significant interaction (SPSS). In or der to determine when differences between groups emerged, 2 way ANOVA was performed at each time point (Prism). If statistical significance was found to exist between groups in the ANOVA, a Bonferroni post test was used to further compare genotype and gend er or genotype and housing as appropriate. Data is presented as Mean SEM. Percent difference was calculated by dividing the higher of the two values by the lesser value, multiplying by 100 and presenting the increase from 100%. Significance was assumed f or P values < 0.05.

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71 Table 2 1. Mice used to study the physiological effects of exercise on male MC3R, MC4R, and DKO mice. Studies presented in Chapters 3 and 4. Genotype Housing Gender Number of mice MC3R WT Conventional Male 4 MC3R WT Running Whee l Male 5 MC4R WT Conventional Male 5 MC4R WT Running Wheel Male 4 MC3R KO Conventional Male 5 MC3R KO Running Wheel Male 5 MC4R KO Conventional Male 5 MC4R KO Running Wheel Male 5 MC3R/MC4R KO Conventional Male 5 MC3R/MC4R KO Running Wheel Male 5 Table 2 2. Mice used to study the physiological effects of exercise on male and female MC4R KO mice. Study presented in Chapter 5. Genotype Housing Gender Number of mice MC4R WT Conventional Male 8 MC4R WT Running Wheel Male 5 MC4R WT Conve ntional Female 6 MC4R WT Running Wheel Female 10 MC4R KO Conventional Male 8 MC4R KO Running Wheel Male 10 MC4R KO Conventional Female 8 MC4R KO Running Wheel Female 11

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72 Figure 2 1. Breeding schemes used to generate experimental mice. Tab le 2 3. Proteinase K tail digest buffer for DNA extraction for making 1 L of solution. Ingredient Amount Source Catalog number EDTA 1.862 g BioRad 161 0729 NaCl 11.688 g Fisher BP 358 1 SDS 2 g BioRad 161 0302 Tris Base 12.114 g Fisher BP 154 1 pH adjusted to 7.5 Proteinase K (added fresh) 10 mL of 10 mg/mL Fisher BP 1700 100 Table 2 4. Primers used for genotyping melanocortin receptor KO mice. 9,10 Primer Sequence MC3RKO F GAT GAG AGA AGA CTG GAG AGA GAG GGT C MC3RKO R GAA GAA GTA CAT GGG AGA GTG CAG GTT MC3RKO NEO TAC CGG TGG ATG TGG AAT GTG TGC MC4RKO F TTC CCA GCC TCT GAG CCC AGA MC4RKO R GAC GAT GGT TTC CGA CCC ATT MC4RKO PGK GGA AGA TGA ACT CCA CCC ACC

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73 Table 2 5. Ge notyping PCR mastermix solution for DNA amplification per reaction. Ingredient Volume Source Catalog number RedTaq Polymerase Sigma D4309 250UN RedTaq 10x Buffer Sigma D4309 250UN DNTP Mix Invitrogen 10297 018 Forward Primer Invitrogen Custom Ordered Reverse Primer Invitrogen Custom Ordered Insert Primer (NEO or PGK) Invitrogen Cu stom Ordered ddH 2 O Table 2 6. Polymerase chain reaction mastercycler program for MC3R genotyping. Step Temperature Length of time Lid 105C 1 94C 2 minutes 2 94C 30 seconds 3 68C 1 minute 4 Go to Step 2, Repeat 35 times 5 6 8C 10 minutes 6 15C, Hold Table 2 7. Polymerase chain reaction mastercycler program for MC4R genotyping. Step Temperature Length of time Lid 105C 1 94C 1 minute 2 92C 45 seconds 3 64C 45 seconds 4 72C, Ramping at 3C/second 45 s econds 5 Go to Step 2, Repeat 39 times 6 72C 10 minutes 7 4C, Hold Table 2 8. Volume of RNAlater used for storage of fresh tissue until RNA extraction. Tissue Volume of RNAlater Brain 6 mL Liver 8 mL Pancreas 5 mL Skeletal Muscle 4 mL

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74 Figure 2 2. Example of a good and poor RNA quality visualized on 1% agarose gels. (A) is an image of good quality RNA extracted from liver samples. (B) is an image of poor quality RNA extracted from pancreas samples. Table 2 9. cDNA synthesis masterm ix solution recipe provided with High Capacity cDNA Archive Kit for cDNA synthesis per reaction. Ingredient Volume 10X RT Buffer 25X dNTP Mix (100mM) 10X Random Primers Nuclease Free H 2 O Table 2 10. Polymerase chain reaction mastercycler program for cDNA synthesis. Step Temperature Length of time Lid 105C 1 25C 10 minutes 2 37C 2 hours 3 95C 5 seconds 4 4C, Hold

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75 Table 2 11. Gene name, gene symbol, and Applied Biosystems catalog number of TaqMan probes and associated genes. Gene name Gene symbol Catalog number Agouti related protein AGR P Mm00475829_g1 5' AMP activated protein kinase PRKAA1 Mm01296695_m1 Carnitine palmitoyltransferase 1a, Liver CPT1A Mm00550438_m1 Carnitine palmitoyltransferase 1b, Muscle CPT1B Mm00487200_m1 Carnitine palmitoyltransferase 2 CPT2 Mm00487202_m1 Co caine and Amphetamine regulated transcript prepropeptide CART Mm00489086_m1 Diglyceride acyltransferase 1 DGAT1 Mm00515643_m1 Diglyceride acyltransferase 2 DGAT2 Mm00499530_m1 Fatty acid synthase FASN Mm00662319_m1 Fructose 1,6 biphosphatase FBP1 M m00490181_m1 Glucokinase (hexokinase 4) GCK Mm00439129_m1 Glucose 6 phophatase G6PC3 Mm00616234_m1 Glucose Transporter 2 SLC2A2 Mm00446224_m1 Glucose Transporter 4 SLC2A4 Mm00436615_m1 Glycogen phosphorylase, Liver PYGL Mm00500078_m1 Glycogen p hosphorylase, Muscle PYGM Mm00478582_m1 Glycogen synthase 2, Liver GYS2 Mm00523953_m1 Glycogen synthase 1, Muscle GYS1 Mm00472712_m1 Hormone sensitive lipase LIPE Mm00495359_m1 Hypocretin (orexin) HCRT Mm01964030_s1 Hypoxanthine guanine phosphori bosyl transferase 1 HPRT1 Mm00446968_m1 Insulin receptor INSR Mm00439693_m1 Interleukin 6 IL 6 Mm99999064_m1 Leptin receptor LEPR Mm01265583_m1 Melanocortin 3 receptor MC3R Mm00434876_s1 Melanocortin 4 receptor MC4R Mm00457483_s1 Neuropeptide Y NPY Mm00445771_m1 Neuropeptide Y 1 receptor NPY1R Mm00650798_g1 Peptidylprolyl isomerase A PPIA Mm03302254_g1 Phosphofructokinase, liver PFKL Mm00435587_m1 Phosphofructokinase, muscle PFKM Mm00445461_m1 Proopiomelanocortin POMC Mm00435874_m1 S uppressor of Cytokine Signaling 3 SOCS3 Mm01249143_g1 Uncoupling protein 2 UCP2 Mm00627597_m1 Uncoupling protein 3 UCP3 Mm00494077_m1

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76 Table 2 12. RT PCR mastermix solution for TaqMan Gene Expression Assay per reaction well. Table 2 13. Real time polymerase chain reaction cycler program for evaluating gene expression by TaqMan Gene Expression Assay. Step Temper ature Length of time Lid 105C 1 50C 2 minutes 2 95C 10 minutes 3 95C 15 seconds 4 60C 1 minute 5 Sample Read 6 Go to Step 3, Repeat 39 times Ingredient Source Catalog number Gene Expression Mastermix 12.5 ABI 4370074 TaqMan Probe 1.25 ABI See 2 9 ddH 2 O 6.25

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77 CHAPTER 3 EFFECT OF VOLUNTARY EXERCISE ON MALE, CE NTRAL MELANOCORTIN RECEPTOR KNOCKOUT MICE Breeding and genotyping of the experimental mice was done by Amy M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, and Erin B. Bruce. Mice cages were changed and cleaned by Amy M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, and Erin B. Bruc e. Measurements and blood draws were done by Amy M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, Erin B. Bruce, and Jay Schaub. Running wheel activity data was compiled by Sarah B. Carey and Jay Schaub. All plasma hormone assays were run by Jay Schau b. Sacrifice and dissection of organs was performed by Dr. Zhimin Xiang, Dr. Sally Litherland, and Jay Schaub. Data analysis was performed by Jay Schaub. Introductory Remarks Obesity is a significant healthcare concern worldwide with nearly one in three ad ult Americans possessing a BMI of greater than 30 (clinical definition of obesity). 1 Populations of obese human patients with mutations in their MC4R disrupting function have been identified with an estimated 2% of obese Americans possessing a mutation to the MC4R. 47 The phenotypes of the MC3R KO and MC4R KO mice have been identified and characterized. 8 10 The DKO mice have also been described as possessing increased body weights. 9 The obese phenotype and high adiposity of the MC4R KO mice make them ideal models for study ing obesity. 10,37,39,63,64,80 The MC3R KO mice possess normal body weights, and slightly increased fat masses compared to WT littermates. 8,9 Obese human patients with mutations in their MC3R have also been identified. 49 54,116 DKO mice that lack both functional MC3 and MC4 r eceptors are more obese than even

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78 MC4R KO mice, supporting the hypothesis that the MC3 and MC4 receptors are not redundant. 9 Exercise is a method for affecting weight loss and preventing weight gain that has fe w detrimental side effects, barring potential injury. Along with diet modification, exercise is the only universally prescribed treatment for obesity. 62 Previous experiments in the Haskell Luevano lab have demonstrated that voluntary exercise is able to prevent or delay the onset of the obese phenotype of male MC4R KO mice. 37,63,64 Herein, experiments designed to study the effects of voluntary exercise on MC3R KO, MC4R KO, and DKO mice are described. To test the hypothesis that voluntary exercise can prevent the onset of the metabolic dysfunctio n previously described in central melanocortin receptor mice, male mice lacking the central melanocortin receptors were allowed access to running wheels in their home cages for a period of seven or eight weeks and their phenotypes and physiology were monit ored. 8 10 Methods and Results Brief Overview of Experiments Male mice were genotyped using DNA from tail tip biopsy at four weeks of age and assigned to either conventional or exercise groups based on body weight. The MC3R and the MC4R mice were housed in conventional cages during the fifth week of age in order to measure baseline values. The D KO mice were housed in running wheel cages during the fifth week of age. At the beginning of the sixth week of age, mice in the exercise group were transferred to cages equipped with Mini Miter running wheels while mice in the control groups were transferr ed to new, clean conventional cages. Animals were given ad libitum access to food and water for the duration of the experiment. Body

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79 weight, body length, and food intake were measured twice a week and averaged for future statistical analysis. Body composit ion was measured once a week in duplicate by quantitative MRI machine and fat mass and lean mass values were averaged for future statistical analysis. Running wheel activity data, as measured by magnetic switch breaks by the Mini Miter VitalView software, was collected (each time the computer recording was stopped to handle the animals) to determine total number of wheel revolutions for statistical analysis. Blood was drawn two (MC3RKO RW 1) or three times a week (RW 11, RW 12, RW 13, DKO RW 2), depending o n individual experimental design, spun, and plasma aliquoted for future use to determine circulating concentrations of insulin and leptin by Luminex assay. At the end of the designed experimental length (seven weeks for mice from experiment RW 11 and eight weeks for all other mice), animals were sacrificed by decapitation, and trunk blood and organs were collected for future study. Animal Selection Male mice of the appropriate genotypes and housing were selected from prior running wheel experiments conduct ed by the Haskell Luevano lab for inclusion in the expanded gene expression study presented in Chapter 4. In order to evaluate the effects of exercise on the physiology and phenotype of these mice, the relevant measurements were compiled for statistical an alysis. A total of 48 male mice were selected across five genotypes (MC3R WT, MC3R KO, MC4R WT, MC4R KO, and DKO) and two housing conditions (conventional and running wheel). Mice were pooled from five different experiments titled: RW 11 (10/24/07 12/17/20 07), RW 12 (1/29/2008 4/1/2008), RW 13 (6/28/2008 8/4/2008), DKO RW 2 (5/13/2008 7/12/2008), and MC3RKO RW 1 (6/29/2009 8/30/2009). The MC3R WT and the MC3R KO mice were

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80 from MC3RKO RW 1. The MC4R WT and the MC4R KO mice were pulled from RW 11 and RW 12. M ale DKO mice were pooled from the RW 13 and DKO RW 2 experiments. There were a total of five mice in each of the experimental groups for all measurements and analyses except for the MC3R WT in conventional housing and the MC4R WT mice in running wheel cage s groups where there were four mice. Mice were selected for inclusion in this dissertation based on final body weight for MC4R and DKO mice and final fat mass for MC3R mice. Statistical Analysis Measured values were analyzed with the PASW Statistics (Versi on 18.0.0, SPSS Inc.) statistical software using the general linear model with repeated measures to determine the effects of age, genotype, housing, and the interaction between genotype and housing. If a statistical difference (P<0.05) was found due to gen otype, housing, or their interaction, further tests were run at each time point. If a difference was found, a secondary ANOVA (one way for running wheel activity and two way for all other measurements) was carried out at each time point using the Prism 4.0 (GraphPad Software, Inc.) statistical software with Bonferroni posttest. Due to the nature of repeated measure analysis, mice who were missing one or more value at any time point were excluded from the analysis. It is because of this data omission that th e results of the statistical tests (ANOVA) conducted at individual time points might differ from the repeated measures test if excluded animals had a valid measurement value included by the second statistical test.

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81 Body Size and Composition Weight Increase d body weight is a known phenotypic characteristic of both MC4R KO and DKO mice. 9,10 To test the hypothesis that voluntary exercise will prevent the overweight phenotype of male MC4R KO and DKO mice, body weig hts was measured twice a week and averaged for statistical analysis (Figure 3 1). Repeated measures ANOVA was performed using SPSS to determine the effect, if any, that genotype and housing had on body weight. Age, genotype, and housing were all found to h ave significant effects on body weight (P<0.001), with a significant interaction between genotype and housing (P<0.001). In order to determine the point in time where significance began, two way ANOVA was performed at the individual time points using Graph Pad Prism with Bonferroni post tests to determine differences between KO genotypes and the appropriate WT control mice and the effects of exercise on the individual genotypes. Effects of genotype within housing. At five weeks of age genotype already had a significant effect on body weight (P<0.001). A significant difference also existed between MC3R WT (17.62g 0.26g) and DKO (20.58g 1.28g ) conventionally housed mice (P<0.05) (Figure 3 2). At six weeks of age genotype had a significant effect on body wei ght (P<0.001), with MC3R WT (17.13g 0.21g) and DKO (23.51g 1.39g) conventional groups significantly different from each other (P<0.001) (Figure 3 3). At seven weeks of age both genotype and housing had a significant effect on body weight (P<0.01) with a interaction between genotype and housing also existing (P<0.01) (Figure 3 4). There was a significant difference between the conventionally housed DKO (28.11g 1.32g) and both MC3R WT (17.65g 0.29g) and MC4R WT groups (20.71g 1.14g) (P<0.001) (Figur e 3 4). A significant difference between MC4R WT

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82 (20.71g 1.14g) and MC4R KO (25.18g 1.04) mice in conventional cages was also found (P<0.01) (Figure 3 4). After the third week of the experiment (eight weeks of age), a significance of P<0.001 had been r eached for housing, genotype, and the interaction between genotype and housing (Figure 3 5). The significant difference between conventionally housed DKO mice (32.33g 1.34g) and the MC3R WT (18.26g 0.23g) and MC4R WT (21.11g 1.15g) groups remained (P <0.001), with the additional emergence of a significance between the DKO (23.03g 2.26g) and MC3R WT (19.30g 0.99g) groups in running wheel cages (P<0.05) (Figure 3 5). The difference between MC4R WT (21.11g 1.15g) and MC4R KO (27.47g 0.98g) mice in conventional housing reached P<0.001 (Figure 3 5). There was a significant difference due to genotype, housing, and the interaction between genotype and housing (P<0.001) in body weights at nine weeks of age (Figure 3 6). The significance between the DKO (35.32g 0.99g) and MC3R WT (18.78g 0.22g) and MC4R WT (21.48g 1.20g) mice in conventional housing continued (P<0.001), with the difference between DKO (24.99g 2.28g) and MC3R WT (19.53g 0.89g) mice in running wheel cages increasing to P<0.01 (Fig ure 3 6). The difference between MC4R WT (21.48g 1.20g) and MC4R KO (23.04g 0.34g) mice in conventional housing remained (P<0.001) (Figure 3 6). There was a significant difference in body weights at ten weeks of age due to genotype, housing and their i nteraction (P<0.001) as seen in Figure 3 7. There was a significant difference in body weight between the DKO mice (38.74g 0.84) and MC3R WT (19.60g 0.19g) and MC4R WT ( 21.87g 1.12g) groups in conventional housing (P<0.001) (Figure 3 7). There was al so a difference in body weights between the DKO mice (26.94g 2.38g) and both strains of WT (MC3R 20.05g 0.88g; MC4R 21.60g

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83 0.47g) mice in running wheel cages (P<0.01) (Figure 3 7). A significant difference in body weights also existed between the MC4 R WT ( 21.87g 1.12g) and MC4R KO (31.45g 1.19g) groups in the conventional cages (P<0.001) (Figure 3 7). A statistically significant difference in body weights existed at 11 weeks of age due to genotype, housing, and the interaction between the two (P<0 .001) (Figure 3 8). Body weights for DKO (Conv. 40.61g 0.86g; RW 28.29g 2.18g) mice were significantly different from those of both MC3R WT (Conv. 20.01g 0.22g ; RW 20.37g 0.87g) and MC4R WT (Conv. 22.09g 1.17g; RW 21.76g 0.40g) mice in both ho using conditions (P<0.001) (Figure 3 8). There was also a significant difference between MC4R KO (33.37g 1.15g) and MC4R WT (22.09g 1.17g) mice housed in conventional cages (P<0.001) (Figure 3 8). Body weights were affected by genotype, housing, and th e interaction between genotype and housing (P<0.001) at 12 weeks of age (Figure 3 9). DKO (Conv. 42.48g 0.35g; RW 29.94g 0.35g) mice weighed significantly more than both the MC3R WT (Conv. 20.73g 0.30g; RW 20.81g 0.95) and MC4R WT (Conv. 22.14g 1 .03g; RW 21.93g 0.37) mice in both conventional and running wheel cages (P<0.001) (Figure 3 9). MC4R KO (34.79g 1.16g) mice weighed significantly more than MC4R WT (22.14g 1.03g) mice in conventional cages (P<0.001) (Figure 3 9). During the final wee k of the experiment (13 weeks of age), housing, genotype, and the interaction between genotype and housing had a significant effect on body weights (P<0.001) (Figure 3 10). DKO (Conv. 44.02g 0.66g; RW 31.41g 1.94g) mice weighed significantly more than either MC3R WT (Conv. 21.21g 0.27g; RW 21.01g 0.90g) or MC4R WT (Conv. 22.06g 0.73; RW 22.42g 0.37) mice in both conventional and running wheel housing (P<0.001) (Figure 3 10). Additionally, MC4R

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84 WT (22.06g 0.73) mice weighed significantly less t han MC4R KO (35.12g 1.55g) mice in conventional cages (P<0.001) (Figure 3 10). Summary. Genotype had a significant effect on body weights at the beginning of the experiment (five weeks of age) that lasted the duration of the experiment (P<0.001). Housing began to have a statistically significant effect on body weights during the second week of treatment (seven weeks of age) (P<0.01) that lasted throughout the rest of the experiment. The interaction between genotype and housing began to have an effect on b ody weight at seven weeks of age (second week of treatment) that lasted until the conclusion of the experiment (P<0.01). The DKO mice weighed 17% more than MC3R WT mice at the start of the experiment (P<0.05) and progressed to being 59% and 36% heavier tha n the MC3R and MC4R WT conventionally housed mice, respectively, by the seventh week of age (second week of treatment) (P<0.001). A statistically 19% difference in body weights emerged between DKO and MC3R WT mice in running wheel cages at eight weeks of a ge (P<0.05) and a 25% difference between DKO and MC4R WT mice in running wheel cages at ten weeks of age (P<0.01). MC4R KO mice weighed 22% more than MC4R WT mice (P<0.01) during the second week of treatment (seven weeks of age). At no point in time did a significant difference in body weights emerge between the two WT groups in either housing condition. Body weights of MC3R WT and MC3R KO mice were not significantly different from one another in either housing type during the experiment. Finally, no differ ence between MC4R KO and MC4R WT body weights was seen in the cages equipped with running wheel equipment.

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85 Effect of housing within genotypes. The effect of voluntary exercise on body weights on the specific mice strains was evaluated by statistical post t est to see if phenotype was significantly changed compared to sedentary animals. No significant difference was seen between any of the sedentary and exercising groups at five weeks of age before treatment began (Figure 3 11). Exercising DKO (18.93g 2.00g ) mice weighed significantly less than DKO mice in conventional cages (23.51g 1.39g) at six weeks of age as shown in Figure 3 12 (P<0.05). At seven weeks of age, there continued to be a difference in body weights between sedentary (28.11g 1.32g) and ex ercising (20.62g 2.14g) DKO mice (P<0.001) (Figure 3 13). Exercise resulted in a significant difference in weight gain for both MC4R KO (Conv. 27.47g 0.98g; RW 22.46g 0.36g) (P<0.05) and DKO (Conv. 32.33g 1.34g; RW ) mice (P<0.001) at eight weeks of age (Figure 3 14). During the fourth week of treatment, access to running wheel equipment resulted in differences in body weights for both MC4R KO (Conv. 29.74g 1.02g; RW 23.04g 0.34g) and DKO (Conv. 35.32g 0.99g; RW 24.99g 2.78g) groups (P<0.0 01) compared to mice of the same genotype in conventional cages (Figure 3 15). There was a significant difference in body weights between groups in conventional and running wheels cages (Figure 3 16) for the MC4R KO (Conv. 31.45g 1.19g; RW 23.77g 0.45g ) and DKO (Conv. 38.74g 0.84g; RW 26.94g 23.38g) genotypes (P<0.001) at ten weeks of age. Figure 3 17 shows the differences in body weights for 11 week old MC4R KO (Conv. 33.37g 1.15g; RW 24.33g 0.37g) and DKO (Conv. 40.61g 0.86g; RW 28.29g 2.1 8g) mice allowed to exercise compared to the same genotype in conventional cages (P<0.001). The significant difference between housing conditions continued at 12 weeks of age for the

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86 MC4R KO (Conv. 34.79g 1.16; RW 25.16g 0.51g) and DKO (Conv. 42.48g 0.35g; RW 29.96g 1.97g) genotypes (P<0.001) as shown in Figure 3 18. During the final week of the experiment there was a difference between sedentary and exercise MC4R KO (Conv. 35.12g 1.55g; RW 25.78g 0.53) and DKO (Conv. 44.02g 0.66g; RW 31.41g 1.94g) mice and their respective sedentary controls (P<0.001) as shown in Figure 3 19. Summary. Voluntary exercise was able to prevent an increase in body weight during the first week of the experimental treatment (six weeks of age) in DKO mice compared t o control DKO mice in conventional cages (P<0.05) with sedentary DKO mice weighing 24% more than exercising DKO mice. This trend in DKO mice continued for the rest of the experiment with DKO mice in running wheel cages consistently weighing less than their conventionally housed counterparts (P<0.001). MC4R KO mice also benefited from the protective properties of exercise with a 22% difference in body weight seen as early as eight weeks of age (after three weeks of treatment) (P<0.05). The difference between sedentary and exercising MC4R KO mice lasted until the conclusion of the experiment with the exercising MC4R KO mice always weighing significantly less than the inactive mice in conventional cages (P<0.001). Exercise did not result in significant differen ces in body weights for MC3R WT, MC4R WT, or MC3R KO mice at any point in time during the experiment. Fat mass Increases in adipose tissue mass compared to WT controls are characteristic of the MC3R KO and MC4R KO mice. 8,9,36,37,39 In order to test the hypothesis that voluntary exercise would prevent the increase in fat mass associated with central melanocortin receptor dys function, MC3R KO, MC4R KO, and DKO mice were allowed

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87 access to running wheels in the home cage environment. Adipose tissue was quantifiably measured by MRI measurement of fat mass once a week in duplicate. If measured fat mass values were off by more than one gram, the measurement was repeated a third time. Values were averaged for graphing and statistical analysis (Figure 3 20). When analyzed by repeated measures ANOVA, age, genotype, housing and the interaction between housing and genotype all had a sign ificant effect on fat mass (P<0.001). To determine at which point in time these factors became significant, two way ANOVA was performed at the individual time points using GraphPad Prism with Bonferroni posttests performed to determine differences between KO genotypes and the appropriate WT control mice. Effects of genotypes within housing. Genotype and housing both had a significant effect on fat mass at five weeks of age (P<0.05) (Figure 3 21). The DKO mice (3.16g 0.53g) in the conventional group alrea dy possessed a greater amount of fat than both MC3R WT (1.57g 0.09g) and MC4R WT (1.94g 0.02g) control mice in conventional housing (P<0.05) (Figure 3 21) even though the DKO mice were housed in running wheel cages from birth until five weeks of age. D uring the first week of treatment (six weeks of age) genotype, housing, and the interaction between the two all had a statistically significant effect on fat mass in male mice (Figure 3 22) (P<0.01). Both WT (MC3R 1.28g 0.05g; MC4R 1.64g 0.26g ) convent ional control groups had a smaller fat mass than the conventionally housed DKO (5.20g 0.91g) mice (P<0.001) (Figure 3 22). At seven weeks of age housing, genotype, and their interaction all had a contributed significantly to differences in fat mass (P<0. 001) (Figure 3 23). The DKO (8.89g 1.10g) mice in conventional cages had a higher fat mass than both WT (MC3R

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88 2.34g 0.20g; MC4R 1.93g 0.26g ) groups (P<0.001) (Figure 3 23). While MC4R WT (1.52g 0.16g) mice in running wheel cages had a significantly lower fat mass than DKO mice (3.48g 1.03g) (P<0.05) (Figure 3 23). The MC4R KO mice (4.44g 0.42g) were also found to have a higher adiposity than MC4R WT mice (1.93g 0.26g ) in conventional cages (P<0.01) (Figure 3 23). Housing, genotype and the inte raction between genotype and housing all had an effect on fat mass at eight weeks of age (P<0.001) (Figure 3 24). DKO mice (Conv. 12.62g 1.15g; RW 4.68g 1.27g ) had significantly more adipose tissue mass than both WT control groups in conventional cages (MC3R 1.36g 0.19g; MC4R 1.93g 0.22g ) (P<0.001) and in the running wheel cages (MC3R 1.10g 0.27g; MC4R 1.90g 0.22g ) (P<0.01) after three weeks of treatment (Figure 3 24). Sedentary MC4R KO (6.08g 0.58g) mice were found to have a significantly hig her fat mass than conventionally housed MC4R WT (1.93g 0.22g ) mice (P<0.001) (Figure 3 24). After the fourth week of treatment (nine weeks of age) genotype, housing, and the interaction between them all had a significant effect on the adiposity of the ex perimental animals (P<0.001) (Figure 3 25). Both MC3R WT (Conv. 1.42g 0.26g; RW 1.40g 0.16g) and MC4R WT (Conv. 2.00g 0.26g; RW 2.06g 0.21g) control mice in both housing conditions had significantly lower fat masses than those of the DKO mice (Conv 15.06g 0.95g; RW 6.16g 1.41g) (P<0.001) (Figure 3 25). Fat mass was also significantly different between sedentary MC4R KO (7.66g 0.69g) and MC4R WT (2.00g 0.26g ) mice (P<0.001) (Figure 3 25). Housing, genotype, and the interaction between genoty pe and housing all contributed significantly to the variation in fat mass between male mice at ten weeks of age (P<0.001) (Figure 3 26). DKO (Conv. 17.52g 0.70g; RW 7.59g 1.53g) mice

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89 continued to possess a greater fat mass than both MC3R WT (Conv. 1.54 g 0.26g; RW 1.53g 0.16) and MC4R WT (Conv. 2.17g 0.27g; RW 1.97g 0.18g) mouse groups under both sedentary and exercise conditions (P<0.001) (Figure 3 26). There was a significant difference in fat mass between MC4R WT (2.17g 0.27g) and MC4R KO (9 .27g 0.78g) mice in standard cages (P<0.001) (Figure 3 26). There was a significant difference in fat mass due to genotype, housing, and their interaction (P<0.001) at 11 weeks of age (Figure 3 27). The DKO (Conv. 19.05g 0.63g; RW 8.42g 1.47g) mice h ad a significantly higher fat mass than either MC3R WT (Conv. 1.99g 0.37g; RW 1.12g 0.16g) or MC4R WT (Conv. 1.96g 0.24g; RW 1.97g 0.18g) mice in either conventional or running wheel cages (P<0.001) (Figure 3 27). Conventionally housed MC4R WT (1.9 6g 0.24g ) mice had a significantly lower fat mass than sedentary MC4R KO (10.53g 0.73g) mice (P<0.001) (Figure 3 27). Housing, genotype, and their interaction were all found to have a significant effect on total fat mass in male mice at 12 weeks of age (P<0.001) (Figure 3 28). The DKO mice (Conv. 20.29g 0.57g; RW 9.68g 1.29g) had a significantly different amount of fat mass compared to either sedentary or exercised MC3R WT (Conv. 2.11g 0.36g; RW 1.07g 0.15g) or MC4R WT (Conv. 2.09g 0.22g; RW 1 .82g 0.11g) mice from all control groups (P<0.001) (Figure 3 28). The MC4R KO mice (11.63g 0.86g) had a significantly higher adiposity than MC4R WT (2.09g 0.22g ) mice in conventional caging (P<0.001) (Figure 3 28). During the final week of the experi ment (13 weeks of age) genotype, housing, and the interaction between genotype and housing all had a significant effect on total fat mass (P<0.001) (Figure 3 29). The MC3R WT (Conv. 2.33g 0.25g; RW 1.50g 0.11g) and the MC4R WT (Conv. 2.12g 0.14g; RW 1.85g 0.10g ) mice had a significantly lower fat masses

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90 than DKO mice (Conv. 21.14g 0.69g; RW 10.83g 1.21g) regardless of housing condition (P<0.001) (Figure 3 29). The MC4R WT (2.12g 0.14g ) mice in conventional cages possessed a significantly lowe r fat mass than sedentary MC4R KO (12.50g 1.11g) mice (P<0.001) (Figure 3 29). Summary. Genotype had a significant effect on fat mass at five weeks of age (P<0.05) that lasted the entire experiment. Housing had a significant effect on adiposity of the e xperimental mice beginning in the fifth week of age (P<0.05) and lasting the duration of the experiment. An interaction between genotype and housing appeared at six weeks of age (first week of treatment) that lasted until the end of the experiment (P<0.01) Sedentary DKO mice had a 101% and 63% higher fat mass than MC3R WT and MC4R WT control groups, respectively, in conventional housing at five weeks of age (P<0.05) that increased in significance and lasted the duration of the experiment. At seven weeks o f age DKO mice had a 129% higher fat mass than MC4R WT mice in running wheel cages (P<0.05). A 326% difference between DKO and MC3R WT exercised mice was seen at eight weeks of age (third week of treatment). The MC4R KO mice had a 130% higher fat mass than MC4R WT mice in conventional cages starting at seven weeks of age (P<0.01) and lasting until the conclusion of the experiment. At no point in time was a significant difference in fat mass seen between MC3R KO and MC3R WT mice in conventional or MC3R WT an d MC3R KO mice in running wheel cages. No difference was seen in fat mass between MC4R KO and MC4R WT mice in the running wheel cages. Effect of housing within genotypes. To determine if voluntary exercise using a running wheel was able to prevent the incr ease in fat mass to levels seen in sedentary

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91 mice, sedentary and exercise groups were compared by post test within the same genotype. A significant difference in fat mass already existed at five weeks of age between DKO mice in conventional (3.16g 0.53g) and running wheel (1.87g 0.51g) treatment groups (P<0.05), even though both DKO groups were in running wheel cages from birth (Figure 3 30). After the first week of treatment, DKO mice in running wheel cages (2.18g 0.74g) exhibited a lower fat mass th an DKO mice in conventional cages (5.20g 0.91g) (P<0.001) (Figure 3 31). At seven weeks of age a difference in fat mass between sedentary (4.44g 0.42g) and exercising (2.02g 0.15g) MC4R KO mice emerged (P<0.05) (Figure 3 32). A difference between DKO mice in conventional (8.89g 1.10g) and running wheel (3.48g 1.03g) cages also existed (P<0.001) (Figure 3 32). Exercise resulted in a decreased fat mass compared to conventionally housed mice for both the MC4R KO (Conv. 6.08g 0.58g; RW 2.30g 0.27g ) and DKO (Conv. 12.62g 1.15g; RW 4.68g 1.27g) genotypes (P<0.001) for eight week old male mice (Figure 3 33). Nine week old sedentary MC4R KO (Conv. 7.66g 0.69g; RW 2.69g 0.37g) and DKO (Conv. 15.06g 0.95g; RW 6.16g 1.41g) mice had significant ly higher fat masses than mice of the same genotype allowed to exercise (P<0.001) (Figure 3 34). A decrease in fat mass compared to sedentary mice was seen in exercising MC4R KO (Conv. 9.27g 0.78g; RW 2.93g 0.41g) and DKO (Conv. 17.52g 0.70g; RW 7.59 g 1.53g) mice at ten weeks of age (P<0.001) (Figure 3 35). A statistically significant difference was seen in adiposity between MC4R KO (Conv. 10.53g 0.73g; RW 3.14g 0.33g) and DKO (Conv. 19.05g 0.63g; RW 8.42g 1.47g) mice in conventional cages a nd cages equipped with running wheels (P<0.001) at 11 weeks of age (Figure 3 36). During the second to last week of treatment (12 weeks of age), there was a

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92 difference in total fat mass as determined by quantitative MRI scan in sedentary and exercising MC3 R KO (Conv. 3.56g 0.51g; RW 1.11g 0.11g), MC4R KO (Conv. 11.63g 0.86g; RW 3.47g 0.36g), and DKO (Conv. 20.39g 0.57g; RW 9.77g 1.29g) mice (P<0.05) (Figure 3 37). Voluntary exercise was able to prevent the full onset of the increased fat mass in MC3R KO (Conv. 4.13g 0.63g; RW 1.47g 0.09g), MC4R KO (Conv. 12.50g 1.11g; RW 3.45g 0.34g), and DKO (Conv. 21.14g 0.69; RW 10.83g 1.21g) mice (P<0.05) in running wheel cages compared to the phenotypes seen in the sedentary mice (Figure 3 38) S ummary. The DKO mice in conventional cage group possessed 68% higher fat masses than DKO mice in the running wheel cage group beginning with the first week of treatment and lasting the entire experiment (P<0.001). After two weeks of access to running wheel equipment (seven weeks of age), fat mass values of the sedentary MC4R KO were 119% higher than exercising MC4R KO mice (P<0.05). a 221 % difference in fat mass was seen between MC3R KO mice in conventional and running wheel cages starting with 12 weeks of age (P<0.05). No differences in fat masses were seen between sedentary or exercising MC3R WT or MC4R WT mice during the experimental timeline. Lean mass Increase in fat free mass (or lean mass) has been reported in MC4R KO mice. 37,39 To test the hypothesis that voluntary exercise would be able to prevent this increase, mice were given free access to running wheel exercise equipment and lean masses were measured over a peri od of nine weeks. Lean mass was measured once a week in duplicate (along with fat mass) by quantitative MRI. If measured lean mass values were different between duplicate scans by more than one gram, a third scan was performed.

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93 Lean mass values were averag ed for graphing (Figure 3 39) and statistical analysis. Though lean mass has been shown to correlate with body length for MCR4 WT and MC4R KO mice in previous experiments, that correlation was not studied within this dissertation. 37 Age and genotype both had a significant effect on the amount of lean mass in each m ouse (P<0.001), whereas housing did not significantly contribute to the variation seen in lean masses (Figure 3 39). To determine at which point in time genotype caused a difference in lean mass between the experimental groups, two way ANOVA was performed at each individual time point using Graph Pad Prism with Bonferroni post test to determine differences between KO genotypes and the appropriate WT control mice. Effects of genotypes within housing. Genotype had a significant effect on amount of lean mass at five weeks of age (P<0.001) (Figure 3 40). A significant difference in lean mass was found between conventionally housed MC3R WT (13.31g 0.12g) and MC4R WT (16.56g 0.98g) mice at five weeks of age (P<0.05) (Figure 3 40), indicating a potential diffe rence between the WT strains. The DKO (Conv. 14.30g 0.38g; RW 13.64g 0.75g) mice had significantly less lean mass than MC4R WT (Conv. 16.56g 0.98g; RW 15.88g 0.34g) mice for both housing groups (P<0.05) (Figure 3 40). Genotype had an effect on lean mass composition at six weeks of age (P<0.001), with a difference in lean mass also seen between sedentary MC3R WT (12.98g 0.22g) and MC4R WT mice (15.47g 0.66g) (P<0.05) (Figure 3 41). At seven weeks of age (second week of treatment) genotype signifi cantly contributed to variation in lean mass (P<0.001) (Figure 3 42). A difference between MC3R WT (13.17g 0.25g) and MC4R WT (15.59g 0.62g) mice was seen in conventional caging (P<0.05) (Figure

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94 3 42). DKO mice (15.81g 0.37g) possessed a greater lean mass than sedentary MC3R WT mice (13.17g 0.25g) (P<0.05) (Figure 3 42). Genotype had a statistically significant effect on lean mass at eight weeks of age (P<0.001) (Figure 3 43). There was a difference between MC3R WT (14.11g 0.19g) and DKO (16.48g 0.37g) mice in conventional cages (P<0.05) (Figure 3 43). Additionally, MC4R KO (17.94g 0.53g) mice had a higher lean mass than MC4R WT (16.05g 0.67g) mice at eight weeks of age (P<0.05) (Figure 3 43). After four weeks of treatment (nine weeks of age) genotype had a significant effect on lean mass (P<0.001) (Figure 3 44). At nine weeks of age DKO mice (16.90g 0.32g) had a greater lean mass than MC3R WT mice (14.68g 0.28g) (P<0.05) in standard cages (Figure 3 44). Total lean masses of MC4R KO (18.56 g 0.55g) and MC4R WT (16.33g 0.73g) were significantly different (P<0.05) in conventional cages at nine weeks of age (Figure 3 44). Lean mass was significantly affected by genotype at ten weeks of age (P<0.001) (Figure 3 45). A difference in lean mass was seen between MC3R WT (15.50g 0.39g) and DKO (17.66g 0.40g) mice in conventional cages (P<0.05) (Figure 3 45). Sedentary MC4R WT (16.27g 0.76g) and MC4R KO (18.95g 0.44g) mice had statistically significant differences in lean mass (P<0.01) (Figu re 3 45). Genotype had a significant effect on lean mass of male mice at 11 weeks of age (P<0.001) (Figure 3 46). The MC3R WT mice (16.33g 0.73) in running wheel cages had a significantly higher lean mass than MC3R KO mice (14.55g 0.25g) in the same ho using condition (P<0.05) (Figure 3 46). The DKO mice (18.15g 0.34g) possessed a higher lean mass than MC3R WT mice (14.96g 0.30g) in conventional cages (P<0.001) (Figure 3 46). Sedentary MC4R WT (16.65g 0.73g) and MC4R KO (19.23g 0.41g) mice had st atistically significant differences in lean mass

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95 (P<0.01) (Figure 3 46). Genotype continued to have an effect on total lean mass in male mice at 12 weeks of age (P<0.001) (Figure 3 47). Sedentary DKO mice (18.76g 0.28g) had a statistically higher lean ma ss than either WT control group (MC3R 15.44g 0.25g; MC4R 16.87g 0.78g) in similar housing (P<0.05) (Figure 3 47). The MC4R WT mice (16.87g 0.78g) had a lower lean mass than the corresponding MC4R KO mice (19.72g 0.46g) in conventional housing (P<0. 001) (Figure 3 47). During the final week of the experiment (13 weeks of age) genotype contributed significantly to the variation seen in lean mass in male mice (P<0.001) (Figure 3 48). DKO mice (19.40g 0.33g) continued to have increased lean masses comp ared to both MC3R WT and MC4R WT mice (MC3R 15.95g 0.27g; MC4R 16.62g 0.49g) in conventional housing (P<0.01) (Figure 3 48). MC4R KO mice (Conv. 19.00g 0.36g; RW 18.50g 0.26 ) had higher lean mass than MC4R WT mice (Conv. 16.62g 0.49g; RW 16.60g 0.13g ) in both housing conditions during the last week of the experiment (P<0.05) (Figure 3 48). Summary. The MC4R WT mice had 24% higher lean masses than MC3R WT mice in conventional cages from the start of the experiment through seven weeks of age (P<0 .05) suggesting a possible difference between the WT strains of mice. The MC4R WT mice had 16% higher lean masses than DKO mice in both conventional and running wheel cages at five weeks of age (P<0.05), though the effect was temporary. Beginning at seven weeks of age, DKO mice in conventional cages had 20% more lean mass than MC3R WT control mice (P<0.05). Starting at eight weeks of age, MC4R KO mice had 12% more lean mass than MC4R WT mice in conventional cages (P<0.05). Finally, DKO mice had 11% and 17% higher lean masses than sedentary MC4R WT mice during the last 12 and 13 weeks of age (P<0.05), respectively.

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96 Effect of housing within genotypes. To determine if voluntary exercise caused any changes in lean mass within the genotypes, lean masses were eval uated by post test at each time point where a significant difference due to genotype or housing was found. No differences in lean masses were found between the housing conditions for any of the genotypes during the experiment (Figure 3 49 through Figure 3 56) with the exception of in DKO during the final week (Conv. 19.40g 0.33g; RW 17.15g 0.63g) (P<0.01) (Figure 3 57). Body length Increased linear growth (height) has been reported in human patients with dysfunctional melanocortin signaling pathways. 48 Body length has also been reported to be increased in MC4R KO mice compared to MC4R WT mice. 10,37 To test the hypothesis that voluntary exercise by running wheel would be able to prevent in the increase in linear growth, body lengths (measured from nose to anus) was measured twice a week and averaged for graphing and statistical analysis (Figure 3 58). Age and genotype both had a significant effect on body length in male mice over the course of the experiment (P<0.001) while housing d id not (Figure 3 58). Effects of genotypes within housing. Genotype had a significant effect on body length at five weeks of age (P<0.001) (Figure 3 59). There was a highly significant difference in body lengths between MC3R WT (Conv. 78.3mm 0.6mm ; RW 79 .5mm 0.9mm) and MC4R WT (Conv. 87.0mm 1.7mm ; RW 86.2mm 0.9mm) mice in both housing condition groups (P<0.001) (Figure 3 59). Sedentary DKO (82.6mm 1.0mm ) mice were significantly different in terms of length compared to both the MC3R WT (78.3mm 0. 6mm ) and MC4R WT (87.0mm 1.7mm ) groups (P<0.05) (Figure 3 59). Body lengths of exercising DKO (80.0mm 1.5mm ) mice were also significantly

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97 different from MC4R WT (86.2mm 0.9mm ) mice (P<0.01) during the fifth week of age (Figure 3 59). At six weeks of age (first week of treatment) genotype significantly contributed to length in male mice (P<0.001) (Figure 3 60). A difference in length existed in both housing conditions between MC3R WT (Conv. 78.1mm 1.4mm ; RW 80.5mm 0.6mm) and MC4R WT (Conv. 87.0mm 1.6mm ; RW 85.9mm 0.4mm) groups (P<0.01), with the MC4R WT mice possessing the greater length in both types of cages (Figure 3 60). DKO mice (85.3mm 0.9mm ) in conventional cages were significantly longer than MC3R WT mice (78.1mm 1.4mm ) (P<0.001) (Fi gure 3 60). Genotype significantly contributed to variation in male mouse body length at seven weeks of age (P<0.001) (Figure 3 61). Male MC4R WT mice (Conv. 87.7mm 1.4mm ; RW 86.7mm 0.3mm) were longer than MC3R WT (Conv. 78.9mm 0.3mm ; RW 81.4mm 1.0 mm) mice in both housing groups (P<0.01) after the second week of treatment (Figure 3 61). Sedentary MC3R WT mice (78.9mm 0.3mm ) were found to be shorter in length that DKO mice (86.5mm 0.7mm ) (P<0.001) (Figure 3 61). Both genotype and housing had a si gnificant effect on body length of male mice at eight weeks of age (P<0.05) (Figure 3 62). The MC4R WT mice (Conv. 89.5mm 1.4mm ; RW 88.0mm 0.4mm) were significantly longer that MC3R WT mice (Conv. 80.8mm 0.5mm ; RW 81.9mm 0.7mm) in both conventional and running wheel cages (P<0.001) (Figure 3 62). The body lengths of DKO mice (Conv. 87.2mm 1.0mm ; RW 83.8mm 1.7mm) were significantly different from MC3R WT mice (80.8mm 0.5mm ) in conventional cages (P<0.001) and from MC4R WT mice (88.0mm 0.4mm) in running wheel cages (P<0.05) (Figure 3 62). Genotype, housing, and the interaction between housing and genotype all had a statistically significant effect on body length in male

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98 mice at nine weeks of age (P<0.05) (Figure 3 63). The MC3R WT mice (Conv. 8 0.7mm 0.2mm ; RW 82.8mm 0.9mm) possessed shorter body lengths than MC4R WT mice (Conv. 89.5mm 1.3mm ; RW 88.6mm 0.6mm) in both housing conditions (P<0.001) (Figure 3 63). The DKO mice (88.4mm 0.9mm ) housed in conventional cages had different body l engths than MC3R WT mice (80.7mm 0.2mm ) in the same housing condition (P<0.001) (Figure 3 63). Genotype had a significant effect on nasal anal body length in ten week old male mice (P<0.001) (Figure 3 64). The MC3R WT (Conv. 81.8mm 0.4mm ; RW 83.5mm 0 .9mm) and the MC4R WT (Conv. 89.6mm 1.4mm ; RW 89.0mm 0.5mm) mice were significantly different from each other in both types of housing (P<0.01) (Figure 3 64). Sedentary DKO mice (88.5mm 0.9mm ) were significantly longer than conventionally housed MC3R WT mice (81.8mm 0.4mm ) (P<0.001) (Figure 3 64). Male MC4R WT mice (89.6mm 1.4mm ) had reduced body lengths compared to male MC4R KO mice (93.2mm 1.0mm ) in conventional caging (P<0.05) (Figure 3 64). Body lengths of eleven week old male mice were sign ificantly influenced by genotype (P<0.001) (Figure 3 65). A significant difference in body length was observed between MC3R WT (Conv. 82.6mm 0.6mm ; RW 84.2mm 0.6mm) and MC4R WT (Conv. 90.5mm 1.2mm ; RW 89.5mm 0.3mm) mice in both conventional and run ning wheel cages (P<0.01) (Figure 3 65). Male DKO mice (88.3mm 0.5mm ) in conventional cages had greater body lengths than similarly housed MC3R WT mice (82.6mm 0.6mm ) (P<0.001) (Figure 3 65). The MC4R WT mice (90.5mm 1.2mm ) continued to possess decre ased body length compared to MC4R KO mice (94.4mm 0.9mm ) in standard housing (P<0.05) (Figure 3 65). During the second to last week of treatment genotype significantly contributed to variation in body length in male mice

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99 (P<0.001) (Figure 3 66). The MC4R WT mice (Conv. 90.0mm 1.1mm ; RW 89.5mm 0.9mm) had an increased body length compared to MC3R WT mice (Conv. 83.5mm 0.5mm ; RW 85.0mm 0.9mm) in both running wheel and conventional cages (P<0.01) (Figure 3 66). The DKO mice (89.0mm 0.5mm ) in convent ional cages had statistically different body lengths compared to MC3R WT mice (83.5mm 0.5mm ) (P<0.001) (Figure 3 66). A difference was found to exist between MC4R KO (94.9mm 0.6mm ) and MC4R WT (90.0mm 1.1mm ) mice in conventional cages (P<0.001). (Fi gure 3 66) Genotype had a statistically significant effect on body length in 13 week old male mice (P<0.001) (Figure 3 67). The MC4R WT (Conv. 88.1mm 1.0mm ; RW 89.2mm 0.4mm) mice remained longer than MC3R WT mice (Conv. 83.5mm 0.6mm ; RW 85.6mm 0.5m m) in both housing conditions (P<0.05) (Figure 3 67). The MC3R KO mice (82.7mm 0.2mm ) were found to have shorter body lengths compared to MC3R WT mice (85.6mm 0.5mm) in running wheel housing (P<0.05) (Figure 3 67). Male DKO mice (89.5mm 0.7mm ) had si gnificantly different body lengths than MC3R WT mice (83.5mm 0.6mm ) in conventional cages (P<0.001) (Figure 3 67). Sedentary male MC4R KO mice (94.3mm 0.3mm ) had longer body lengths than their corresponding MC4R WT control mice (88.1mm 1.0mm ) (P<0.01 ) (Figure 3 67). Summary. The MC3R WT mice had significantly shorter body lengths compared to MC4R WT mice in both housing conditions at all time points during the experiment (P<0.05). The DKO mice were 5% and 1% longer than MC3R WT mice in conventional an d running wheel housing respectively at five weeks of age (before treatment began), and in conventional cages for the rest of the experiment (P<0.001). The MC4R WT mice were 5% longer than DKO mice at five weeks of age in conventional cages and 5%

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100 longer a t eight weeks of age in running wheel cages (P<0.05). The MC4R KO mice were 6% longer than MC4R WT mice in conventional cages beginning at 10 weeks of age (P<0.05). Effect of housing within genotypes. Body lengths within the same genotype, but different ho using conditions were compared to determine the effect, if any, of voluntary exercise on the lengths of the individual genotypes. No significant differences in body lengths were observed from the beginning of measurements (five weeks of age) through eight weeks of age (Figure 3 68 through Figure 3 71). Starting at nine weeks of age (fourth week of treatment), exercise resulted in shorter body lengths in MC4R KO mice (88.2mm 0.5mm ) compared to sedentary MC4R KO mice (92.6mm 0.9mm ) (Figure 3 72) that was also seen at 10, 11, and 12 weeks of age (P<0.05) (Figure 3 73 through Figure 3 75). During the final week of the experiment, no differences were seen between the housing conditions for any of the genotypes (Figure 3 76). Food Intake Previous research has postulated that the obesity associated with the MC4R KO phenotype is cause primarily by hyperphagia, although a metabolic disorder has been revealed by pair feeding studies. 38,117 Haskell Luevano et al. have also reported an increase in food intake in MC4R WT and MC4R KO mice allowed to exercise. To test the hypothesis that both genotype and housing would significan tly effect chow consumption, food intake was measured twice a week by subtracting the amount of chow left in the food hopper from the amount of chow that was initially placed there are the beginning of the week. For mice that were pair housed in convention al cages, food intake values were assumed to be equal between the mice and divided by two. Food consumed was divided by seven for each week and average daily food intake was

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101 averaged by group for graphing and statistical analysis (Figure 3 77). Repeated me asures analysis found that both genotype and age had a significant effect on average daily food intake (P<0.001). Effects of genotypes within housing No significant differences in food intake were observed during the fifth week of age due to either genotyp e or housing group (Figure 3 78). Genotype significantly influenced food intake in six week old male mice (P<0.001) (Figure 3 79). A significant difference in food intake was observed between sedentary DKO mice (4.70g 0.29g) and both MC3R WT (3.29g 0.1 5g) and MC4R WT (3.51g 0.25g) mice in conventional cages (P<0.01) (Figure 3 79). A significant difference in food consumption was seen due to genotype in seven week old male mice (P<0.001) (Figure 3 80). Sedentary DKO mice (5.23g 0.19g) ate significan tly more than either MC3R WT (3.21g 0.20g) or MC4R WT (3.13g 0.12g) control groups in conventional housing (P<0.001) (Figure 3 80). Food intake of male eight week old mice was significantly affected by genotype (P<0.001) (Figure 3 81). The DKO mice (5. 07g 0.12) continued to eat more than MC3R WT (2.78g 0.21g) or MC4R WT (3.04g 0.12g) mice in conventional cages (P<0.001) (Figure 3 81). The DKO mice (4.13g 0.34g) also consumed more food than MC4R WT mice (3.49g 0.15g) in running wheel cages (P<0 .05) (Figure 3 81). Male MC4R KO mice (3.86g 0.20g) were hyperphagic compared to MC4R WT mice (3.04g 0.12g) in conventional cages (P<0.01) (Figure 3 81). Genotype affected a significant difference in the average daily food consumed in nine week old mal e mice (P<0.001) (Figure 3 82). Conventionally housed DKO mice (4.95g 0.19g) were hyperphagic compared to similarly housed MC3R WT (3.48g 0.32g) and MC4R WT (2.96g 0.11g) control mice (P<0.001) (Figure 3 82). The DKO mice (4.37g 0.37g) also ate

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102 sig nificantly more than male MC4R WT mice (3.52g 0.17g) in running wheel cages (P<0.05) (Figure 3 82). The MC4R KO mice (3.79g 0.16g) in conventional cages continued to consume more chow than MC4R WT control mice (2.96g 0.11g) (P<0.05) (Figure 3 82). Th e average amount of food eaten daily was significantly affected by genotype in male mice at ten weeks of age (P<0.001) (Figure 3 83). A difference in food intake was observed between DKO (5.00g 0.18g) and both MC3R WT (3.39g 0.19g) and MC4R WT (2.93g 0.12g) mice in standard housing (P<0.001) (Figure 3 83). The MC4R KO mice (3.67g 0.21g) ate significantly more food than MC4R WT mice (2.93g 0.12g) in conventional cages (P<0.05) (Figure 3 83). The DKO mice (4.45g 0.37g) in running wheel cages were hyperphagic compared to MC4R WT mice (3.45g 0.12g) also allowed to exercise (P<0.01) (Figure 3 83). Genotype significantly contributed to the variation in the amount of food eaten in male mice at 11 weeks of age (P<0.001) (Figure 3 84). Mice from the MC3 R WT strain (Conv. 3.54g 0.29g; RW 4.01g 0.16 ) were hyperphagic compared to those from the MC4R WT strain (Conv. 2.91g 0.11g; RW 3.41g 0.05g ) at 11 weeks of age in both housing conditions (P<0.05) (Figure 3 84). The DKO mice (4.66g 0.24g) ate sig nificantly more than both the MC3R WT (3.54g 0.29g ) and MC4R WT mice (2.91g 0.11g ) in conventional cages (P<0.001) (Figure 3 84). Male DKO mice (4.31g 0.23g) also consumed more chow than MC4R WT mice (3.41g 0.05) in running wheel cages (P<0.01) (Fi gure 3 84). The hyperphagia of sedentary MC4R KO mice ( ) remained compared to MC4R WT mice ( ) (P<0.01) (Figure 3 84). Both genotype and housing significantly contributed to the amount of food eaten by male mice in the twelfth week of age (P<0.01) (Fi gure 3 85). Male MC3R WT mice (3.79g 0.12g) ate significantly more food than MC4R WT mice

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103 (2.93g 0.16g) in conventional housing (P<0.001) (Figure 3 85). The DKO mice (Conv. 4.52g 0.09; RW 4.48g 0.18g ) remained hyperphagic compared to both MC3R WT ( Conv. 3.79g 0.12g ; RW 3.86g 0.14g ) and MC4R WT (Conv. 2.93g 0.16g ; RW 3.47g 0.08g ) control groups under both housing conditions (P<0.01) (Figure 3 85). The MC4R WT mice (2.93g 0.16g) consumed significantly less chow that MC4R KO (3.72g 0.09g) m ice in conventional cages (P<0.001) (Figure 3 85). During the final week of the experiment, genotype and housing both significantly affected food consumption in male mice (P<0.01) (Figure 3 86). Exercising MC3R WT mice (4.27g 0.16g) consumed more food on average compared to MC4R WT mice (3.57g 0.05g) in running wheel cages (P<0.01) (Figure 3 86). Both MC3R WT (3.44g 0.20g) and MC4R WT (3.11g 0.13g) mice in conventional cages ate less food that DKO mice (4.52g 0.13g) (P<0.001) (Figure 3 86). The DK O mice (4.52g 0.20g) were hyperphagic compared to MC4R WT mice (3.57g 0.05g) in running wheel cages (P<0.001) (Figure 3 86). Summary Beginning at six weeks of age, DKO mice in conventional cages ate 43% and 34% more than MC3R WT and MC4R WT mice (P<0 .01), respectively (Figure 3 79). Exercising DKO mice consumed 18% more food on average per day than MC4R WT mice in running wheel cages starting at eight weeks of age (P<0.05). Male MC4R KO mice consumed 27% more food than MC4R WT mice in conventional cag es beginning with the third week of treatment (eight weeks of age) (P<0.05). Finally, MC3R WT mice ate more on average than MC4R WT mice in conventional cages at 11 and 12 weeks of age (P<0.05) and in running wheel cages at 11 and 13 weeks of age (P<0.05).

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104 Effect of housing within genotypes To determine if voluntary exercise increased, decreased, or had no effect on food consumption for the individual genotypes, statistical post tests were conducted when a significant difference was found. No differences w ere seen between the housing groups at five weeks of age for any genotype (Figure 3 87). Six and seven week old DKO mice (Six Weeks 3.63g 0.49g; Seven Weeks 3.82g 0.42g ) in running wheel cages ate significantly less than conventionally housed DKO mice (Six Weeks 4.70g 0.29g ; Seven Weeks 5.23g 0.19g) (P<0.05) (Figure 3 88 and Figure 3 89). At eight weeks of age MC3R WT mice (3.69g 0.22g) in running wheel cages ate significantly more than MC3R WT mice (2.78g 0.21g) in conventional cages (P<0.01) (Figure 3 90). In contrast, DKO mice ate significantly less in running wheel cages (4.13g 0.34g) than in conventional cages (5.07g 0.12g) at eight weeks of age (P<0.01) (Figure 3 90). No differences in food intake between housing conditions were seen f or any of the genotype from nine weeks of age through 11 weeks of age (Figure 3 91 through Figure 3 93). At 12 weeks of age, MC4R WT mice in conventional cages (2.93g 0.16g) ate significantly less than MC4R WT mice in running wheel cages (3.47g 0.08g) (P<0.05) (Figure 3 94). During the final week of the experiment (13 weeks of age), MC3R WT mice in running wheel cages (4.27g 0.16g) consumed more chow than MC3R WT mice in conventional cages (3.44g 0.20g) (P<0.01) (Figure 3 95). Summary Food intake f or DKO mice in running wheel cages was lower than that of DKO mice in conventional cages for weeks six, seven, and eight of age (P<0.05). Increases in food intake were seen in running wheel caged mice compared to conventionally

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105 housed mice for MC3R WT at e ight and 13 weeks of age (P<0.01) and for 12 week old MC4R WT mice (P<0.05). Whole Blood Glucose Blood glucose was measured by glucometer using one microliter of whole blood after each draw before blood was spun for plasma. Whole blood glucose levels are not presented in this work due in part to the large amount of variability within groups. Due to the design of the experiments from which the included mice were selected, fasting was not done prior to blood draws to prevent the possibility of refeeding beha viors in the mice that could affect food intake measurements. Without a mandatory fast for all animals, it is not possible to accurately know whether the mouse in question had recently consumed food which can directly alter circulating glucose levels. Pla sma Hormone Concentrations Experimental mice were bled from the anterior facial vein on the cheek without prior fasting. Whole blood was collected in a K 2 EDTA coated tube, transferred to a clean 1.5 mL microcentrifuge tube and spun for 10 minutes at 10,000 rpm at room temperature. Plasma was transferred to a new, labeled tube and frozen and stored at 20C For some experiments (RW 11, RW 12, RW 13, and DKO RW 2), plasma was determine hormone concentration. Serial samples from one other experiment (MC3RKO RW 1) were not c ombined at time of collection, and were thawed and pooled prior to assay. by running wheel access in male MC4R KO mice, it was decided not to assay blood samples from each week, rather just from the start (five weeks of age) and conclusion of

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106 the experiment (12 or 13 weeks of age depending on experiment). 37,64 At the time when the samples were thawed it was discovered that the majority of the MC4R WT and MC4R KO samples had dried out and potentially degraded. Following the advice of Millipore technical support (commercial suppliers of the Linco mouse endocrine panel kits), samples were reconstituted in ddH 2 O and watched for signs of degradation. Plasma concentrations of both insul in and leptin were compared to data previously published by the Haskell Luevano lab, and found to diverge from accepted values. 37 After this discovery, all samples that had been rehydrated or diluted with water were excluded from further analysis. Exclusion of the mice whose plasma samples had been diluted resulted in the dramatic reduction of sample size in some experimental groups, including leaving no viable mice in the MC4R WT running wheel group, thereby preventing the two way ANOVA method for statistical analysis. As an alternative, mice from the MC3RKO RW 1 e xperiment (MC3R WT and KO mice) were analyzed using a two way ANOVA for genotype and housing. Plasma hormone concentrations for DKO mice were evaluated using unpaired t test. To compare plasma hormone concentrations of the MC3R WT, MC3R KO, and DKO mice t o those of MC4R WT and MC4R KO mice, previously published values were used for 13 weeks of age to provide a general comparison to evaluate the effects of genotype and voluntary exercise on insulin and leptin concentrations. 37 Comparisons were not made at all time points because the values for MC4R WT and MC4R KO wo uld not correspond to the mice used for the measurements presented in Chapter 3 or in the RT PCR experiments presented in Chapter 4.

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107 Insulin Insulin is secreted by the cells of the pancreas and is responsible for the translocation of insulin sensitive glucose transporters to the cell surface. 87,89 Insulin also modulates many other metabolic processes including glycolysis, lipolysis, glycogenolysis, gluconeogenesis, lipogenesis, glycogenesis and food intake. 83,118 Insulin secretion is also been shown to be influenced by melanocortin signaling and activity. 119,120 Analysis of MC3R WT and MC3R KO mi ce in both conventional and running wheel cages by repeated measures analysis found no significant effect due to either genotype (P=0.228) or housing (P=0.068) on plasma insulin concentrations (Figure 3 96). This finding is consistent with the results publ ished by Butler et al., but contrasts with the increase in plasma insulin in male MC3R KO mice reported by Chen et al.. 8,9 These finding are further confounded by the fact that both the Butler and Chen groups fasted their mice prior to blood draw, which was not done for the mice included in these experiments. 8,9 Plasma insulin concentrations for DKO mice (Figure 3 96) could not be analyzed by repeated measures analysis due to each mouse missing data for at least one time point, so t tests were performed at each time point. Effects of housing on DKO insulin. No differences in circulating insulin concentrations were seen between DKO mice in conventional and running wheel cages at five, si x, seven, or eight weeks of age (Figures 3 97 through 3 100). Starting at nine weeks of age, a statistical difference was seen between exercising and sedentary DKO groups (P<0.05) that lasted until the conclusion of the experiment at 13 weeks of age (Figur es 3 101 through 3 105).

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108 Comparison of MC3R WT, MC3R KO, and DKO insulin concentrations to published values. Plasma insulin concentrations at 13 weeks of age from this experiment were compared to previously published values by the Haskell Luevano lab. 37 Two way ANOVA analysis revealed that genotype, housing, and th e interaction between these two variables were all statistically significant influences on plasma insulin levels (P<0.001) (Figure 3 106). A significant difference existed between DKO mice (3591pM 909pM) in conventional cages and both MC3R WT (258pM 46 pM) and MC4R WT (90pM 6pM) groups in standard cages (P<0.001) (Figure 3 106). A significant difference between conventional (3591pM 909pM) and running wheel (500pM 156pM) housed groups were seen for the DKO genotype (P<0.001) (Figure 3 107). A second two way ANOVA was conducted without the DKO mice to see if their large values were masking differences in and between the other genotypes (Figure 3 108). Genotype, housing, and the interaction between the two were all significant (P<0.001), with significa nt differences between the MC3R WT (258pM 46pM) and MC4R WT (90pM 6pM) groups in conventional housing existing (P<0.01) (Figure 3 108). There were also differences in insulin concentration between sedentary MC3R WT (258pM 46pM) and MC3R KO (457pM 1 03pM) groups (P<0.01) and also between MC4R WT (90pM 6pM) and MC4R KO (308pM 28pM) groups (P<0.001) in conventional cages (Figure 3 108). Mice with access to running wheels (compared to those in conventional cages) had significantly lower circulating i nsulin for the MC3R KO (Conv. 457pM 103pM; RW 146pM 29pM) and MC4R KO (Conv. 308pM 28pM; RW 121pM 11pM) genotypes (P<0.001) (Figure 3 109).

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109 Leptin total fat stores. 105,106 Plas ma samples acquired from weekly bloods draws of experimental mice were analyzed for leptin concentration using Linco mouse endocrine panel kits available from Millipore. Analysis of MC3R WT and MC3R KO mice in both conventional and running wheel cages foun d that both age and housing had significant effects on circulating leptin (P<0.01) (Figure 3 110). Similar to insulin, not enough viable readings were available for DKO mice (Figure 3 110) to perform a repeated measures test, so t tests were performed for each time point. Effects of genotypes within housing. No significant differences in leptin concentrations were seen at five weeks of age before treatment began for either DKO or MC3R mice (Figure 3 111 and Figure 3 120). The DKO mice in conventional cages (554pM 16pM) possessed greater plasma leptin concentrations at six weeks of age compared to DKO mice in running wheel cages (117pM 54pM) (P<0.05) (Figure 3 121). A significant difference due to genotype was seen in plasma leptin concentrations in MC3R mice (P<0.05) at seven weeks of age (Figure 3 113). At eight weeks of age, genotype had a significant effect on plasma leptin concentration in MC3R mice (P<0.05) (Figure 3 114). Housing significantly contributed to variation in leptin concentration in MC3R mice (P<0.05) at nine weeks of age (Figure 3 115). Nine week old sedentary DKO mice (791pM 165pM) had higher leptin concentrations than DKO mice allowed to exercise (285pM 106pM) using running wheel cages (P<0.05) (Figure 3 124). Housing had a signifi cant effect on circulating leptin levels in MC3R mice (P<0.001) at ten weeks of age (Figure 3 116). Sedentary DKO mice (716pM 120pM) had higher leptin levels than exercising DKO mice (343pM 77pM) at 10 weeks of age (P<0.05)

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110 (Figure 3 125). Both housing and genotype had significant effects on leptin levels in 11 week old MC3R mice (P<0.05) with a difference seen between MC3R WT (80pM 24pM) and MC3R KO (165pM 40pM) mice in conventional caging (P<0.05) (Figure 3 117). The DKO mice with access to runnin g wheels (333pM 30pM) had significantly lower leptin levels than DKO mice in conventional cages (1490pM 238pM) (P<0.01) during the eleventh week of age (Figure 3 126). Leptin concentrations in the plasma of twelve week old male mice from the MC3R backg round were different due to housing (P<0.01) (Figure 3 118). Housing conditions significantly contributed to differences in leptin concentrations of MC3R mice during the final week of the experiment (P<0.01) (Figure 3 119). There was also a significant dif ference in circulating leptin levels between DKO mice in conventional (1694pM 228pM) and running wheel (538pM 91pM) cages (P<0.01) during the final week of the experiment (Figure 3 128). Summary. The only time point that there was a difference in lept in concentrations between the MC3R genotypes was at 11 weeks of age when sedentary MC3R KO mice possessed a 106% higher circulating leptin concentrations than the similarly housed MC3R WT group (P<0.05). Sedentary DKO mice had higher plasma leptin levels t han exercising DKO mice at six, nine, ten, eleven, and thirteen weeks of age (P<0.05) Effect of housing within genotypes. Plasma leptin concentrations were analyzed by Bonferroni post test to determine the effect, if any, that voluntary exercise had on lep tin concentrations in the individual MC3R genotypes. No significant differences between the housing groups were seen in either MC3R WT or MC3R KO groups from five weeks of age through eight weeks of age (Figure 3 129 through Figure 3 132). Beginning at nin e weeks of age (Figure 3 133), exercising MC3R KO mice

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111 (28pM 5pM) had lower leptin levels compared to sedentary MC3R KO mice (87pM 17pM) (P<0.05). Comparison of MC3R WT, MC3R KO, and DKO leptin concentrations to published values. Plasma leptin concentr ations at 13 weeks of age from this experiment were compared to previously published values by the Haskell Luevano lab. 37 Two way ANOVA analysis revealed that genotype, housing, and the interaction between these two variables were all statistically significant influences on plasma leptin levels (P<0.001) at 13 week s of age (Figure 3 138). A significant difference existed between DKO mice (Conv. 1694pM 228pM; RW 538pM 91pM) and the MC3R WT (Conv. 116pM 45pM; RW 23pM 5pM) and MC4R WT (Conv. 64pM 5pM; RW 54pM 6pM) groups in both housing conditions (P<0.001 ) (Figure 3 138). A significant difference between conventional and running wheel housing groups were seen for both the MC4R KO (Conv. 454pM 45pM; RW 104pM 17pM) and DKO (Conv. 1694pM 228pM; RW 538pM 91pM) genotypes (P<0.001) (Figure 3 139). A seco nd two way ANOVA was conducted with the DKO mice excluded to see if their large values were masking differences in and between the other genotypes. Genotype, housing, and the interaction between the two were all significant (P<0.001), but no differences be tween the genotypes that were not already seen in Figure 3 138 were found. Mice with access to running wheels (compared to those in conventional cages) had significantly lower circulating leptin for the MC3R KO (Conv. 182pM 42pM; RW 29pM 8pM) and MC4R KO (Conv. 454pM 45pM; RW 104pM 17pM) genotypes (P<0.001) when DKO mice were not included in the statistical analysis (Figure 3 140).

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112 Running Wheel Activity Animals in running wheel cages had access to exercise equipment at all times when they were in t heir home cage environment. Running wheels were constructed of stainless steel and had a diameter of 24 cm, resulting in circumference of approximately 75.4 cm. Data presented within Chapter 3 is done so in terms of number of running wheel rotations, howev er conversion to distance run would be a simple conversion by multiplying number of turns by 75.4 cm/turn and then converting the distance in cm to the desired unit. Due to the primarily nocturnal nature of the experimental mice, only running wheel activit y recorded during the dark phase was analyzed. Data was analyzed by repeated measures ANOVA to determine the effects of age and genotype on amount of running wheel activity. Both age and genotype were found to have a statistically significant effect on run ning wheel activity during the dark cycle (P<0.001) (Figure 3 141). Average daily number of running wheel rotations during the dark cycle were further analyzed using one way ANOVA with Bonferroni posttest. Data is presented as average number of running whe el rotations per dark cycle during each week of the experiment. Running wheel activity was not recorded or analyzed for mice that were five weeks of age since the majority of the mice were in conventional cages (i.e. no running wheels). There was no signif icant difference in running wheel activity due to genotype at six or seven weeks of age (Figure 3 142 and Figure 3 143). Genotype had a significant effect on running wheel activity at eight weeks of age (P<0.05) (Figure 3 144). Genotype contributed signif icantly to the variation in running wheel activity for male mice at nine weeks of age (P<0.05) (Figure 3 145). Running wheel activity during the dark cycle was significantly influenced by genotype at ten week old male mice (P<0.05)

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113 (Figure 3 146). At eleve n weeks of age, the running wheel activity of male mice was effected by genotype (P<0.05) (Figure 3 147). The MC3R WT mice (6218 turns 829 turns) ran significantly more on their running wheel cages than male DKO mice (2174 turns 763 turns) of the same age (P<0.05) (Figure 3 147). Genotype significantly influenced nocturnal running wheel activity in twelve week old male mice (P<0.01) (Figure 3 148). The MC3R WT mice (6629 turns 615 turns) ran significantly farther using the running wheels in their home cages that DKO mice (2129 turns 695 turns) of the same age (P<0.01) (Figure 3 148). During the final week of experimentation (13 weeks of age) genotype had a statistically significant effect on running wheel activity (P<0.001) (Figure 3 149). The MC3R W T mice (6860 turns 585 turns) used their running wheels significantly more than either the MC4R WT (2721 turns 392 turns) or the DKO (2091 turns 672 turns) mice (P<0.01) (Figure 3 149). Summary. No significant differences in dark cycle running wheel activity was seen in mice aged six, seven, eight, nine, or ten weeks. Beginning at 11 weeks of age MC3R WT mice ran 186% more than DKO mice in running wheel cages (P<0.05) as measured by wheel turns during the dark cycle. During the final week of the exper iment MC3R WT mice also completed 152% more running wheel revolutions than MC4R WT mice (P<0.01) (Figure 3 149). Minimum Amount of Exercise Needed to Prevent Obesity beneficial of wheel rotations for the entire experiment (used as a measure of total exercise). A linear regression line was plotted for each genotype and the slope of the resulting line was statistically analyzed to see if its slope was significantly different than zero.

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114 Correlation of exercise and body weight When final body weights were plotted against total number of wheel turns done over the course of the experiment and best fit lines were plotted, none possessed a slope that was significantly different than zero (Figure 3 150). Body weights for MC3R KO mice correlated best with amount of exercise (r 2 = 0.5914) while those from the MC3R WT group had the worst correlation (r 2 = 1.504 x 1 0 4 ). Body weights of MC4R WT, MC4R KO, and DKO did not strongly correlate with running wheel activity with r 2 values of 0.1164, 0.2943, and 6.235 x 10 2 respectively. Correlation of exercise and fat mass When fat masses from the final MRI scan were plot ted against total number of running wheel turns and best fit lines were plotted, the slope of the line for the MC4R KO group was significantly different from zero (P<0.05) (Figure 3 151). The slope of the line for the MC4R KO mice was 5.66 x 10 3 1.01 x 10 3 with an Y intercept of 4.870.251, and an X intercept of 860.4. The fat masses of MC4R KO mice correlated well with total exercise with an r 2 value of 0.912. The slopes of the lines of best fit for the other groups were not significantly different f rom zero. Other than the MC4R KO genotype, fat masses of the MC3R WT mice correlated best with exercise (r 2 = 0.615), fat masses for the MC3R KO, MC4R WT, and DKO did not correlate well with amount of exercise performed (r 2 = 0.101, 0.220, and 4.24 x 10 2 respectively). Additional MC3R Data and Statistics Due to low sample sizes (n= 4 5) used in the statistical analyses presented above, and pronounced phenotype differences seen in MC4R KO and DKO mice, it is possible that differences in the measured variabl es were present between the MC3R WT and MC3R KO genotypes that would be too subtle to be identified. In order to determine if

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115 minor, yet significant, differences between MC3R WT and MC3R KO mice exist, statistical analyses were carried out on MC3R KO and M C3R WT mice in conventional and running wheel cages for a larger sample size (n= 7 13). Seven MC3R WT mice from conventional cages, eight MC3R WT mice from running wheel cages, 13 MC3R KO mice in conventional cages, and 12 MC3R KO mice that were allowed to exercise in running wheel cages were used for the more comprehensive statistical analysis. All mice were from experiment MC3RKO RW 1 (6/29/2009 8/30/2009).Insulin and leptin concentrations were not reanalyzed since plasma was assayed only for mice that we re included in the above and RT PCR studies. After statistical analysis by repeated measures using a general linear model, it was found that neither genotype nor housing had an effect on body weight or food intake for mice of a MC3R background and that gen otype did not significantly affect running wheel activity (Figures 3 152, 3 153, and 3 154 respectively). Statistically significant differences were found for body length (Figure 3 155), fat mass (Figure 3 165), and lean mass (Figure 3 184) for MC3R WT and KO mice in conventional and running wheel cages. MC3R body length Genotype and age both had significant effects on body length of mice from a MC3R background (P<0.05) over the course of the entire running wheel experimental treatment (Figure 3 155). Genot ype had a significant effect on body length in five week old male mice from a MC3R background (P<0.05) (Figure 3 156). Body lengths were significantly affected by genotype in mice of a MC3R background at six weeks of age (P<0.05) (Figure 3 157). Seven week old male mice from a MC3R background had differences in body length due to genotype (P<0.05) (Figure 3 158). There were no

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116 significant effects on body length at eight weeks of age for mice of a MC3R background (Figure 3 159). Genotype significantly contri buted to variation in body lengths for nine week old male mice of a MC3R background (P<0.05) (Figure 3 160). The body lengths of ten week of mice from a MC3R background were significantly affected by genotype (P<0.05) (Figure 3 161). Genotype had a signifi cant effect on nasal anal body length in male mice from a MC3R background at 11 weeks of age (P<0.05) (Figure 3 162). Genotype, but not housing, had a significant influence on body length for 12 week old male mice from a MC3R strain background (P<0.05) (Fi gure 3 163). Neither genotype nor housing had a significant effect on body length during the final week of treatment (13 weeks of age) in mice from a MC3R background (Figure 3 164). Summary. Although genotype had an overall effect on body length for mice f rom a MC3R background, no specific differences were found to exist between the MC3R WT and MC3R KO genotypes during the experimental timeframe. There was also no significant difference in body lengths between the two housing conditions for either the MC3R WT or MC3R KO genotypes at any of the ages studied in this experiment. MC3R fat mass Genotype, housing condition, and age all had a significant effects on fat mass in mice of a MC3R background (P<0.05) (Figure 3 165). There were no significant differences in total fat mass during the first two weeks of measurement (five and six weeks of age) (Figure 3 166 and Figure 3 167). Housing had a significant effect on adiposity of mice from a MC3R background (P<0.05) after only two weeks of treatment (seven weeks of age) (Figure 3 168). A significant difference in fat mass between conventionally housed (2.54g 0.24g) and exercising (1.95g 0.13g) MC3R KO mice was seen at seven weeks of age (P<0.05) (Figure 3 177). Both genotype and housing

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117 had a significant effect on total fat mass in eight week old male mice from MC3R strains (P<0.01) (Figure 3 169). MC3R KO mice (2.21g 0.16g) in conventional cages had higher fat masses than MC3R WT mice (1.37g 0.10g) in similar housing (P<0.001) (Figure 3 169). Exercising MC3 R KO mice (1.52g 0.10g) had lower fat masses than conventionally housed MC3R KO mice (2.21g 0.16g) at eight weeks of age (P<0.01) (Figure 3 178). At nine weeks of age genotype and housing continued to significantly affect fat mass levels in male mice o f a MC3R background (P<0.05) (Figure 3 170). The MC3R WT mice (1.55g 0.19g) had lower total body fat compared to MC3R KO mice (2.30g 0.20g) in conventional caging (P<0.05) (Figure 3 170). The MC3R KO mice in conventional cages (2.30g 0.20g) had highe r fat masses than MC3R KO mice in running wheel cages (1.34g 0.12g) at nine weeks of age (P<0.01) (Figure 3 179). Housing and genotype both significantly contributed to variation in fat mass as quantified by MRI scan (P<0.05) in ten week old mice from a MC3R background (Figure 3 171). Sedentary MC3R WT mice (1.76g 0.20g) continued to have lower fat masses than MC3R KO mice (2.55g 0.22g) in conventional cages (P<0.01) (Figure 3 171). The MC3R KO mice in running wheel cages (1.62g 0.09g) had lower tot al body fat mass values than MC3R KO mice in conventional cages (2.55g 0.22g) (P<0.01) for ten week old mice (Figure 3 180). Fat mass was significantly influenced by housing condition in male MC3R mice that were 11 weeks old (P<0.01) (Figure 3 172). A si gnificant difference in fat masses existed between MC3R KO mice in conventional (2.44g 0.29g) and running wheel (1.38g 0.12g) cages at 11 weeks of age (P<0.01) (Figure 3 181). Housing remained a significant factor for adiposity levels in 12 week old mi ce of a MC3R background (P<0.01) (Figure 3 173). Fat masses of sedentary MC3R

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118 KO mice (2.59g 0.31g) were higher than fat masses of exercising MC3R KO mice (1.38g 0.10g) during the second to last week of the experiment (P<0.01) (Figure 3 182). During th e final week of treatment, housing continued to possess a significant influence over total fat mass in male MC3R mice (P<0.001) (Figure 3 174). The MC3R KO mice in conventional cages (3.04g 0.36g) possessed greater fat masses than MC3R KO mice allowed to exercise in running wheel cages (1.75g 0.12g) (P<0.01) (Figure 3 183). Summary. At eight, nine, and ten weeks old, MC3R KO mice had significantly higher fat masses compared to MC3R WT mice in conventional cages (P<0.05). MC3R KO mice in conventional ca ges had fat masses than 30% higher than MC3R KO mice in running wheel cages starting at seven weeks of age (Figure 3 177), lasting until the conclusion of the experiment (Figure 3 183). MC3R lean mass Genotype and age were both found to have a significant influence on lean mass for male mice from a MC3R background (P<0.05) (Figure 3 184). Genotype had a significant effect on total lean mass measured by MRI scan in MC3R mice at five weeks of age (P<0.05) (Figure 3 185). The lean mass of male MC3R mice was si gnificantly affected by genotype during the first week of treatment (six weeks of age) (P<0.05) (Figure 3 186). No significant differences due to either genotype or housing type were seen at seven weeks of age for male mice from a MC3R background (Figure 3 187). Genotype significantly contributed to variation in lean mass for eight week old male mice from a MC3R background (P<0.01) (Figure 3 188). The MC3R KO mice (13.60g 0.32g) in running wheel cages possessed a statistically lower amount of lean mass th an MC3R WT mice (15.08g 0.77g) in the same type of housing (P<0.05) at eight weeks

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119 of age (Figure 3 188). Lean mass quantities of male mice from a MC3R background were significantly influenced by genotype at nine weeks of age (P<0.05) (Figure 3 189). Gen otype had a statistically significant effect on lean mass of ten week old MC3R mice (P<0.05) (Figure 3 190). A significant difference due to genotype was seen in lean mass for male MC3R mice at 11 weeks of age (P<0.05) (Figure 3 191). Genotype had a signif icant effect on total amount of lean mass in 12 week old male mice from a MC3R background (P<0.05) (Figure 3 192). During the final week of treatment (13 weeks of age), genotype had a significant effect on lean mass of male MC3R mice (P<0.05) (Figure 3 193 ). Summary. Genotype had a significant effect on lean mass at most time points measured while housing had no statistical influence over lean mass. A significant difference in lean mass between MC3R KO and MC3R WT mice was only seen at eight weeks of age an d only in running wheel cages, with MC3R WT mice having 11% more lean mass than MC3R KO mice. At no point was a significant difference in lean mass quantity seen between conventionally and running wheel housed mice for either the MC3R KO or MC3R WT genotyp es. Discussion Obesity is a troubling disease since it significantly increases the likelihood of the onset of another disease including type 2 diabetes and cardiovascular disease. 3 The melanocortin system has been clearly linked to metabolic dysfunction through the identification of obese human patients with disruptive mutations in their receptors and the phenotypes of the MC3R KO, MC4R KO, and DKO mice. 8 10,45 54,116 Diet and exercise are both widely accepted methods of controlling energy balance and affecting weight loss. 62

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120 The beneficial effects of exercise on male MC4R KO mice which are genetically predisposed to hyperphagia and obesity have been previously described by the Haskell Luevano laboratory. 37,63,64 The results presented in Chapter 3 detailed the effects of exercise thoroughly for MC3R KO and DKO mice as well as the MC4R KO mice. Discussion of Results Body weight Consistent with previous work, in standard conventional cages, MC4R KO mice had a higher body weight compared to MC4R WT mice while DKO mice had higher body weights than both MC3R WT and MC4R WT groups (Figure 3 1). 9,10,37,63,64 Similarly, MC3R KO mice did not possess significantly different body weights from MC3R WT mice in either housing condition (Figure 3 1 and Figure 3 152) as expected from previous descriptions of t his genotype. 8,9 The MC4R KO mice weighed significantly more than MC4R WT mice in convention al cages beginning at seven weeks of age (Figure 3 4). This difference in body weights between the MC4R KO and WT mice was not seen in running wheel cages until the thirteenth week of age (Figure 3 10). This supports the hypothesis that voluntary exercise can significantly slow, if not prevent, the onset of the obese phenotype in MC4R KO mice. 37,64 Voluntary exercise was able to delay, but not prevent the onset of the obese phenotype of DKO mice. Though voluntary exercise by running wheel was able to prevent the onset of the obese phenotype of the MC4R KO mice within the experimental time frame, it merely delayed the onset of the obese phenotype of the DKO mice (Figures 3 5 and Figure 3 7). While DKO mice weighed more than both WT groups, voluntary exercise did r esult in diminished weight gain compared to sedentary DKO

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121 mice (Figure 3 19). This supports the hypothesis that voluntary exercise is capable of blunting the severe obese phenotype of the DKO mice. Fat mass Increased fat mass compared to wildtype mice is a result of both MC3R and MC4R deletion in mice. 8 10,36,37,39 Consistent with the literature both MC4R KO mice and DKO mice in conventional cages had increased fat mass compared to the appropriate WT groups (Figure 3 20). 9,10,37 Although preliminary analysis MC3R KO mice did not have increased fat masses compared to MC3R WT mice in conventional cages (Figure 3 29), increasing sample size did result in a difference in fat masses being seen between MC3R WT and MC3R KO mice (Figure 3 165) consistent with previous reports. 8,9 Exercise was able to result in a decrease in adiposity in MC3R KO mice compared to sedentary MC3R KO mice (Figure 3 38). Consistent with previous reports, exercising on running wheels was able to prevent the increase in fat mass in MC4R KO mice within the experimental time course (Figure 3 29). 37 Exerci se was also able to delay the onset of increased adiposity in DKO compared to MC3R WT and MC4R WT mice (Figures 3 23 and 3 24) and decrease the total amount of fat deposition compared to sedentary DKO mice (Figure 3 38). The fact that there was already a significant difference in fat mass between DKO mice in the conventional and running wheel groups at five weeks of age (Figure 3 30) indicates an experimental design error since all DKO mice had been in running wheel cages since birth. This error also accou nts for the fact that the conventional DKO group had higher fat masses than MC3R WT and MC4R WT groups, but DKO mice in the running wheel group did not (Figure 3 21). This error could be accounted for and

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122 corrected in the future by doing an MRI scan of any experimental DKO mice prior to making the group assignments. Eight weeks of voluntary exercise resulted in lower fat masses in male MC3R KO, MC4R KO, and DKO mice at 13 weeks of age compared to conventionally housed mice of the same genotypes (Figure 3 38 ). Using the illustration shown Figure 1 1 as a general model for body weight and fat mass homeostasis, with the decrease in fat mass seen either a decrease in energy intake or expenditure would be predicted. Since other than at six, seven, and eight weeks of age in DKO mice (Figures 3 88, 3 89, and 3 90), food intake for mice in running wheel cages was equal to or greater than mice in conventional cages, meaning that a decrease in energy intake is not responsible for the changes in body size and compositio n. This would indicate the differences in fat mass are due to increased energy expenditure caused by voluntary exercise by running wheel. Based on the design and limitations of this experiment, it is impossible to tell the mechanism that resulted in differ ent adiposities in conventionally housed KO mice compared to KO mice allowed to exercise. It is possible that the increase in energy expenditure resulted in fewer calories that needed to be stored as fatty acids in adipose tissue. The lack of a need for e xtensive storage of excess calories in white adipose tissue might also reduce proliferation of adipocytes, further contributing the difference in adipose tissue mass seen between the exercising and sedentary KO mice groups. Lean mass Both MC4R KO and DKO m ice possessed increased lean masses in conventional cages compared to WT mice (Figure 3 39). This is consistent with the Haskell Luevano et al. report of increased lean mass in MC4R KO mice compared to MC4R WT mice. 37 No significant differences in lean mass measured by MRI were found between MC3R

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123 WT and MC3R KO mic e (Figure 3 39) contrary to previous reports of the MC3R KO mouse phenotype have described an increased in fat mass accompanied by no change in total body weight, resulting in an overall decrease in lean body mass. 8,9 Since a significant difference was seen in fat masses, but not in body weights between MC3R WT and MC3R KO mice, this indicates a trend of decreased lean mass in MC3R KO mice. The difference in lean mass that existed between conventionally housed MC3R WT and MC4R WT mice from five to seven weeks of age (Figure 3 40 through 3 42) suggests an underlying difference between the backgrou nd strains of these mice. Exercise was able to delay the increase in lean mass seen in MC4R KO mice compared to MC4R WT mice from eight weeks of age in conventional cages (Figure 3 43) to 13 weeks of age in running wheel cages (Figure 3 48). Voluntary exe rcise was also able to prevent the increase in lean mass seen in DKO mice (disregarding the difference seen before treatment began (Figure 3 40)) compared to WT mice also in running wheel cages during the experiment (Figure 3 48). Only for DKO mice at 13 w eeks of age was a difference in lean mass seen between mice in conventional and running wheel cages (Figure 3 57). These findings support the hypothesis that voluntary exercise is able to delay the onset of increased growth in male MC4R KO and DKO mice. B ody length The MC3R WT were significantly shorter than MC4R WT mice in both housing conditions during the experimental time frame (Figure 3 58). This difference in body length hints at the possibility of a strain difference between the MC3R WT and MC4R WT mice. The difference in body length also corresponds to the differences in lean mass between the same groups (Figure 3 40). A correlation between body length and

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124 lean mass has been previously reported. 37 A genotype effect was seen between male MC3R KO and MC3R WT mice, though no specific differences were seen. This is consistent with reports by Butler et al. that no differences in body length was seen between MC3R WT and MC3R KO mice, but conflicts with Chen et al. that did see a difference in body lengths between the MC3R genotypes. 8,9 The increase in MC4R KO body length compared to MC4R WT mice is consistent with the literature. 10,37 Unlike body weight (Figure 3 1), fat mass (Figure 3 20), or lean mass where s edentary DKO mice had greater or equal values to MC4R KO mice in conventional cages, MC4R KO mice were the genotype that possessed the greatest body lengths (Figure 3 58). This might be the result of a strain difference, since the DKO mice were derived fro m both the MC3R and MC4R breeding colonies, or it could be a result of a functioning MC3R in MC4R KO mice, but not in DKO mice. It is possible that the difference in body lengths seen between the MC3R and MC4R strains is due to differences in measurement t echnique used to stretch the mice for measurement. The body lengths of the MC4R mice from the RW 11 and RW 12 experiments were measured by Laurie Koerper and Amy Andreasen, while the lengths of the MC3R mice from MC3RKO RW 1 were measured by Jay Schaub. E xercise by running wheels had a significant influence on body lengths of male mice (Figure 3 58). Similar to previously published results, MC4R KO mice allowed to exercise never reached lengths significantly different from those of exercising MC4R WT mice (Figure 3 67). 37 Likewise, DKO mice allowed to exercise were not significantly longer than MC3R WT mice in running wheel cages during the experiment (Figure 3 67). Exercise significantly reduced MC4R KO body length compared to conventionally

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125 housed MC4R KO mice (Figure 3 73 through Figure 3 75). Since this change w as seen only in MC4R KO mice, and not in either MC3R KO or DKO mice, it could possibly indicate that the MC3R is necessary for preventing the change in body length seen in MC4R KO mice. Food intake As expected, MC4R KO mice were hyperphagic compared to MC4 R WT mice, although this difference was not observed until eight weeks of age (Figure 3 81). 10,37,38,63,64 Not surprisingly, DKO mice were also hyperphagic compared to MC3R WT and MC4R WT mice in conventional cages beginning at six weeks of age (Figure 3 77). No difference in food intake was seen between the MC4R KO a nd WT mice in the running wheel cages during the experiment (Figure 3 77). These results conflict with previous findings that voluntary exercise increases food intake. 37 Voluntary exercise only increased food intake in MC4R WT mice at 12 weeks of age (Figure 3 94) and in MC3R WT mice at 13 weeks of age (Figure 3 95 ). Voluntary exercise was able to delay the onset of hyperphagia in DKO mice (compared to MC4R WT mice) until eight weeks of age (Figure 3 81). The DKO mice in running wheel cages actually consumed less food than DKO mice in conventional cages (Figures 3 8 8 through 3 90). All DKO mice were housed in running wheel cages from birth, and once the DKO mice in the conventional group were transferred from running wheel cages at six weeks of age, a significant increase in food intake in seen in the conventional gr oup compared to the exercising group. This effect is similar to that reported by Irani et al. when they moved MC4R KO mice from running wheel to conventional cages and observed hyperphagic behavior after the transfer. 64 Interpretation of DKO food intake compared to the other

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126 genotypes is further confounded by the fact that DKO mice were housed in running wheel cages for the first five weeks life while the other genoty pes were all in conventional cages at that time point. This means that it is possible that the DKO mice would have been hyperphagic compared to the other genotypes at that early age in conventional cages, but were not since there had access to exercise equ ipment. Plasma hormone concentrations Insulin. Insulin is an extremely important metabolic hormone that affects a multitude of metabolic processes. 83 Due to sample degradation, plasma insulin levels for the MC4R WT and KO mice in conventional and running wheel cages were not able to be determined. Genotype and housing did not significantly affect plasma insulin levels in MC3R WT and KO mice in running wheel or conventional cages (Figure 3 96). This finding contrasts with the increase in plasma insulin in male MC3R KO mice reported by Chen et al., but is consist ent with the results published by Butler et al.. 8,9 Exercise was found to result in a signif icant difference between sedentary and exercising DKO mice starting at nine weeks of age (Figure 3 101). The large increase in circulating insulin concentrations over time seen in sedentary male DKO mice can most likely be explained by peripheral insulin r esistance, one of the indicators of type 2 diabetes. Insulin resistance is where the tissues in the body are less responsive to insulin, as a result blood glucose levels do not decrease and the pancreas progressively secretes increasing amounts of insulin. Comparing insulin levels from the experiments described above and by the Haskell Luevano lab showed a significant difference in insulin between DKO and both WT groups in conventional housing (Figure 3 106). 37 The difference found between DKO mice in conventional and running wheel cages by t test was also seen aft er

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127 ANOVA (Figure 3 107). A second ANOVA test without inclusion of the DKO mice revealed significant differences between both MC3R WT and KO mice and MC4R WT and KO mice in conventional cages (Figure 3 108). A difference in the plasma concentrations of insu lin between MC3R WT and MC4R WT mice in conventional cages also support the hypothesis of the presence of a strain difference (Figure 3 108). What is surprising is that mice from the MC3R background have higher levels of insulin compared to the published v alues for MC4R KO mice using the same assay. 37 The differenc e in plasma insulin levels between the MC4R WT and MC3R WT mice are evidence that a metabolic difference exists between these two strains of mice. However, since the values used for MC4R mice for statistical analysis of plasma insulin concentration are not from mice from the presented experiment, further comparisons are not appropriate. Leptin. Leptin is a polypeptide hormone secreted from adipocytes that influences metabolism and eating behavior. 41,43,59,106,121,122 Li ke insulin, leptin concentrations were not determined for the MC4R WT and MC4R KO mice in this experiment due to sample degradation. Housing significantly influenced leptin concentrations in MC3R mice, but genotype did not have a significant effect on MC3R plasma leptin concentrations. This is different from previous reports of MC3R KO mice having increased plasma leptin values compared to MC3R WT mice. 9 Significant differences were also seen between the DKO mous e groups (Figure 3 110). Exercise resulted in significantly lower leptin levels in exercising MC3R KO mice compared to sedentary MC3R KO mice beginning at nine weeks of age (Figure 3 133) Plasma leptin concentrations varied widely in sedentary DKO mice ov er the course of

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128 the experiment (Figure 3 110) which resulted in intermittent differences from exercising DKO mice. DKO mice allowed to exercise had significantly lower leptin concentrations at six, nine, ten, eleven, and thirteen weeks of age (Figures 3 1 21, 3 124, 3 125, 3 126, and 3 128 respectively). The decrease in plasma leptin corresponded with the decrease in fat mass seen in exercising MC3R KO and DKO mice compared to sedentary MC3R KO and DKO mice respectively. This is consistent with the literatu re in that leptin secretion correlates with adipose tissue mass. 106 Comparing leptin levels from the experiments described above to those previously published by the Haskell Luevano lab showed a significant difference in insulin between DKO and both WT groups in b oth housing conditions as well as a difference between MC4R KO and MC4R WT mice (Figure 3 138). 37 It is not surprising that leptin concentrations were elevated in MC4R KO or DKO mice as this been tissue content, which was elevated in both the MC4R KO and DKO mice (Figure 3 29). 10,37,106 The fact that increases in circulating leptin were seen for MC4R KO and DKO mice but not for MC3R KO mice in conventional cages might be explained by the fact that, compared to the MC4R KO and DKO mice, MC3R KO mice only have modest increases in body fat mass (Figure 3 20). A significant difference in plasma leptin concentration might be seen if the number of samples run for MC3R mice were increased as was the case for fat mass (Figure 3 165). The fact that voluntary exercise prevented the increase in circulating leptin was previous reported by Haskel l Luevano et al., and this phenomena is expected to have been confirmed had the plasma samples for the MC4R KO mice been viable. 37 Even

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129 though DKO mice in running wheel cages had significantly increased leptin values from WT controls, voluntary exercise was able to blunt the increase in circulating leptin compared to sedentary DKO mice (Figure 3 139). Additionally, exercise was shown to decrease circulating leptin in MC3R KO mice (Figure 3 140) when DKO mice were not included in the statistical analysis. Luminex data limitations. As discussed in the results section whole blood glucose levels were not reported due to the inability to account for meal affects since mice were not fasted prior to when blood was drawn. The lack of a fast prior to a blood draw also might have significantly influenced circulating insulin levels since they are also linked to meal consumption and incretin release by the gut. 103,104 Taken together with the limitations on blood glucose signify the need for fasting prior to blood draws in future experiments. Running wheel activity Ru nning wheel activity was monitored by magnetic switch breaks as measured by the Mini Mitter VitalView software program. 8,37,63,64,80 Instead of reporting activity in average number of turns per minute, it was instead presented as average number of rotations per day (Figure 3 141). 37,64 By converting this data to daily average distance run, it can be compared to other inbred mouse strains. 78,79 Genotype was found to significantly influence the amount of physical activity performed on the running wheels (Figure 3 142) with MC3R WT mice running more than both DKO (Figure 3 148) and MC4R WT mice (Figure 3 149). When the number of running wheel turns were converted into distance (by multiplying by 75.4 cm), these values were within the range of daily distance run for various inbred strains of mice reported by Lightfoot et al. with DKO mice running the least (on average 1.2 km per d ay) at seven weeks of age (Figure

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130 3 143) and MC3R WT the most (on average 5.2 km per day) at 13 weeks of age (Figure 3 149). 79 Previous studies have reported MC3R KO mice to be hypoactive compared to MC3R WT mice. 8,9 Butler et al. reported decreased running wheel activity in male MC3R KO mice compared to WT littermates. 8 Chen et al. reported a decrease in locomotor activity in female MC3R KO, but not male MC3R KO mice. 9 These reports contrast with the data presented in this dissertat ion where MC3R KO mice did not exercise any more or any less than MC3R WT mice (Figures 3 141 and 3 154). Similarly, MC4R KO running wheel activity has not previously been reported to be significantly different than running wheel exercise performed by MC4R WT mice, which is consistent with the data presented herein. 37,80 The differences seen between the MC3R WT and MC4R WT mice cannot be explained solely by a strain difference since Lightfoot et al. did not observe a significant difference in daily distance run between th e C57BL/6J and 129x1/SvJ strains. 79 The drawback to the activity monitoring system use d was that the wheels are rather large and rotations were measured by a magnet mounted in one place, meaning that if the mice rocked the wheel multiple rotations could be recorded without a full rotation of the wheel being recorded. Another observation mad e by experimental animal staff was that due to the size (25 cm diameter) of the wheel, and its stainless steel construction, at young ages not all mice were heavy enough to cause the wheel to spin. The running wheel monitoring software was not able to moni tor or measure levels of ambulatory activity, making it necessary to assume that ambulatory activity levels in mice were not different between the conventional and running wheel cage groups.

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131 These concerns will be addressed in the future through use of cal orimetric cages from TSE systems that are equipped with ambulatory activity monitoring equipment as well as smaller running wheels capable of measuring half turns of the running wheel, making analysis of exercise behaviors potentially more accurate. Correl ations One of the interesting questions that can be answered by this research is do mice that exercise more have increased protection from the obese phenotypes and metabolic disorders associated with central melanocortin receptor dysfunction compared to th ose who exercise less? To address this question body weight and fat mass values from the final measurement were plotted against the total number of running wheel turns during the experiment for mice in running wheel cages. When fat mass or body weights wer e plotted against running wheel activity, the best fit lines were plotted and their slopes were analyzed. If the slopes were significantly different from zero, it would mean that increased exercise reduced body weight if the slope was negative, or increase d exercise increased body weight the if the slope was positive. The same conclusions could be made from the plots of fat masses. Because none of slopes of the lines were significantly different from zero on the body weight versus running wheel activity pl ot, it can be concluded that increasing the amount of exercise does not increase the amount of protection from the obese phenotypes (Figure 3 149). While these results are not surprising for the WT mice or MC3R KO mice which are not obese, this is signific ant for the MC4R KO and DKO mice. These results imply the presence of an minimum exercise threshold that is required to provide the beneficial effects of exercise seen on body weight (Figure 3 19).

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132 The slopes of the lines from the plot of fat mass versus r unning wheel rotations were not significantly different from zero for any of the genotypes except for the MC4R KO group (Figure 3 150). Since the slope of the line for the MC4R KO mice was negative, it implies that increased exercise by mice in this genoty pe actually decreases fat mass (Figure 3 150). With an Y intercept of 4.870.251, the line of best fit for MC4R KO fat mass versus running wheel activity predicts a fat mass of approximately five grams with no running wheel rotations. Since the fat masses of conventionally housed MC4R KO mice is roughly double that, it can be assumed that exercise provides an overall prevention from increases in fat mass before the effects of additional exercise take effect. These results imply the presence of an minimum e xercise threshold that is required to provide the beneficial effects of exercise seen on fat mass for DKO, MC3R KO, and MC4R KO mice (Figure 3 38), with increasing amounts of exercise further decreasing the fat mass of MC4R KO mice. Because the running whe el exercise studied within this experiment was a voluntary behavior for the mice it is difficult to quantifiably determine the benefits of exercise. If a treadmill experimental paradigm was used in which the exercise activity of the mice could more be easi ly monitored and controlled it might be easier to determine what level of exercise activity is required to gain the beneficial effects of exercise. Additionally, due to small sample size (n = 4 5), mice with unusual levels of exercise, body weights, or fat masses might skew the results of a best fit plot. Looking at the DKO group in both the body weight (Figure 3 150) and fat mass (Figure 3 151) plots, there is one mouse that had a lower body weight and fat mass regardless of the fact it also ran the least over the course of the experiment. Exclusion of this point would appear to

PAGE 133

133 cause the line to shift resulting in a negative slope for both plots. However, further examination of this mouse revealed that while it ran significantly less than other mice of the same genotype, there was no plausible reason to exclude the mouse from analysis. It can therefore be concluded that in order to better understand the relationship between exercise and body weight and fat mass in the central melanocortin receptor KO mice, group sizes must be increased in order to reduce the amount of random error that can adversely affect this analysis. Strain Differences Between MC3R and MC4R Breeding Colony Mice For several of the measured variables statistically significant differences were found between the MC3R WT and MC4R WT groups (Figures 3 40, 3 59, 3 85, and 3 108). Some of these differences in phenotype and metabolism might be explained by differences in background strain. Originally, the Haskell Luevano laboratory obtained mice to generate MC3R heterozygous and KO mice from Merck Research Laboratories. 9 The mice used to generate a breeding colony for MC4R KO mice were obtained from Dennis Huszar of Millennium Pharmaceuticals. 10 Both strains of mice were originally generated using embryonic stem cells from the 129/Sv inbred mouse strain that were injected into C57BL/6J blastocysts and then chimeric males were bred back to female C57BL/6J female mice to generate offspring heterozygous for the appropriate receptor. 9,10 When the Haskell Luevano laboratory received these mice, they were on a mixed background of both the 129/Sv and C57BL/6J mice. Since no eff orts to back cross these strains to one of the parent strains were made during the maintenance of the Haskell developed into separate strains. These strains were intermixed when permission was obtain to cross the two KO lines to breed mice lacking both the MC3R and MC4R, or

PAGE 134

134 DKO mice. The WT mice that would be generated from crossing MC3R +/ and MC4R +/ mice were not used as controls in the described experiments. Data support the hypothesis that an inh erent metabolic and phenotypic difference exists between the MC3R and MC4R strains is supported by body weight, lean mass, body length, food intake, and plasma insulin concentration data. Even though there was no significant difference between the MC3R WT and MC4R WT groups based on direct comparison, the fact that the DKO mice weighed significantly more than the MC3R WT mice two weeks before they weighed more than the MC4R WT mice in both housing conditions hints at the possibility of a strain difference b etween the MC3R and MC4R backgrounds. The difference in lean mass that existed between MC3R WT and MC4R WT mice in conventional cages from five to seven weeks of age (Figure 4 40 through 3 42) suggests there might be an underlying difference in the backgro und strains of these KO mice lines. This theory is further supported by the difference in lean mass also seen between MC4R WT and DKO mice in both housing groups (Figure 3 40). However, since the difference between MC3R WT and MC4R WT mice was only present in one group (conventionally housed) and not the other before treatment began (Figure 3 40), this difference might also be attributo experimental design error. Body length is significantly different between the MC3R and MC4R WT groups in both conventional and running wheel cages for the entire experiment (Figure 3 67), further contributing to the pool of evidence that there is a strain difference between the MC3R and MC4R inbred strains. Further supporting the theory that the MC3R and MC4R lines are inhere ntly different from one another due to strain differences are differences in food intake seen between MC3R WT and MC4R WT in conventional (Figures 3 84 and 3 85)

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135 and running wheel cages (Figure 3 86). Figure 3 108 shows the significant difference that exis ted between insulin concentrations of MC3R WT and MC4R WT mice in conventional cages. The hypothesis that the MC3R and MC4R strains are different is supported by data illustrating the significant differences in glucose metabolism and homeostasis of the 129 /Sv and C57BL/6 founder strains. 123 Without backcrossing the MC3R KO and MC4R KO genotypes back to an inbred strain an d regenerating the DKO breeding colony for generation of future experimental animals, it might be difficult to attribute small, but significant, differences between the genotypes to differences in genotype versus background strain unless the results can be confirmed by other laboratory groups with their own lines of KO mice. Method By Which Exercise Causes Changes Increasing physical activity by exercise increases energy expenditure which in theory can affect weight loss if energy intake is not also increas ed to match. The discovery of more and more genes and proteins that have great regulatory control over the energy homeostasis of the body have raised just as many questions as they have answered. In order to better understand the mechanism by which volunta ry exercise is able to prevent the onset of the obese phenotypes of the MC4R KO and DKO mice and the increased adiposity of the MC3R KO mice, gene expression was measured and analyzed in metabolically relevant tissues in Chapter 4. Concluding Remarks The o bese phenotype of MC4R KO mice, increased body weight of DKO mice, and increased adiposity of MC3R KO mice have all been well characterized. 8 10,35,37 39,64,80 The effects of voluntary exercise independent of diet or other pharmaceutical intervention on these genotypes have been preliminarily investigated by the Haskell

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136 Luevano group in the past focusing on the effects of exer cise on male MC4R KO mice. 37,63,64 The experiments shown in Chapter 3 present and discuss the effects of voluntary exercise by running wheel on the MC3R KO and DKO genotypes which have not been analyzed before in this manner. To conclude that voluntary exercise reduces the obese phenotype of MC4R KO and DKO mice and also decreases in adiposity in MC3R KO mice would be incorrect. Voluntary exercise slows, or prevents, the onset of these phenotypes. Previously published research by the Haskell Luevano lab showed that when exercised MC4R KO mice were placed in conventional cages, weight was gained back to levels of MC4R KO that were in conventional cages the ent ire experiment. 64 Furthermore, it was shown that if these MC4R KO mice were placed back in running wheel cages that weight loss was not achieved. 64 It can consequently be extrapolated to this experiment and the other melanocortin receptor KO genotypes that the same outcome is possible if not likely. The findings of Chapter 3 sup port the hypothesis that the metabolic dysfunction seen in all the central melanocortin KO mice can be prevented or significantly delay by exercise intervention at an early age.

PAGE 137

137 Figure 3 1. Body weights of male mice during running wheel experiments. R epeated measures ANOVA determined that age, genotype, and housing all had significant effects on body weight during the experimental timeline (P<0.001). A significant interaction between genotype and housing was seen for body weights of male mice (P<0.001)

PAGE 138

138 Figure 3 2. Body weights at five weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

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139 Figure 3 3. Body weights at six weeks of age. Two way ANOVA significance achieved for genotype (P<0.001 ). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 140

140 Figure 3 4. Body weights at seven weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.01). A significant interaction between genotype and housing also existed (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 141

141 Figure 3 5. Body weights at eight weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

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142 Figure 3 6. Body weigh ts at nine weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 143

143 Figure 3 7. Body weights at ten weeks of age. Two way ANOVA significanc e achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 144

144 Figure 3 8. Body weights at 11 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interacti on between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 145

145 Figure 3 9. Body weights at 12 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0. 05; **, P<0.01; ***, P<0.001)

PAGE 146

146 Figure 3 10. Body weights at 13 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 147

147 Figure 3 11. Body weights at five weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 148

148 Figure 3 12. Body weights at six weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P <0.01; ***, P<0.001)

PAGE 149

149 Figure 3 13. Body weights at seven weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.01). A significant interaction between genotype and housing also existed (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0. 001)

PAGE 150

150 Figure 3 14. Body weights at eight weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 151

151 Figure 3 15. Body weights at nine weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 152

152 Figure 3 16. Body weights at ten weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 153

1 53 Figure 3 17. Body weights at 11 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between gen otype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 154

154 Figure 3 18. Body weights at 12 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.0 1; ***, P<0.001)

PAGE 155

155 Figure 3 19. Body weights at 13 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 156

156 Figure 3 20. Fat masses of mal e mice during running wheel experiments. Repeated measures ANOVA determined that age, genotype, and housing all had significant effects on fat mass during the experimental timeline (P<0.001). A significant interaction between genotype and housing was seen for the fat masses of male mice (P<0.001).

PAGE 157

157 Figure 3 21. Fat masses at 5 weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 158

158 Figure 3 22. Fat masses at 6 weeks of age. Two way ANOV A significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 159

159 Figure 3 23. Fat masses at 7 weeks of age. Two way ANOVA significance achieved for genotype, housing, and th e interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 160

160 Figure 3 24. Fat masses at 8 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001 ). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 161

161 Figure 3 25. Fat masses at 9 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 162

162 Figure 3 26. Fat masses at 10 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 163

163 Figure 3 27. Fat masses at 11 weeks of age. Two way ANOVA s ignificance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 164

164 Figure 3 28. Fat masses at 12 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 165

165 Figure 3 29. Fat masses at 13 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001 ). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 166

166 Figure 3 30. Fat masses at 5 weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 167

167 Figure 3 31. Fat masses at 6 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 168

168 Figure 3 32. Fat masses at 7 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 169

169 Figure 3 33. Fat masses at 8 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 170

170 Figure 3 34. Fat masses at 9 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 171

171 Figure 3 3 5. Fat masses at 10 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 172

172 Figure 3 36. Fat masses at 11 weeks of age. Two way ANOVA sig nificance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 173

173 Figure 3 37. Fat masses at 12 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the i nteraction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 174

174 Figure 3 38. Fat masses at 13 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 175

175 Figure 3 39. Lean masses of male mice during running wheel experiments. Repeated measures ANOVA determined that age and genotype had significant effects on lean mass during the experimental timeline (P<0.001).

PAGE 176

176 Figure 3 40. Lean masses at 5 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 177

177 Figure 3 41. Lean masses at 6 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P <0.05; **, P<0.01; ***, P<0.001)

PAGE 178

178 Figure 3 42. Lean masses at 7 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 179

179 Figure 3 43. Lean masses at 8 weeks of age. Two way ANOVA significance ac hieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 180

180 Figure 3 44. Lean masses at 9 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 181

181 Figure 3 45. Lean masses at 10 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 182

182 Figure 3 46. Lean masses at 11 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 183

183 Figure 3 47. Lean masses at 12 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 184

184 Figure 3 48. Lean masses at 13 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 185

185 Figure 3 49. Lean masses at 5 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 186

186 Figure 3 50. Lean masses at 6 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 187

187 Figure 3 51. Lean masses at 7 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 188

188 Figure 3 52. Lean masses at 8 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 189

189 Figure 3 53. Lean masses at 9 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 190

190 Figure 3 54. Lean masses at 10 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 191

191 Figure 3 55. Lean masses at 11 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 192

192 Figure 3 56. Lean masses at 12 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 193

193 Figure 3 57. Lean masses at 13 weeks of age. Two way ANOVA significanc e achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 194

194 Figure 3 58. Body Lengths of male mice during running wheel experiments. Repeated measures ANOVA determined that age and genotype had significant effects on body length during the experimental timeline (P<0.001).

PAGE 195

195 Figure 3 59. Body Lengths at 5 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 196

196 Figure 3 60. Body Lengths at 6 weeks of age. Two way ANOVA significance ac hieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 197

197 Figure 3 61. Body Lengths at 7 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 198

198 Figure 3 62. Body Lengths at 8 weeks o f age. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 199

199 Figure 3 63. Body Lengths at 9 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genot ype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 200

200 Figure 3 64. Body Lengths at 10 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 201

201 Figure 3 65. Body Lengths at 11 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 202

202 Figure 3 66. Body Lengths at 12 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 203

203 Fig ure 3 67. Body Lengths at 13 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 204

204 Figure 3 68. Body Lengths at 5 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0 .05; **, P<0.01; ***, P<0.001)

PAGE 205

205 Figure 3 69. Body Lengths at 6 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 206

206 Figure 3 70. Body Lengths at 7 weeks of age. Two way ANOVA significance achi eved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 207

207 Figure 3 71. Body Lengths at 8 weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 208

208 Figure 3 72. Body Lengths at 9 weeks of age. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 209

209 Figure 3 73. Body Lengths at 10 weeks of age. Two way ANOVA significance achieve d for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 210

210 Figure 3 74. Body Lengths at 11 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 211

211 Figure 3 75. Body Lengths at 12 weeks of a ge. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 212

212 Figure 3 76. Body Lengths at 13 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 213

213 Figu re 3 77. Food intake of male mice during running wheel experiments. Repeated measures ANOVA determined that age and genotype had significant effects on food consumption during the experimental timeline (P<0.001).

PAGE 214

214 Figure 3 78. Average daily food intake f or 5 weeks of age. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 215

215 Figure 3 79. Average daily food intake for 6 weeks of age. Two way ANOVA significance achieved for genotype (P<0. 001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 216

216 Figure 3 80. Average daily food intake for 7 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 217

217 Figure 3 81. Average daily food intake for 8 week s of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 218

218 Figure 3 82. Average daily food intake for 9 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ** *, P<0.001)

PAGE 219

219 Figure 3 83. Average daily food intake for 10 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 220

220 Figure 3 84. Average daily food intake for 11 weeks of age. Two way ANOVA signif icance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 221

221 Figure 3 85. Average daily food intake for 12 weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 222

222 Fig ure 3 86. Average daily food intake for 13 weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 223

223 Figure 3 87. Average daily food intake for 5 weeks of age. No significant differences d ue to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 224

224 Figure 3 88. Average daily food intake for 6 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 225

225 Figure 3 89. Average daily food intake for 7 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 226

226 Figure 3 90. Average daily food intake for 8 weeks of age. Two way ANOVA significance achieved f or genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 227

227 Figure 3 91. Average daily food intake for 9 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 228

228 Figure 3 92. Average daily food intake for 10 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 229

229 Figure 3 93. Average daily food intake for 11 weeks of age. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0 .05; **, P<0.01; ***, P<0.001)

PAGE 230

230 Figure 3 94. Average daily food intake for 12 weeks of age. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 231

231 Figure 3 95. Average daily food intake for 13 week s of age. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 232

232 Figure 3 96. Average plasma insulin concentration male MC3R and DKO mice. Repeated measures ANOVA determined that neither genotype or housing had a significant effect on plasma insulin for MC3R mice during the experimental timeline. Plasma insulin values for DKO mice were analyzed by t test at each individual time point.

PAGE 233

233 Figure 3 97. Plasma insulin concentration for 5 week old male DK O mice in conventional and running wheel cages. Plasma insulin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 234

234 Figure 3 98. Plasma insulin concentration for 6 week old male DKO mice in conventional and r unning wheel cages. Plasma insulin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 235

235 Figure 3 99. Plasma insulin concentration for 7 week old male DKO mice in conventional and running wheel cages. Plasma i nsulin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 236

236 Figure 3 100. Plasma insulin concentration for 8 week old male DKO mice in conventional and running wheel cages. Plasma insulin concentration compar isons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 237

237 Figure 3 101. Plasma insulin concentration for 9 week old male DKO mice in conventional and running wheel cages. Plasma insulin concentration comparisons for DKO mice were mad e by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 238

23 8 Figure 3 102. Plasma insulin concentration for 10 week old male DKO mice in conventional and running wheel cages. Plasma insulin concentration comparisons for DKO mice were made by t test. (*, P<0.05; *, P<0.01; ***, P<0.001)

PAGE 239

239 Figure 3 103. Plasma insulin concentration for 11 week old male DKO mice in conventional and running wheel cages. Plasma insulin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 240

240 Figure 3 104. Plasma insulin concentration for 12 week old male DKO mice in conventional and running wheel cages. Plasma insulin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 241

241 Figure 3 105. Plasma ins ulin concentration for 13 week old male DKO mice in conventional and running wheel cages. Plasma insulin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 242

242 Figure 3 106. Plasma insulin of 13 week old male M C3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. 37 Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 243

243 Figure 3 107. Plasma insulin of 13 week old male MC3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. 37 Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 244

244 Figure 3 108. Plasma insulin o f 13 week old male MC3R WT and MC3R KO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. 37 Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 245

245 Figure 3 109. Plasma insul in of 13 week old male MC3R WT and MC3R KO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. 37 Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 246

246 Figure 3 110. Average plasma leptin concentration for male MC3R and DKO mice in running wheel experiments. Repeated measures ANOVA determined that age and housing had significant effects on plasma leptin concentration for MC3R mice during the experimental timeline (P<0.01). Pl asma leptin values for DKO mice were analyzed by t test at each individual time point.

PAGE 247

247 Figure 3 111. Plasma leptin concentration for 5 week old male mice from MC3R strain in conventional and running wheel cages. No significant differences due to genotyp e or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 248

248 Figure 3 112. Plasma leptin concentration for 6 week old male mice from MC3R strain in conventional and running wheel cages. No significant differences due to genotype or ho using were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 249

249 Figure 3 113. Plasma leptin concentration for 7 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0. 05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 250

250 Figure 3 114. Plasma leptin concentration for 8 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ** *, P<0.001)

PAGE 251

251 Figure 3 115. Plasma leptin concentration for 9 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 252

252 Figure 3 116. Pla sma leptin concentration for 10 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 253

253 Figure 3 117. Plasma leptin concentration for 11 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 254

254 Figure 3 118. Plasma leptin concentration for 12 week old male m ice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 255

255 Figure 3 119. Plasma leptin concentration for 13 week old male mice from MC3R strain in convent ional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 256

256 Figure 3 120. Plasma leptin concentration for 5 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 257

257 Figure 3 121. Plasma leptin concentration for 6 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 258

258 Figure 3 122. Plasma leptin concentration for 7 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 259

259 Figure 3 123. Plasma leptin concentration for 8 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; *** P<0.001)

PAGE 260

260 Figure 3 124. Plasma leptin concentration for 9 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 261

261 Figure 3 125. P lasma leptin concentration for 10 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 262

262 Figure 3 126. Plasma leptin concentration f or 11 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 263

263 Figure 3 127. Plasma leptin concentration for 12 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 264

264 Figure 3 128. Plasma leptin concentration for 13 week old male DKO mice in conventional and running wheel cages. Plasma leptin concentration comparisons for DKO mice were made by t test. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 265

265 Figure 3 129. Plasma leptin concentration for 5 week old male mice from MC3R strain in conventional and running wheel cages. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 266

266 Figure 3 130. Plasma leptin concentration for 6 week old male mice from MC3R strain in conventional and running wheel cages. No signif icant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 267

267 Figure 3 131. Plasma leptin concentration for 7 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA si gnificance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 268

268 Figure 3 132. Plasma leptin concentration for 8 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for genotyp e (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 269

269 Figure 3 133. Plasma leptin concentration for 9 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.05). (*, P<0.05; **, P<0. 01; ***, P<0.001)

PAGE 270

270 Figure 3 134. Plasma leptin concentration for 10 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 271

271 Figure 3 135. Plasma leptin concentration for 11 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 272

272 Figure 3 136. Plasma lepti n concentration for 12 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 273

273 Figure 3 137. Plasma leptin concentration for 13 week old male mice from MC3R strain in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 274

274 Figure 3 138. Plasma leptin of 13 week old male MC3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. 37 Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 275

275 Figure 3 139. Plasma leptin of 13 week old male MC3R WT, MC3R KO, and DKO mice from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. 37 Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 276

276 Figure 3 140. Plasma leptin of 13 week old male MC3R WT and MC3R KO mi ce from this series of experiments and MC4R WT and MC4R KO from Haskell Luevano et al. 37 Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 277

277 Figure 3 141. Average number of running wheel turns of male mice du ring the dark cycle of running wheel experiments. Repeated measures ANOVA determined that age and genotype had significant effects on running wheel activity during the experimental timeline (P<0.001).

PAGE 278

278 Figure 3 142. Average number turns of running wheel during dark cycle for 6 week old mice. No significant differences due to genotype were found by ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 279

279 Figure 3 143. Average number turns of running wheel during dark cycle for 7 week old mice. No significant differ ences due to genotype were found by ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 280

280 Figure 3 144. Average number turns of running wheel during dark cycle for 8 week old mice. One way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01 ; ***, P<0.001)

PAGE 281

281 Figure 3 145. Average number turns of running wheel during dark cycle for 9 week old mice. One way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 282

282 Figure 3 146. Average number turns of running wheel during dark cycle for 10 week old mice. One way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 283

283 Figure 3 147. Average number turns of running wheel during dark cycle for 11 week old mice. One way ANOVA sign ificance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 284

284 Figure 3 148. Average number turns of running wheel during dark cycle for 12 week old mice. One way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 285

285 Figure 3 149. Average number turns of running wheel during dark cycle for 13 week old mice. One way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 286

286 Figure 3 150. Linear regression plots of fina l body weights versus total amount of exercise performed by male mice in running wheel cages. None of the slopes of the lines of best fit were significantly different from zero.

PAGE 287

287 Figure 3 151. Linear regression plots of final masses versus total amount o f exercise performed by male mice in running wheel cages. Only the slope of the line for the MC4R KO was significantly different from zero. The slope of the line for the MC4R KO mice is presented above the plotted line.

PAGE 288

288 Figure 3 152. Body weights of mal e MC3R WT and KO mice in conventional and running wheel cages. Repeated measures ANOVA determined that neither genotype nor housing had significant effects on body weights of male MC3R mice during the experimental time course.

PAGE 289

289 Figure 3 153. Average dail y food intake of male MC3R WT and KO mice in conventional and running wheel cages. Repeated measures ANOVA determined that neither genotype nor housing had significant effects on food intake of male MC3R mice during the experimental time course.

PAGE 290

290 Figure 3 154. Dark cycle activity as measured by turns of a running wheel for male MC3R WT and KO mice in running wheel cages. Repeated measures ANOVA determined that genotype did not have a significant effect on running wheel activity of male MC3R mice during th e experimental time course.

PAGE 291

291 Figure 3 155. Body lengths of male MC3R WT and KO mice in conventional and running wheel cages. Repeated measures ANOVA determined that age and genotype had significant effects on body length during the experimental timeline (P<0.05).

PAGE 292

292 Figure 3 156. Body lengths of 5 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 293

293 Figure 3 157. Body lengths of 6 week old male mice from a MC3R backg round. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 294

294 Figure 3 158. Body lengths of 7 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 295

295 Figure 3 159. Body lengths of 8 week old male mice from a MC3R background. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 296

296 Figure 3 160. Body lengths of 9 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 297

297 Figure 3 161. Body lengths of 10 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 298

298 Figure 3 162. Body lengths of 11 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 299

299 Figure 3 163. Bo dy lengths of 12 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 300

300 Figure 3 164. Body lengths of 13 week old male mice from a MC3R background. No significant diffe rences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 301

301 Figure 3 165. Fat masses of male MC3R WT and KO mice in conventional and running wheel cages. Repeated measures ANOVA determined that age, genotype, and housing all had significant effects on body length during the experimental timeline (P<0.05).

PAGE 302

302 Figure 3 166. Fat Masses of 5 week old male mice from a MC3R background. No significant differences due to genotype or housing were found by two way ANOVA. (* P<0.05; **, P<0.01; ***, P<0.001)

PAGE 303

303 Figure 3 167. Fat Masses of 6 week old male mice from a MC3R background. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 304

304 Figure 3 168. Fat Ma sses of 7 week old male mice from a MC3R background. Two way ANOVA significance achieved for housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 305

305 Figure 3 169. Fat Masses of 8 week old male mice from a MC3R background. Two way ANOVA significance achi eved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 306

306 Figure 3 170. Fat Masses of 9 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P< 0.001)

PAGE 307

307 Figure 3 171. Fat Masses of 10 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 308

308 Figure 3 172. Fat Masses of 11 week old male mice from a MC3 R background. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 309

309 Figure 3 173. Fat Masses of 12 week old male mice from a MC3R background. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 310

310 Figure 3 174. Fat Masses of 13 week old male mice from a MC3R background. Two way ANOVA significance achieved for housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 311

311 Figure 3 175. Fat Masses of 5 week old male mice fro m a MC3R background. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 312

312 Figure 3 176. Fat Masses of 6 week old male mice from a MC3R background. No significant differences due to geno type or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 313

313 Figure 3 177. Fat Masses of 7 week old male mice from a MC3R background. Two way ANOVA significance achieved for housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 314

314 Figure 3 178. Fat Masses of 8 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 315

315 Figure 3 179. Fat Masses of 9 week old male mice from a MC3R backgrou nd. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 316

316 Figure 3 180. Fat Masses of 10 week old male mice from a MC3R background. Two way ANOVA significance achieved for genotype and housing (P<0. 05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 317

317 Figure 3 181. Fat Masses of 11 week old male mice from a MC3R background. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 318

318 Figure 3 182. Fat Masses of 12 week o ld male mice from a MC3R background. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 319

319 Figure 3 183. Fat Masses of 13 week old male mice from a MC3R background. Two way ANOVA significance achieved for housin g (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 320

320 Figure 3 184. Lean masses of male MC3R WT and KO mice in conventional and running wheel cages. Repeated measures ANOVA determined that age and genotype had significant effects on lean mass during the ex perimental timeline (P<0.05).

PAGE 321

321 Figure 3 185. Lean mass of 5 week of male MC3R KO and WT mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 322

322 Figure 3 186. Lean m ass of 6 week of male MC3R KO and WT mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 323

323 Figure 3 187. Lean mass of 7 week of male MC3R KO and WT mice in conventi onal and running wheel cages. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 324

324 Figure 3 188. Lean mass of 8 week of male MC3R KO and WT mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 325

325 Figure 3 189. Lean mass of 9 week of male MC3R KO and WT mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P< 0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 326

326 Figure 3 190. Lean mass of 10 week of male MC3R KO and WT mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 327

327 Figu re 3 191. Lean mass of 11 week of male MC3R KO and WT mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 328

328 Figure 3 192. Lean mass of 12 week of male MC3R KO and W T mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 329

329 Figure 3 193. Lean mass of 13 week of male MC3R KO and WT mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 330

330 CHAPTER 4 EFFECTS OF VOLUNTARY EXERCISE ON GENE EXP RESSION IN ORGANS INVOLVED IN ENERGY H OMEOSTASIS Breeding and genotyping of the experimental mice was done by A my M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, and Erin B. Bruce. Mice cages were changed and cleaned by Amy M. Andreasen, Kimberly R. Haskell, Laurie M. Koerper, and Erin B. Bruce. Measurements and blood draws were done by Amy M. Andreasen, Kimb erly R. Haskell, Laurie M. Koerper, Erin B. Bruce, and Jay Schaub. Running wheel activity data was compiled by Sarah B. Carey and Jay Schaub. All plasma hormone assays were run by Jay Schaub. Sacrifice and dissection of organs was performed by Dr. Zhimin X iang, Dr. Sally Litherland, and Jay Schaub. RNA extraction, cDNA synthesis, and RT PCR of hypothalamus was done by Dr. Zhimin Xiang and Jay Schaub. RNA extraction, cDNA synthesis, and RT PCR of liver and muscle tissue was done by Jay Schaub. Introductory R emarks As long as the obesity pandemic continues, new methods will continue to be sought for weight loss and for the treatment of the diseases associated with obesity. While exercise is a low risk and effective method of increasing energy expenditure, it g enerally requires dedication on the behalf of the individual to maintain a regular training schedule to gain the benefits of regular exercise. A more desirable approach would be the administration of a drug that mimics the effects of exercise. In order to design a drug that mimics the effects of exercise, a target in the body, either a gene or a protein, must be identified. In the most simple experiment, two groups would be used: mice allowed to exercise and mice not given the opportunity to exercise. Gene expression in exercising and sedentary mice would be analyzed and any genes

PAGE 331

331 that experience changes in expression levels would be potential targets (Figure 4 1A). An experiment comparing an obese model to lean WT mice can also be useful since it can show changes in gene expression that are result of obesity. These genes could be targets for the treatment of obesity, but not for mimicking the effects of exercise (Figure 4 1B). Finally, these two types of experiments can be combined to result in a single exp eriment giving the most amount of information. Changes in gene expression can be categorized as changes due to either exercise or obesity, but also allow for the analysis of gene expression in exercising obese mice. Changes in gene expression might be seen in obese mice or mice predisposed to obesity (such as the MC4R KO mice) allowed to exercise that were not seen exercising lean mice (Figure 4 1C). Finally, exercise might result in differences in gene expression seen between sedentary obese and lean mice not occurring in obese mice allowed to exercise (Figure 4 1D). Once a gene target has been identified, a drug can then be designed to affect the specific gene or protein. While obesity is not the direct result of a change in expression of a single gene or protein, this approach would work to correct changes that significantly contribute to the onset of obesity. The many effects of exercise on the body are still being researched. 37,56 65,67,69 7 2 One of the documented effects of exercise in mice is changes in hypothalamic expression of genes involved in energy homeostasis. 37 Central nervous system influence of peripheral organs is controlled in part by the autonomic nervous system. The autonomic nervous system is made up of the sympathetic and parasympat hetic nervous systems. The sympathetic nervous system (SNS) is excitatory in nature and is generally associated with the fight or flight response. The parasympathetic nervous

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332 system (PSNS) is complementary to the SNS and exerts control over function when t he body is at rest including digestion and elimination of waste products. The melanocortin pathway has been linked to the sympathetic nervous system by Song et al. who demonstrated that the MC4R is expressed in sympathetic outflow neurons that innervate bo th white adipose and brown adipose tissue depots. 124,125 This suggests that changes in gene regulation caused by exercise is possible in peripheral tissues involved in energy balance through the autonomic nervous system. The melanocortin system, specifically centrally ex pressed MC3 and MC4 receptors, has been linked to food intake and energy homeostasis through feeding studies and the changes in metabolism in MC3R KO and MC4R KO mice. 8 10,23,29,34 The MC4R KO mice are an accepted model for studying obesity. 36 39,63,64,80,117,126 To test the hypothesis that voluntary exercise causes changes in expression of genes involved in energy homeostasis, experiments were designed to generate tissue samples that would investigate the effects of gen otype (specifically lack of central melanocortin receptors) and voluntary exercise. Methods and Results RNA Extraction Hypothalamus, liver, and muscle Extraction of RNA from hypothalamus, liver and muscle samples was done using the Trizol extraction metho d, precipitation by isopropanol, and redissolved in DEPC treated water. When total RNA from these samples were run on 1% agarose gel and visualized under UV light, distinct bands corresponding to 18s and 28s ribosomal RNA were seen on a 1% agarose gel unde r UV light. Concentrations of the samples were determined using a NanoDrop spectrophotometer.

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333 One muscle sample was completely consumed during RNA extraction process due to its small size, and even so did not produce enough RNA to proceed to cDNA synthesi s (mouse ear tag number 8407, MC4R KO Conv). Without this mouse, there were still four viable subjects in the conventionally housed MC4R KO group, and the experiment proceeded with this group size for muscle RT PCR experiments. Pancreas Extraction of pancr eatic RNA was done using the Trizol extraction method, precipitation by isopropanol, and redissolved in DEPC treated water. When total RNA from pancreas samples were run on 1% agarose gel and visualized under UV light, the majority samples had streaked on the gel indicating sample degradation (Figure 2 2B). Due to poor quality of extracted RNA, it was decided to omit the pancreas from the study design. Selection of Housekeeping Genes Both the peptidylprolyl isomerase A (PPIA) and the hypoxanthine guanine ph osphoribosyl transferase 1 (HPRT1) genes are suggested for use as housekeeping genes by the commercial suppliers of the RT PCR equipment and reagents. 127 Both genes were run as potential control genes and the gene with the least amount of variation due to genotype or housing condition was selected for each tissue. The HPRT1 gene was selected as the housekeepi ng gene used for hypothalamic and muscle samples. The PPIA gene was selected for use as the housekeeping gene for liver samples. Relative Expression Housekeeping genes that have constant levels of expression across experimental groups are useful for the qu antitative analysis of levels of expression for genes of

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334 interest. However, it can be useful to directly compare two genes whose actions oppose or differ from each other giving an indication of which has a larger effect. In order to be able to gauge the ma gnitude of differences in expression levels between two genes in change was calculated as 2 Hypothalamus Agouti related protein (AGRP) Genotype significantly influenced AGRP expression in the hypothalamus of male mice (P<0.01) (Figure 4 2). Expression of the AGRP gene in the hypothalamus of DKO mice (3.43 fold change 1.14 fold change) was 227% higher than in MC4R WT mice (1.05 fold change 0.06 fold change) under runn ing wheel housing conditions (P<0.01) (Figure 4 2). 5' AMP activated protein kinase (PRKAA1) Housing, genotype, and the interaction between housing and genotype significantly influenced hypothalamic expression of the PRKAA1 gene (P<0.01) which encodes the alpha catalytic subunit of the AMPK protein (Figure 4 3). The MC3R WT (1.24 fold change 0.07 fold change) expression of PRKAA1 (AMPK) was 35% higher than MC3R KO (0.92 fold change 0.04 fold change) expression in running wheel cages (P<0.001) (Figure 4 3). Access to running wheels for voluntary exercise increased hypothalamic expression of PRKAA1 (AMPK) for both the MC3R WT (Conv. 1.01 fold change 0.07 fold change; RW 1.24 fold change 0.07 fold change) and the MC4R WT (Conv. 1.00 fold change 0.03 fold change; RW 1.21 fold change 0.04 fold change) groups compared to the conventionally housed groups of the corresponding genotype (P<0.05) (Figure 4 4).

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335 Cocaine and amphetamine regulated transcript (CART) Genotype had a statistically significant effe ct on CART expression in the hypothalamus of male mice (P<0.05) (Figure 4 5). CART gene expression in exercising DKO mice (1.30 fold change 0.08 fold change) was 48% higher than exercising MC4R WT mice (0.87 fold change 0.07 fold change) CART expressio n (P<0.05) (Figure 4 5). Carnitine palmitoyltransferase 2 (CPT2) Hypothalamic expression of the carnitine palmitoyltransferase 2 gene was not affected by either genotype or housing condition in male mice (Figure 4 6). Glucokinase (GCK) Neither genotype nor housing significantly influenced levels of hypothalamic expression of the glucokinase gene in male mice (Figure 4 7). Hypocretin (HCRT) The expression of the hypocretin (orexin) gene in the hypothalamus was influenced by both housing and genotype (P<0.01) in male mice (Figure 4 8). Expression of HCRT was upregulated 101% in male DKO mice (2.05 fold change 0.32 fold change) in running wheel cages compared to MC4R WT mice (1.02 fold change 0.06 fold change) in the same housing condition (P<0.01) (Figure 4 8). Additionally, HCRT expression was 53% higher in exercising male DKO mice (2.05 fold change 0.32 fold change) compared to sedentary DKO mice (1.34 fold change 0.24 fold change) (P<0.05) (Figure 4 9). Insulin receptor (INSR) Hypothalamic expression of the insulin receptor gene was not found to be different among the experimental groups regardless of genotype or housing type (Figure 4 10).

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336 Leptin receptor (LEPR) Genotype significantly contributed to variation in hypothalamic levels of leptin receptor gene in male mice (P<0.001) (Figure 4 11). The MC3R WT mice in running wheel cages (1.25 fold change 0.18 fold change) had 74% higher hypothalamic LEPR expression than MC3R KO mice allowed to exercise (0.72 fold change 0.11 fold change) (P<0.01) (Figu re 4 11). Melanocortin 3 receptor (MC3R) Genotype had a statistically significant effect on MC3R gene expression in the hypothalamus of male mice when all groups were included in the statistical analysis (P<0.001) (Figure 4 12). However, when mice from the MC3R KO and DKO groups were excluded, no significant differences in MC3R gene expression were seen between MC3R WT, MC4R WT, and MC4R KO group regardless of housing (Figure 4 13). Melanocortin 4 receptor (MC4R) Genotype had a statistically significant ef fect on MC4R gene expression in the hypothalamus of male mice when all groups were included in the statistical analysis (P<0.001) (Figure 4 14). However, when mice from the MC4R KO and DKO groups were excluded, no significant differences in MC4R gene expre ssion were seen between MC3R WT, MC4R WT, and MC3R KO groups in either type of cage (Figure 4 15). Proopiomelanocortin (POMC) Hypothalamic expression of the proopiomelanocortin gene was significantly influenced by genotype in male mice (P<0.001) (Figure 4 16). POMC expression was 95% and 107% higher in MC4R KO mice (Conv. 1.97 fold change 0.48 fold change; RW 1.84 fold change 0.12 fold change) for both conventional and running wheel groups, respectively, compared to the MC4R WT mice (Conv. 1.01 fold ch ange 0.09

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337 fold change; RW 0.89 fold change 0.12 fold change) (P<0.05) (Figure 4 17). Expression of the POMC gene was 238% and 119% higher in male DKO mice (1.94 fold change 0.33 fold change) compared to MC3R WT (0.57 fold change 0.13 fold change) or MC4R WT (0.89 fold change 0.12 fold change) mice in running wheel cages (P<0.01), respectively (Figure 4 16). Neuropeptide Y (NPY) Both genotype and housing had significant effects on neuropeptide Y gene expression in the hypothalamus of male mice (P< 0.01) (Figure 4 17). NPY gene expression was 61% and 66% higher in MC3R WT (1.40 fold change 0.07 fold change) or MC4R WT (1.45 fold change 0.09 fold change) mice, respectively, in running wheel cages than in male DKO mice (0.87 fold change 0.12 fol d change) also allowed to exercise (P<0.05) (Figure 4 17). Neuropeptide Y receptor type 1 (NPY1R) Expression of the neuropeptide Y 1 receptor gene in the hypothalamus of male mice was significantly influenced by genotype (P<0.01) (Figure 4 18). No specific differences were found between WT and KO genotypes for either housing type. Suppressor of cytokine signaling 3 (SOCS3) Genotype, housing, and the interaction between housing and genotype all had a significant effect on SOCS3 gene expression in the hypotha lamus of male mice (P<0.05) (Figure 4 19). The MC4R KO (1.89 fold change 0.15 fold change) expression of SOCS3 was upregulated 87% compared to MC4R WT mice (1.01 fold change 0.07 fold change) in conventional cages (P<0.001) (Figure 4 19). Male DKO (0.9 9 fold change 0.10 fold change) expression of hypothalamic SOCS3 was 76% higher than MC3R WT mice (0.56 fold change 0.17 fold change) in running wheel cages (P<0.05)

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338 (Figure 4 19). Expression of SOCS3 in the hypothalamus in sedentary MC4R KO mice (1.89 fold change 0.15 fold change) was 55% higher than SOCS3 expression in MC4R KO mice (1.22 fold change 0.19 fold change) allowed to voluntarily exercise (P<0.01) (Figure 4 20). Uncoupling protein 2 (UCP2) Hypothalamic expression levels of the uncoupling protein 2 gene (UCP2) were not influenced by either genotype nor housing under this experimental protocol (Figure 4 21). Relative expression Melanocortin 3 receptor to melanocortin 4 receptor. Because melanocortin 3 and 4 receptors are both expressed in only the WT mice, those were the only groups that relative expression of the two receptors of interest was examined. Both the MC3R WT and MC4R WT mice had higher hypothalamic MC4R expression compared to MC3R gene expression in both housing conditions (Fig ure 4 22). MC4R WT mice (Conv. 0.69 fold change 0.05 fold change; RW 0.62 fold change 0.04 fold change) had a 83% and 63% higher hypothalamic MC3R to MC4R gene expression ratio compared to MC3R WT mice (Conv. 0.37 fold change 0.08 fold change; RW 0.3 8 fold change 0.05 fold change) in both the conventional and running wheel cages (P<0.05), respectively (Figure 4 22). Voluntary exercise did not change the ratio at which these receptors were expressed for either genotype. Agouti related protein to proo piomelanocortin. POMC was expressed at higher levels in the hypothalamus than AGRP was for all genotypes other than MC4R WT mice where AGRP levels were higher than POMC levels (Figure 4 23). Genotype greatly affected the ratio at which POMC and AGRP were e xpressed in the

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339 hypothalamus of male mice (P<0.001) (Figure 4 23). The AGRP to POMC relative expression ratio in the hypothalamus of MC4R WT mice (Conv. 1.35 fold change 0.29 fold change; RW 1.575 fold change 0.27 fold change) was significantly higher than seen in MC3R WT (Conv. 0.11 fold change 0.02 fold change; RW 0.28 fold change 0.04 fold change), MC4R KO (Conv. 0.20 fold change 0.08 fold change; RW 0.46 fold change 0.02 fold change), and DKO (Conv. 0.53 fold change 0.16 fold change; RW f old change 0.62 0.18 fold change) mice in both housing types (P<0.001) (Figure 4 23). Liver 5' AMP activated protein kinase (PRKAA1) PRKAA1 gene (AMPK) expression was not significantly influence by either genotype or housing in the liver of male mice (F igure 4 24). Carnitine palmitoyltransferase 1a (CPT1A) Genotype significantly affected expression of the carnitine palmitoyltransferase 1a gene in the liver of male mice (P<0.01) (Figure 4 25). The liver CPT1A expression level in MC4R KO mice (1.79 fold ch ange 0.24 fold change) was 69% higher than in the livers than MC4R WT mice (1.06 fold change 0.19 fold change) in conventional cages (P<0.001) (Figure 4 25). The CPT1A gene was also upregulated 60% and 51%, respectively, in the livers of sedentary male DKO mice (1.60 fold change 0.05 fold change) compared to MC3R WT (1.00 fold change 0.04 fold change) or MC4R WT (1.06 fold change 0.19 fold change) mice in conventional cages (P<0.05) (Figure 4 25). Expression of the CPT1A gene was 58% higher in the livers of sedentary MC4R KO mice (1.79 fold change 0.24 fold change) compared to MC4R KO mice allowed to exercise (1.13 fold change 0.07 fold change) (P<0.01) (Figure 4 26).

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340 Carnitine palmitoyltransferase 2 (CPT2) Expression of CPT2 in the livers of m ale mice was significantly influenced by genotype (P<0.001) (Figure 4 27). Expression of the CPT2 gene in the liver was elevated 51% in sedentary MC4R KO mice (1.53 fold change 0.17 fold change) compared to MC4R WT mice (1.02 fold change 0.09 fold chan ge) in conventional cages (P<0.01) (Figure 4 27). Expression of CPT2 in the liver was increased 61% and 60% in conventionally housed male DKO mice (1.630 fold change 0.08 fold change) compared to both MC3R WT (1.01 fold change 0.08 fold change) and MC4 R WT (1.02 fold change 0.09 fold change) mice in the same housing condition (P<0.001) (Figure 4 28). The DKO (1.43 fold change 0.17 fold change) expression of liver CPT2 was elevated 44% in running wheel cages compared to exercising MC3R WT mice (0.99 fold change 0.04 fold change) (P<0.01) (Figure 4 27). Liver expression of CPT2 was 44% higher in MC4R KO mice in conventional cages (1.53 fold change 0.17 fold change) compared to MC4R KO mice allowed to exercise (1.06 fold change 0.07 fold change) ( P<0.05) (Figure 4 28). Diglyceride acyltransferase 1 (DGAT1) Liver expression of diglyceride acyltransferase 1 was not significantly affected by genotype or housing condition in male mice (Figure 4 29). Diglyceride acyltransferase 2 (DGAT2) Both genotype a nd housing significantly contributed to variation in the levels at which DGAT2 was expressed in the liver of male mice (P<0.05) (Figure 4 30). Liver DGAT2 expression was elevated 40% in sedentary DKO mice (1.40 fold change 0.08 fold change) compared to t he MC3R WT mice (1.00 fold change 0.05 fold change) in conventional cages (P<0.05) (Figure 4 30). Liver expression levels of DGAT2 in

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341 sedentary MC4R KO mice (1.34 fold change 0.11 fold change) was elevated 63% compared to MC4R KO mice in running wheel cages (0.82 fold change 0.05 fold change) (P<0.01) (Figure 4 31). Fatty acid synthase (FASN) Genotype significantly influenced expression of the fatty acid synthase gene in the liver of male mice (P<0.001) (Figure 4 32). Expression of FASN in the livers of sedentary male DKO mice (4.30 fold change 0.47 fold change) was upregulated 270% and 314% compared to MC3R WT (1.16 fold change 0.35 fold change) and MC4R WT (1.04 fold change 0.14 fold change) mice in conventional cages (P<0.001) (Figure 4 32). T o address the possibility that the large increase in FASN expression in DKO mice was masking changes in the other groups, the statistical analysis was performed again without the values for the DKO mice (Figure 4 33). Under this set of conditions, housing had a significant effect on liver FASN expression in male mice (P<0.05) (Figure 4 33). Exercising MC4R WT mice (1.33 fold change 0.23 fold change) had a 79% higher expression of FASN in the liver than exercising MC4R KO mice (0.74 fold change 0.13 fold change) (P<0.05) (Figure 4 33). Sedentary MC3R WT mice (1.16 fold change 0.35 fold change) had 130% higher liver FASN expression compared to MC3R WT mice allowed to exercise (0.50 fold change 0.05 fold change) (P<0.05) (Figure 4 34). Fructose 1,6 biph osphatase (FBP1) Expression of the fructose 1,6 biphosphatase gene in the liver of male mice was significantly affected by genotype (P<0.001) (Figure 4 35). Liver FBP1 levels were elevated 47% in sedentary MC4R KO mice (1.50 fold change 0.18 fold change) compared to MC4R WT mice (1.02 fold change 0.10 fold change) in conventional

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342 cages (P<0.01) (Figure 4 35). Expression levels of the FBP1 gene in the liver were upregulated 93% and 94%, respectively, in conventionally housed DKO mice (1.97 fold change 0.12 fold change) compared to MC3R WT (1.02 fold change 0.11 fold change) and MC4R WT (1.02 fold change 0.10 fold change) mice in conventional cages (P<0.001) (Figure 4 35). Liver expression of the FBP1 gene in exercising DKO mice (1.58 fold change 0 .20 fold change) was 36% higher than FBP1 liver expression in MC3R WT mice (1.16 fold change 0.04 fold change) in running wheel cages (P<0.05) (Figure 4 35). Glucokinase (GCK) Both genotype and housing had significant effects on expression levels of the glucokinase gene in the livers of male mice (P<0.05) (Figure 4 36). Liver GCK expression was elevated in 65% sedentary DKO mice (1.66 fold change 0.18 fold change) compared to MC4R WT mice (1.00 fold change 0.04 fold change) in the same housing type (P <0.05) (Figure 4 36). Expression of the GCK gene in the liver of MC3R KO mice in conventional cages (1.67 fold change 0.10 fold change) was 91% higher than that of male MC3R KO mice allowed to exercise (0.87 fold change 0.22 fold change) (P<0.05) (Figu re 4 37). Glucose 6 phosphatase (G6PC3) Glucose 6 phosphatase gene expression in the liver is influenced by genotype (P<0.05) (Figure 4 38). Liver G6PC3 expression in DKO mice in running wheel cages (1.32 fold change 0.20 fold change) was increased 36% c ompared to exercising MC3R WT mice (0.97 fold change 0.03 fold change) (P<0.05) (Figure 4 38).

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343 Glucose transporter 2 (SLC2A2) Genotype had a significant effect on expression of the glucose transporter 2 (GLUT2) gene in the livers of male mice (P<0.001) ( Figure 4 39). Expression of the GLUT2 gene SLC2A2 was upregulated 71% in sedentary MC4R KO mice (1.75 fold change 0.21 fold change) in comparison to MC4R WT mice (1.02 fold change 0.12 fold change) also in conventional cages (P<0.001) (Figure 4 39). Li ver SLC2A2 expression was upregulated 55% in exercising male DKO mice (1.34 fold change 0.16 fold change) compared to MC3R WT mice (0.86 fold change 0.04 fold change) also in running wheel cages (P<0.05) (Figure 4 39). There was a 37% upregulation of l iver GLUT2 expression in sedentary MC4R KO mice (1.75 fold change 0.21 fold change) compared to MC4R KO mice in running wheel cages (1.28 fold change 0.14 fold change) (P<0.05) (Figure 4 40). Glycogen phosphorylase (PYGL) Gene expression levels of live r glycogen phosphorylase were significantly influenced by both genotype and housing (P<0.05) (Figure 4 41). Expression of the PYGL gene was upregulated 41% in the livers of sedentary MC4R KO mice (1.45 fold change 0.08 fold change) compared to MC4R WT (1 .03 fold change 0.12 fold change) controls (P<0.05) (Figure 4 41). Male DKO mice (1.71 fold change 0.06 fold change) in standard housing had 69% and 67% increases in liver PYGL gene expression compared to MC3R WT (1.01 fold change 0.08 fold change) a nd MC4R WT (1.03 fold change 0.12 fold change) mice, respectively, in conventional cages (P<0.001) (Figure 4 41). Expression of the PYGL gene was also upregulated 72% in exercising DKO mice (1.43 fold change 0.23 fold change) compared to MC3R WT

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344 mice ( 0.83 fold change 0.04 fold change) also in running wheel cages (P<0.001) (Figure 4 41). Glycogen synthase 2 (GYS2) Genotype had a statistically significant effect on glycogen synthase 2 gene expression in the livers of male mice (P<0.001) (Figure 4 42). Expression of the GYS2 gene was increased 59% and 61% in conventionally housed MC3R WT (1.00 fold change 0.05 fold change) and MC4R WT (1.01 fold change 0.08 fold change) mice compared to sedentary male DKO mice (0.63 fold change 0.03 fold change) (P <0.01) (Figure 4 42). Hormone sensitive lipase (LIPE) Hormone sensitive lipase gene expression was significantly affected by genotype in the livers of male experimental mice (P<0.01) (Figure 4 43). Liver LIPE expression was increased 84% in sedentary MC4R KO mice (1.90 fold change 0.24 fold change) compared to MC4R WT mice (1.03 fold change 0.14 fold change) in conventional cages (P<0.001) (Figure 4 43). Liver expression of LIPE in sedentary male DKO mice (1.55 fold change 0.14 fold change) was elevat ed 55% and 51% compared to MC3R WT (1.01 fold change 0.06 fold change) and MC4R WT (1.03 fold change 0.14 fold change) mice under the same housing condition (P<0.05) (Figure 4 43). The DKO mice (1.53 fold change 0.17 fold change) in running wheel cag es expressed LIPE at 44% higher levels than MC3R WT mice (1.06 fold change 0.06 fold change) also in running wheel cages (P<0.05) (Figure 4 43). Liver LIPE expression was increased 88% in male MC4R KO mice in conventional cages (1.90 fold change 0.24 f old change) compared to exercising MC4R KO mice (1.01 fold change 0.12 fold change) (P<0.001) (Figure 4 44).

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345 Insulin receptor (INSR) Levels of insulin receptor gene mRNA expression were significantly influenced by genotype (P<0.05) (Figure 4 45). No spec ific differences between WT and KO genotypes or housing conditions were seen in INSR expression levels. Leptin receptor (LEPR) Genotype and housing significantly contributed to the variation seen in leptin receptor gene expression in the liver of male mice (P<0.01) (Figure 4 46). Exercising MC4R WT mice (2.51 fold change 0.68 fold change) had 115% more LEPR mRNA in their liver compared to MC4R KO mice (1.17 fold change 0.41 fold change) in running wheel cages (P<0.05). Additionally, liver LEPR expressio n in MC3R WT (1.81 fold change 0.61 fold change) and MC4R WT (2.51 fold change 0.68 fold change) mice in running wheel cages was 272% and 417% higher, respectively, than DKO mice (0.49 fold change 0.06 fold change) allowed to exercise (P<0.05) (Figur e 4 46). Access to voluntary exercise equipment in the form of running wheels led to 129% higher expression of the LEPR gene in the liver of MC4R WT mice (2.51 fold change 0.28 fold change) compared to MC4R WT mice in conventional cages (1.10 fold change 0.24 fold change) (P<0.05) (Figure 4 47). Phosphofructokinase (PFKL) Phosphofructokinase gene expression in the liver of male mice was significantly influenced by genotype (P<0.05) (Figure 4 48). Sedentary DKO mice (1.38 fold change 0.05 fold change) had 37% and 33% increases in PFKL expression compared to MC3R WT (1.00 fold change 0.05 fold change) and MC4R WT (1.03 fold change 0.15 fold change) mice in conventional housing (P<0.05) (Figure 4 48). The MC4R WT mice (1.40 fold change 0.13 fold ch ange) allowed to exercise in running wheel cages

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346 had 34% higher liver PFKL expression than MC4R KO mice (1.04 fold change 0.06 fold change) also in running wheels (P<0.05) (Figure 4 48). Melanocortin 3 receptor and melanocortin 4 receptor (MC3R and MC4R) Probes for the MC3R and MC4R genes were used to investigate the presence of mRNA for the corresponding receptors in the liver. Neither MC3R nor MC4R probes resulted in amplification for any of the groups, indicating a lack of expression of these genes in the liver. Relative expression Glucokinase to glucose 6 phosphatase. Glucokinase and glucose 6 phosphatase catalyze the opposing mechanisms of glucose phosphorylation and dephosphorylation, regulating glucose flow into and out of the cell. Glucokinase is expressed in higher levels in the liver that glucose 6 phosphatase (Figure 4 49). Genotype significantly influenced the ratio at which these two genes were expressed (P<0.001) (Figure 4 49). The ratio at which GCK was expressed to G6PC3 in the liver was si gnificantly different between MC3R WT (Conv. 11.43 fold change 2.73 fold change; RW 6.09 fold change 1.45 fold change) and MC4R WT (Conv. 40.13 fold change 3.57 fold change; RW fold change 42.36 6.85 fold change) mice in both conventional and runni ng wheel cages (P<0.001) (Figure 4 49). The GCK gene was expressed in higher levels compared to G6PC3 in DKO mice (Conv. 27.20 fold change 2.77 fold change; RW 17.30 fold change 2.58 fold change) compared to MC3R WT mice (Conv. 11.43 fold change 2.73 fold change; RW 6.09 fold change 1.45 fold change) in both housing conditions (P<0.05) (Figure 4 49). The GCK gene was present in higher levels than G6PC3 in liver samples from MC4R WT mice (Conv. 40.13 fold change 3.57 fold change; RW fold change 42. 36 6.85 fold change) compared to

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347 both MC4R KO (Conv. 28.94 fold change 1.90 fold change; RW 29.36 fold change 4.66 fold change) and DKO (Conv. 27.20 fold change 2.77 fold change; RW 17.30 fold change 2.58 fold change) mice in conventional and run ning wheel cages (P<0.05) (Figure 4 49). Glycogen synthase 2 to glycogen phosphorylase. Expression levels of the GYS2 and the PYGL genes in the livers of male mice were approximately the same, with GYS2 being expressed at slightly higher levels than GYS2 ( Figure 4 50). Both genotype and housing significantly influenced the ratio in which the GYS2 and PYGL genes were expressed (P<0.01) (Figure 4 50). Expression of the GYS2 gene relative to PYGL was 75% higher in MC4R WT mice (0.86 fold change 0.06 fold cha nge) compared to MC4R KO mice (0.49 fold change 0.06 fold change) in conventional cages (P<0.01) (Figure 4 50). The relative amount of GYS2 to PYGL expressed in the liver was significantly higher in sedentary MC4R KO (0.49 fold change 0.06 fold change) mice compared to DKO mice (0.31 fold change 0.01 fold change) in conventional housing (P<0.001) (Figure 4 50). The ratio of GYS2 to PYGL was significantly lower in DKO mice (Conv. 0.31 fold change 0.01 fold change; RW 0.54 fold change 0.12 fold chan ge) compared to MC3R WT mice (Conv. 0.81 fold change 0.05 fold change; RW 1.06 fold change 0.09 fold change) for both housing groups (P<0.001) (Figure 4 50). The relative expression of the GYS2 gene compared to PYGL in the liver of male mice was 46% hi gher in exercising MC3R WT mice (1.06 fold change 0.09 fold change) compared to MC4R WT mice (0.73 fold change 0.09 fold change) also in running wheel cages (P<0.05) (Figure 4 50). Voluntary exercise significantly affected the ratio at which GYS2 and P YGL were expressed in the livers of

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348 exercising MC3R KO (0.98 fold change 0.07 fold change) compared to MC3R KO mice in conventional cages (0.67 fold change 0.05 fold change) (P<0.05) (Figure 4 51). Fructose 1,6 biphosphatase to phosphofructokinase. The FBP1 gene is expressed at much higher levels than PFKL in the liver (Figure 4 52). Genotype had a significant effect on the ratio of FBP1 to PFKL expression in the liver (P<0.001) (Figure 4 53). Expression of FBP1 in proportion to PFKL in the liver was lo wer in MC4R WT mice (Conv. 29.10 fold change 1.12 fold change; RW 25.93 fold change 2.22 fold change) in both housing types compared to MC4R KO mice (37.72 fold change 1.43 fold change; RW 31.51 fold change 1.59 fold change) (P<0.05) (Figure 4 52). The ratio of FBP1 to PFKL expression was higher in DKO mice (Conv. fold change fold change; RW fold change fold change) than in either MC3R WT (Conv. 26.90 fold change 2.13 fold change; RW 30.63 fold change 0.45 fold change) and MC4R WT (Conv. 2 9.10 fold change 1.12 fold change; RW 25.93 fold change 2.22 fold change) mice in both types of cages (P<0.01) (Figure 4 52). Voluntary exercise by running wheel resulted in a significant change in the FBP1 to PFKL expression ratio in the livers of exe rcising MC4R KO mice (31.51 fold change 1.59 fold change) compared to sedentary MC4R KO mice(37.72 fold change 0.73 fold change) (P<0.05) (Figure 4 53). Diglyceride acyltransferase 1 to diglyceride acyltransferase 2. The DGAT1 gene was expressed at mu ch lower levels than DGAT2 in the liver of male mice for all genotypes and housing treatments (Figure 4 54). Genotype, housing, and the interaction between housing and genotype all had statistically significant effects on the

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349 ratio of DGAT1 to DGAT2 in the liver (P<0.001) (Figure 4 54). The ratio of DGAT1 to DGAT2 was greater in MC4R WT mice (Conv. 0.03 fold change 0.00 fold change; RW 0.03 fold change 0.00 fold change) in both cage types compared to DKO mice (Conv. 0.02 fold change 0.00 fold change; RW 0.02 fold change 0.00 fold change) (P<0.01) (Figure 4 54). In exercising MC3R WT mice (0.02 fold change 0.00 fold change) the ratio of DGAT1 to DGAT2 was significantly lower than in MC4R WT mice (0.03 fold change 0.00 fold change) also allowed to exercise (P<0.01) (Figure 4 54). The ratio of DGAT1 to DGAT2 was significantly increased in MC4R KO mice (0.03 fold change 0.00 fold change) in running wheel cages compared to MC4R WT mice (0.03 fold change 0.00 fold change) allowed to exercise (P<0. 05) (Figure 4 54).The relative expression of DGAT1 compared to DGAT2 was increased in male MC4R KO mice (0.03 fold change 0.00 fold change) when they were allowed to exercised in comparison to MC4R KO mice in conventional cages (0.02 fold change 0.00 f old change) (P<0.001) (Figure 4 55). Hormone sensitive lipase to diglyceride acyltransferase 1. The genes for hormone sensitive lipase and diglyceride acyltransferase 1 were expressed in similar quantities in the livers of male mice (Figure 4 56). Genotype had a significant effect on the relative expression of LIPE to DGAT1 in male experimental mice (P<0.001) (Figure 4 56). The ratio of LIPE to DGAT1 was significantly higher in MC3R WT mice (Conv. 0.93 fold change 0.04 fold change; RW 0.98 fold change 0 .09 fold change) compared to MC4R WT mice (Conv. 0.52 fold change 0.04 fold change; RW 0.62 fold change 0.03 fold change) in both housing conditions (P<0.01) (Figure 4 56). The relative expression of LIPE in relation to DGAT1 was 62% and 79% higher in sedentary

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350 MC4R KO (0.84 fold change 0.08 fold change) and DKO (0.93 fold change 0.11 fold change) mice compared to MC4R WT mice (0.52 fold change 0.04 fold change) in conventional cages (P<0.05) (Figure 4 56). An 81% increase in the ratio of LIPE to DGAT1 was seen in sedentary MC4R KO mice (0.84 fold change 0.08 fold change) compared to MC4R KO mice (0.46 fold change 0.04 fold change) in running wheel cages (P<0.05) (Figure 4 57). Hormone sensitive lipase to diglyceride acyltransferase 2. Hormone sensitive lipase was expressed at much lower levels than DGAT2 in the liver of male experimental mice (P<0.001) (Figure 4 58). Genotype had a significant effect on the relative expression level of LIPE to DGAT2 (P<0.01) (Figure 4 58). The ratio of LIPE to DGAT2 was 46% higher in sedentary MC3R WT mice (0.02 fold change 0.00 fold change) compared to male MC4R WT mice (0.01 fold change 0.00 fold change) in conventional cages (P<0.05) (Figure 4 58). Fatty acid synthase to carnitine palmitoyltransferase 1 a. Genotype significantly contributed to the variation in the ratio of FASN to CPT1A expression in the livers of male mice (Figure 4 59) (P<0.001). Expression of FASN relative to CPT1A was higher in sedentary DKO mice (Conv. 3.62 fold change 0.45 fold ch ange; RW 2.71 fold change 1.16 fold change) compared to both MC3R WT (Conv. 1.20 fold change 0.35 fold change; RW 0.44 fold change 0.07 fold change) and MC4R WT (Conv. 1.68 fold change 0.17 fold change; RW 2.21 fold change 0.24 fold change) mice also in both types of cages (P<0.05) (Figure 4 59). Fatty acid synthase to carnitine palmitoyltransferase 2. The FASN gene was expressed in greater quantity compared to CPT2 in the livers of male mice (Figure 4

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351 60). Genotype and housing both had significa nt effects on the ratio of FASN to CPT2 expression in the livers of male mice (P<0.05) (Figure 4 60). The ratio of FASN to CPT2 was elevated in DKO mice (Conv. 8.56 fold change 1.01 fold change; RW 5.82 fold change 2.39 fold change) compared to MC3R WT (3.47 fold change 0.85 fold change) and MC4R WT mice (3.36 fold change 0.37 fold change) in conventional cages (P<0.01) and compared to MC3R WT mice in running wheel cages (1.61 fold change 0.12 fold change) (P<0.01) (Figure 4 60). Skeletal Muscle (Gastrocnemius of the Leg) 5' AMP activated protein kinase (PRKAA1) Expression of the PRKAA1 gene (AMPK) in skeletal muscle was not significantly influenced by either genotype or housing condition in male mice (Figure 4 61). Carnitine palmitoyltransferase 1b (CPT1B) Skeletal muscle carnitine palmitoyltransferase 1b gene expression was not affected by housing or genotype in male mice (Figure 4 62). Carnitine palmitoyltransferase 2 (CPT2) Housing significantly affected CPT2 expression in muscle tissue of mal e mice (P<0.01) (Figure 4 64). No specific differences were found to exist between WT and KO genotypes or the two housing types (Figure 4 63). Glucokinase (GCK) Both genotype and housing significantly influenced glucokinase gene expression in the skeletal muscle tissue of male mice (P<0.01) (Figure 4 64). The expression of the GCK gene was upregulated 79% in sedentary MC3R KO mice (1.96 fold change 0.30 fold change) compared to MC3R WT mice (1.00 fold change 0.05 fold change) in the same housing conditi on (P<0.01) (Figure 4 64). Exercise was able to significantly

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352 reduce muscle GCK expression in MC3R KO mice (0.82 fold change 0.25 fold change) compared to MC3R KO mice in conventional cages (1.96 fold change 0.30 fold change) (P<0.001) (Figure 4 65). Glucose transporter 4 (SLC2A4) Expression of the glucose transporter 4 gene (GLUT4) in muscle tissue was significantly affected by genotype in male mice (P<0.05) (Figure 4 66). No specific differences were found between WT and KO genotypes or conventional and running wheel housing conditions. Glycogen phosphorylase (PYGM) Housing had a significant effect on glycogen phosphorylase gene levels in the muscle tissue of male mice (P<0.01) (Figure 4 67). Specific differences between either the genotypes or the ho using conditions were not found. Glycogen synthase 1 (GYS2) Neither genotype nor housing condition significantly affected skeletal muscle expression of the GYS1 gene in male mice (Figure 4 68). Interleukin 6 (IL 6) Interleukin 6 expression in the muscle t issue of male mice was not affected by either housing or genotype (Figure 4 69). Phosphofructokinase (PFKM) Housing had a significant effect on phosphofructokinase gene expression levels in the skeletal muscle tissue of male mice (P<0.01) (Figure 4 70). S pecific differences between the WT and KO genotypes or the running wheel and conventional housing groups were not found.

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353 Uncoupling protein 2 (UCP2) Expression of the UCP2 gene in the muscle of male mice was significantly influenced by genotype (P<0.01) (F igure 4 71). Expression of the UCP2 gene was 185% higher in exercising DKO mice (2.37 fold change 0.68 fold change) than in male MC4R WT mice (0.83 fold change 0.18 fold change) in running wheel cages (P<0.01) (Figure 4 71). Exercise resulted in a 135% increase in UCP2 expression in the muscle of male MC3R WT mice ( 2.42 fold change 0.17 fold change) compared to MC3R WT (1.03 fold change 0.15 fold change) mice in conventional cages (P<0.05) (Figure 4 72). Uncoupling protein 3 (UCP3) Genotype signif icantly affected expression of the UCP3 gene in the skeletal muscle tissue of male experimental mice (P<0.01) (Figure 4 73). Exercising MC3R WT mice (1.93 fold change 0.18 fold change) had 65% higher expression levels of muscle UCP3 than DKO mice (1.17 f old change 0.27 fold change) also in running wheel cages (P<0.05) (Figure 4 73). Voluntary exercise by running wheel resulted in an 84% upregulation of the UCP3 gene in the muscle of MC3R WT mice (1.93 fold change 0.18 fold change) compared to MC3R WT mice in conventional cages (1.05 fold change 0.20 fold change) (P<0.05) (Figure 4 74). Relative expression Uncoupling protein 2 to uncoupling protein 3. Expression of the UCP2 gene is lower in the muscle than UCP3 gene expression for MC3R WT and MC3R KO mice in both conventional and running wheel cages (Figure 4 75). UCP2 is expressed at higher levels than UCP3 in skeletal muscle tissue of MC4R WT, MC4R KO, and DKO mice in both types of housing (Figure 4 75). Genotype had a statistically significant effec t on the

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354 ratio of UCP2 to UCP3 in the skeletal muscle of male mice (P<0.001) (Figure 4 75). The relative expression of UCP2 to UCP3 in muscle was 1346% higher in MC4R WT mice (6.70 fold change 3.06 fold change) in running wheel cages compared to MC3R WT mice (0.46 fold change 0.08 fold change) allowed to exercise (P<0.05) (Figure 4 75). Glycogen synthase 1 to glycogen phosphorylase. Glycogen phosphorylase (PYGM) was expressed in much higher levels than GYS1 in the muscle of male mice (Figure 4 76). Geno type, housing, and the interaction between housing and genotype all had a significant effect on the ratio of GYS1 to PYGM expressed in the muscle of male mice (P<0.05) (Figure 4 76). The ratio of GYS1 to PYGM was significantly higher in the muscle tissue o f DKO mice (Conv. fold change fold change; RW fold change fold change) compared to both MC3R WT (Conv. 0.05 fold change 0.00 fold change; RW 0.09 fold change 0.01 fold change) and MC4R WT (Conv. 0.06 fold change 0.00 fold change; RW 0.06 fold change 0.00 fold change) mice in both conventional and running wheel cages (P<0.05) (Figure 4 76). The relative expression of GYS1 to PYGM was 40% higher in exercising MC3R WT mice (0.09 fold change 0.01 fold change) compared to MC4R WT mice (0.06 fol d change 0.00 fold change) in running wheel cages (P<0.05) (Figure 4 76). The amount of GYS1 mRNA expressed relative to the amount of PYGM mRNA in muscle tissue of exercising MC4R KO mice (0.08 fold change 0.01 fold change) was 33% higher compared to t hat of MC4R WT mice (0.06 fold change 0.00 fold change) in running wheel cages (P<0.05) (Figure 4 76). Voluntary exercise resulted in a change in the ratio of GYS1 to PYGM gene expression in the muscle of both MC3R WT (Conv. 0.05 fold change 0.00 fold change; RW 0.09 fold change 0.01 fold change) and MC3R KO mice (Conv. 0.06 fold change 0.00

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355 fold change; RW 0.09 fold change 0.01 fold change) (P<0.01), with values being higher for mice allowed to exercise compared to those in conventional cages (Fi gure 4 77). Discussion Discussion of Results Pancreas Due to the degradation of the mRNA in the pancreas collected from the experimental mice at the time of sacrifice, RT PCR was not able to be performed (Figure 2 2B). This is unfortunate due to the import control over energy balance, specifically in maintaining safe blood glucose concentrations. It is believed that since this level of degradation was seen in so many of the samples from the pancreas, and so few from the other t issues that mRNA was extracted from, that the poor quality of the pancreatic mRNA was a result of general degradation and not sample handling error. The pancreas is a producer of many digestive enzymes in addition to insulin, glucagon, and somatostatin. Wh en the mice were dissected after sacrifice, brains were removed first, followed by the heart, and then the pancreas. It is possible that in the time that lapsed during the removal of the other organs that the pancreas was not removed and preserved fast eno ugh to ensure mRNA integrity. Hypothalamus No change in hypothalamic gene expression due to either genotype or housing was seen for the CPT2, GCK, INSR, MC3R, MC4R, or UCP2 genes. The lack of change of CPT2 or GCK gene expression indicates that if the proc esses of fatty acid oxidation or glucose phosphorylation are affected in the hypothalamus, they are not

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356 affected at the transcriptional level. Consistent expression levels of the INSR gene across genotypes and housing conditions would indicate that any dif ferences in insulin signaling are not due to differing levels of the insulin receptor in the hypothalamus. Uncoupling protein 2 gene expression in the hypothalamus of male mice might not have been affected by genotype or housing due to relatively low level s of expression of the gene. The UCP2 gene was amplified on average 3.25 cycles (~9.5 fold difference) after the HPRT1 house keeping gene in the hypothalamus, while UCP2 in skeletal muscle tissue was amplified 1.6 cycles (~3 fold difference) before HPRT1. Though difficult to draw any definitive conclusions across tissue types, hypothalamic UCP2 expression would appear to be lower than UCP2 gene expression in muscle tissue. Consistent with previously published data, hypothalamic MC3R expression was not signi ficantly different in WT mice compared to MC4R KO mice. 128 However, the fact that MC3R gene expression was not affected by exercise is different from previously published data by Haskell Luevan o et al.. 37 Patterson et al. also reported a change in MC3R expression in the arcuate nucleus of the hypothalamus (ARC). 129 Since changes were only seen in one nuclei of the hypothalamus, it is possible that since whole hypothalamus was examined for the experiments presented in this dissertat ion that changes in MC3R expression were present in individual nuclei, but were lost when analyzing the entire hypothalamus. 129 Similar to previous reports, MC4R gene expression in the hypothalamus of male mice did not differ betwee n MC3R WT, MC4R WT, and MC3R KO mice. 128 Consistent with previous reports, expression of the neuropeptide receptor Y type 1 was significantly affected by genotype in male mice. 37

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357 As reported previously by Haskell Luevano et al., genotype had a significa nt effect on AGRP expression in the hypothalamus. 37 Consiste nt with Levin et al., exercise did not significantly affect AGRP expression. 130 Upregulation of AGRP, a melanocortin antagonist, should result in increased food intake. Therefore, it would be expected for AGRP expression to be downregulated in obese mice. A tr end of a decrease in AGRP expression was seen in MC4R KO mice as expected. However, AGRP was significantly upregulated in DKO mice in running wheel cages compared to exercising MC3R WT mice. The increase in AGRP expression in exercising DKO mice along with the fact that it appeared that AGRP expression was trending up in MC3R WT and MC3R KO mice could be a result of the increased energy expenditure due to exercise resulting in an increase in AGRP expression. The increase in AGRP expression in MC3R WT and MC 3R KO mice in running wheel cages is consistent with the trend of an increase in food eaten by the exercising groups compared to those in conventional cages (Figure 3 77). Genotype, housing, and their interaction all had significant effects on expression o f the alpha catalytic subunit of the AMPK protein (Figure 4 4). The AMPK protein has been demonstrated to play an important role in glucose sensing of POMC and AGRP expressing neurons in the hypothalamus. 131 The AMPK protein generally activates catabolic pathways to promote the generation of ATP and to inhibit non essential p rocesses that consume ATP. The difference seen between exercising MC3R WT and MC3R KO mice appears to be due to an increase in expression in MC3R WT mice and not a decrease in the KO mice (Figure 4 5). The upregulation of PRKAA1 (AMPK) seen in exercising M C3R WT and MC4R WT mice could be the outcome of an increase in

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358 energy expenditure due to running wheel activity resulting in the need to increase catabolic function. Genotype significantly contributed to variation in expression levels of the CART gene in t he hypothalamus of male mice, consistent with previous reports. 37 Hypothalamic expression of the cocaine and amphetamine regulated transcript was higher in exercising DKO mice compared MC4R WT mice in running wheel cages (Figure 4 6). Further examination showed that DKO expression of CART remained constant while a decrease in CART expression was seen in MC4R WT mice allowed to exercise (Figure 4 6). Since CART has an anorectic effect, it is possible that the downregulation seen in male MC4R WT mice resulted in the slight (but non significant) increase in food intake seen in exercising MC4R WT mice compared to the sedentary MC4R WT group (Figure 3 77). Hypocretin (orexin) has been linked to physical activity through experiments that have shown that central administration of orexin A peptide results in spontaneous phys ical activity in male Sprague Dawley rats. 132,133 Haskell Luevano et al. found that voluntary exercise had a significant effect on HCRT gene expression that was also seen here (Figure 4 9). 37 Examination of the graphs in Haskell publication show a trend of voluntary exercise decreasing HCRT gene expression which is contrary to the results shown in Figures 4 9 and 4 10. 37 One would expect the increase in hypothalamic HCRT gene expression in DKO mice to correspond to an increase in running wheel activity, however this was not the case with DKO mice exercisi ng significantly less than MC3R WT mice for several weeks (Figure 3 141). Because the increase in hypocretin gene expression was not linked to an increase in

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359 running wheel activity, it is possible that HCRT is involved in another regulatory pathway. Hypocr etin has been linked to hypothalamic control of glucose homeostasis and food intake through studies that studied the effects of orexin A administration. 134,135 Leptin signals through the leptin receptor which is co expressed with AGRP and POMC in neurons, affecting the activity of those neurons and transcription of melanocortin agonist and antagonist mRNA. 4 1,121,122,136 The significant effect genotype had on leptin receptor expression in the hypothalamus of male mice (Figure 4 12) could be in part due to differences in leptin levels seen between the genotypes (Figure 3 138). The fact that exercise did not h ave a significant effect on hypothalamic LEPR gene expression is consistent with previous reports. 129,130 Both genotype and exercise significantly affected NPY expression in male mice, which is similar to the report that there was a significant interaction between the two f actors. 37 A decrease in hypothalamic NPY mRNA was not seen i n sedentary MC4R KO mice compared to MC4R WT mice contrary to previously published data. 37,38 The decrease in DKO hypothalamic NPY expression could be the result of the body trying to decrease body weight by decreasing food int ake (Figure 4 18). Though DKO NPY expression was lower than the WT groups in running wheel cages, there was no significant difference from sedentary DKO expression, indicating that the differences are probably due to increases in NPY expression in the WT g roups (Figure 4 18). Patterson et al. reported a change in NPY mRNA expression in the dorsomedial hypothalamic nucleus (DMN) due to exercise, while Levin et al. did not see a significant change in NPY expression in the ARC. 129,130

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360 Similar to previously published reports, ex pression of POMC in the hypothalamus was upregulated in MC4R KO mice in conventional housing. 37,38 Exercise did not have a significant effect on POMC gene expression, consistent with some, but not other, previous studies. 129,130 Increases in POM C expression in the hypothalamus of MC4R KO and DKO mice is consistent with the hypothesis that POMC expression would increase to counter the obese and hyperphagic phenotypes. The suppressor of cytokine signaling 3 (SOCS3) protein can block leptin signal transduction by binding to the tyrosine 985 residue of the leptin receptor. 137,138 The increase in SOCS3 expression in MC4R KO and DKO mice corresponds with seen and expected increases in circulating leptin (Figure 3 138). Patterson et al reported that exercise for three weeks postweaning decreased SOCS3 expression in the DMN of the hypothalamus in male DIO rats. 129 The lack of change due to exercise in hypothalamic SOCS3 expression in this experiment could be because of a difference between the MCR KO mouse and DIO rat models, or because the RNA expression was studied in the entire hypothalamus and not in the individual nuclei. Liver Neither genotype nor housing condition significantly affected expression of the PRKAA1 (AMPK) or DGAT1 genes in the livers of male mice. The conclusion that can be drawn from these results is that if the concentrations of these proteins are s ignificantly different in the different experimental groups, they are not regulated at the transcriptional level. Genotype had a significant effect on the expression of the insulin receptor gene in the livers of male mice (Figure 4 46). Insulin released f rom the pancreas acts through the insulin receptors to increase peripheral glucose uptake through the glucose

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361 transporter 4 (GLUT4). 87 89 Insulin also promotes glycogenesis and lipogenesis while inhibiting gluconeogenesis and lipolysis. 83 Changes in insulin receptor mRNA levels suggests the possibility that differences in insulin signaling potential exist between t he genotypes that might result in altered glucose homeostasis.. The carnitine palmitoyltransferase proteins are responsible for the transport of fatty acids across the mitochondrial membranes for oxidation. 94,95 Expression of both the CPT1A and the CPT2 genes were found to be upregulated in the livers o f sedentary MC4R KO and DKO mice indicating the potential for increased fatty acid oxidation. Expression of the CPT1A gene in the liver was not reported to be upregulated in MC4R KO mice compared to MC4R WT mice in contrast to the results shown in Figure 4 26. 39 Expression of CPT1A in the liver has been reported to be increased by peripheral melanocortin receptor agonist administration in DIO mice. 139 It is poss ible that the increase in POMC expression seen in Figure 4 17 resulted in the upregulation of these genes in the liver. Voluntary exercise significantly reduced both CPT1A and CPT2 gene expression in the livers of MC4R KO mice (Figures 4 27 and 4 29), indi cating that exercise can reduce liver fat oxidation potential independent of the increased POMC expression in the hypothalamus (Figure 4 17). Albarado et al. reported an upregulation of liver DGAT2 mRNA in MC4R KO mice as the result of eating a high fat di et. 39 While no change in DGAT2 expression was seen in MC4R KO mice, liver D GAT2 expression was increased in male DKO mice (Figure 4 31). It is possible that the increase in DGAT2 in DKO mice might be the result of the need to be able to store excess energy gained from hyperphagia (Figure 3 77) as triglycerides which further resul ts in the high amount of adipose tissue seen in DKO

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362 mice (Figure 3 20). No change in liver FASN gene expression was seen in MC4R KO mice compared to MC4R WT mice unlike previously published results. 39,139 Expression of FASN was upregulated in DKO mice in both housing conditions, perhaps to respond to the fact that the hyperphagia of DKO mice (Figure 3 77) results in excess calories that can be stored as fatty acids, necessitating the increase in fat ty acid synthase expression in the liver. Increases in genes involved in fatty acid and triglyceride synthesis in DKO mice are consistent with the drastic increases in fat mass seen compared to the other genotypes in the experiment (Figure 3 20). Fatty aci ds can be released from triglycerides for oxidation by hormone sensitive lipase, levels of which are controlled by circulating hormones including ACTH, glucagon, epinephrine, norepinephrine, and thyroid stimulating hormone. 99 The increase in LIPE expression in the livers of MC4R KO and DKO mice indicates an increase in the capability to break down triglycerides (Figure 4 44). The action of LIPE opposes that of DGAT2, which was also upregulated in the livers of male mice (Figure 4 31). This discrepancy would pro bably best be resolved through protein expression and activity studies which could indicate which process is dominantly upregulated. The increase of LIPE expression in the liver in the presence of elevated insulin levels might indicate insulin resistance i n the liver since one of the effects of insulin is decrease lipogenesis (Figure 3 106). The glucose transporter 2 is expressed in the cell membrane of hepatocytes and allows for glucose quickly diffuse in or out of the cell depending on concentration. 85,86 The increase in SLC2A2 gene (GLUT2) expression in the livers of MC4R KO mice indicate the capacity to handle a greater amount of glucose uptake and release (Figure

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363 4 40). Glu cokinase phosphorylation of glucose to glucose 6 phosphate allows for control over intracellular glucose concentrations to facilitate the diffusion of glucose into or out of the cell through GLUT2 in the liver. The upregulation of GCK gene expression in th e liver of MC4R KO and DKO mice (Figure 4 37) along with the increase of SLC2A2 gene (GLUT2) expression in the livers of MC4R KO mice (Figure 4 40) might indicate glu cose into the blood stream for use by other organs, glucose 6 phosphate must be dephosphorylated by glucose 6 phosphatase. The increase of G6PC3 gene expression in the livers of exercising DKO mice shows that the livers of these mice can accommodate releas ing larger amounts of glucose for other tissue use. The increase in both G6PC3 and GCK in exercising DKO mice might be the result of a need for the uptake or release of large amounts of glucose to maintain safe blood glucose concentrations. An experiment t o examine the activities of these enzymes under different conditions involving high or low glucose levels would answer the question of which was more active in the exercising DKO mice. Liver expression of fructose 1,6 biphosphatase, which is involved in th e gluconeogenic pathway, was upregulated in both MC4R KO and DKO mice. This is counterintuitive considering both genotypes are expected to be hyperinsulinemic and hyperleptinemic which should suppress this pathway. One potential explanation for this discre pancy is that the liver glucose production could be increased in these genotypes to supply glucose to meet the demand of the increased lean mass seen in these genotypes at the end of the experiment (Figure 3 48).

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364 Glucose can be stored by the liver as glyco gen for release into circulation to maintain blood glucose levels. A decrease in the liver expression of the GYS2 gene in DKO mice, and an increase in PYGL gene expression in both sedentary MC4R KO and DKO mice indicates a decrease in glycogenesis and incr ease in glycogenolysis to increase glucose availability for use or release by the liver cells. Another possibility could be that liver cells have to be able rapidly break down glycogen stores to respond to increased energy demand, but can replenish glycoge n stores over time. Phosphofructokinase is the rate limiting enzyme in the glycolysis pathway and is upregulated in sedentary DKO mice and exercising MC4R WT mice (Figure 4 49). It is possible that the increase of hepatic PFKL gene expression seen in exerc ising MC4R WT mice was needed to maintain appropriate levels of energy for the mice given access to running wheels. Hepatic leptin receptor gene expression was significantly affected by genotype and housing. While sedentary MC4R KO and DKO mice had slightl y lower LEPR expression than WT mice, exercise did not significantly change their levels of LEPR gene expression (Figure 4 47). The differences seen between the KO and WT mice in running wheel cages was the result of an increase in leptin receptor expressi on in the WT mice (Figure 4 48). Muscle No change was seen in skeletal muscle expression of PRKAA1 gene for the alpha catalytic subunit of the AMPK protein in male mice (Figure 4 62). Taking into consideration the increased plasma leptin concentrations of MC4R KO and DKO mice (Figure 3 138), this is not consistent with reports that chronic administration of leptin has been shown to increase both expression and phosphorylation of AMPK in the muscle of

PAGE 365

365 female rats. 140 It is possible that this discrepancy is due to a spe cies difference between rats and mice, or perhaps the result of dysfunctional melanocortin signaling which have been shown to be necessary for proper central leptin signaling. 43 No changes in CPT1B, GYS1, or IL 6 gene expression were seen in skeletal muscle of male mice regardless of housing type or genotype (Figures 4 63, 4 69, and 4 70). Interleukin 6 has been linked to increased insulin and leptin sensitivity after exercise. 59 It is possible that IL 6 expression is an acute response to exercise, and since chronic exercise was the method used in this exper iment and that is why changes in expression were not seen between sedentary and exercising groups. Housing had a significant effect on gene expression of the CPT2, PYGM, and PFKM genes in the skeletal muscle tissue of male mice. The slight increase in CPT2 expression in running wheel cages along with the slight decreases in PYGM and PFKM gene expression in exercising male mice might indicate a shift away from glycolysis and an increase in fatty acid oxidation as a source of energy in muscle tissue. Expressi on of SLC2A4 gene which codes for the glucose transporter 4 protein was significantly influenced by genotype. Non significant decreases in mRNA coding for GLUT4 seen in the MC4R KO and DKO mice might indicate an inhibition of the glucose transporter due to increases in plasma insulin compared to WT mice (Figure 3 106). Skeletal muscle expression of glucokinase mRNA was significantly affected by both genotype and housing with sedentary MC3R KO mice having higher levels than MC3R WT mice in conventional cages (Figure 4 65). It is possible that the increase of GCK expression seen in muscle tissue would also be seen in adipose tissue. If this were true, and increase in glucose phosphorylation, resulting in higher intracellular

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366 energy availability might be partia lly responsible for the increased adiposity seen in MC3R KO mice. 8,9 The fact that the incre ase in GCK expression is prevented by exercise in MC3R KO mice in skeletal muscle supports this hypothesis since exercise also resulted in a decrease in adiposity in male MC3R KO mice (Figure 3 183). Genotype had a significant effect on the expression leve ls of both the UCP2 and UCP3 genes in muscle tissue of male mice (Figures 4 72 and 4 74). Voluntary exercise resulted in significant increases in both UCP2 and UCP3 gene expression in MC3R WT mice compared to MC3R WT mice in conventional cages (Figures 4 7 3 and 4 75). A trend of an increase in uncoupling protein gene expression was also seen in MC3R KO mice allowed to exercise compared to those kept in conventional cages (Figures 4 73 and 4 75). Relative Expression Housekeeping genes that have constant lev els of expression across experimental groups are useful for the quantitative analysis of levels of expression for genes of interest. However, it is sometimes useful to directly compare two genes whose actions oppose or differ from each other giving an indi cation of which has a larger effect. Hypothalamus Higher amounts of MC4R mRNA were found than that of the MC3R gene in the hypothalamus of MC3R WT and MC4R WT mice with MC3R expression significantly higher in MC4R WT mice compared to MC3R WT mice in both c onventional and running wheel cages (Figure 4 23). This could be indicative of a difference between the strains in the melanocortin signaling pathway. POMC derived agonists and AGRP are the primary endogenous ligands of the melanocortin receptors in the ce ntral nervous system, and knowing the relative

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367 abundance of these ligands can give an indication of the state of the melanocortin signaling pathway. With the exception the MC4R WT mice, POMC gene mRNA was expressed in higher quantities than the AGRP gene i n the hypothalamus for all genotypes (Figure 4 24) indicating the possibility of the dominance of the anorectic influence of POMC over AGRP. The ratio of AGRP to POMC expression in the hypothalamus has been reported to be affected by time of day in female rats. 141 Hypothalamic release of AGRP and POMC derived ligands are also influenced by fed or fasting state. 122 The increase in AGRP expression rela tive to POMC gene expression in the hypothalamus of MC4R WT mice supports the hypothesis that there is a difference in the melanocortin signaling pathways in strains between the MC3R WT and MC4R WT groups. Liver Messenger RNA for the DGAT2 gene was found i n much higher amounts (30 to 50 fold difference) than the DGAT1 gene in the liver of male mice (Figure 4 55). Even though both genotype and housing had significant effects on the relative expression of these two genes, DGAT2 was always expressed in much h igher amounts. Because of the large difference in the expression levels of these two genes, it can be concluded that DGAT2 is likely the more active and metabolic relevant isoform of this protein in the liver. While the diglyceride acyltransferase catalyze the addition of the final fatty acid to diglycerides to form triglycerides, hormone sensitive lipase catalyzes the release of fatty acids from the triglyceride molecules. Liver LIPE gene expression was found to be approximately equal to that of DGAT1 (Fig ure 4 57), while DGAT2 expression was much higher than LIPE expression (Figure 4 59). While DGAT2 expression is greater than both DGAT1 and LIPE, it is also possible that there is additional modification of the

PAGE 368

368 involved proteins concentration or activity a t the translational or post translational level resulting in the ratio of their relative activities to be different. The other explanation for this is that the high level of DGAT2 expression compared to LIPE could be from the livers synthesizing triglyceri des for transport to and use by other peripheral tissues. The fatty acid synthesis protein builds fatty acids from acetyl CoA while CPT1 and oxidation to form acetyl CoA to use for gluconeo genesis or ATP production through the citric acid cycle. The increased FASN expression compared to CPT1A and CPT2 in the livers of DKO mice might indicate that these mice have an excess of energy substrates that can be converted to fatty acids (Figures 4 6 0 and 4 61). The decrease in the relative expression of FASN to CPT1A and CPT2 in exercising MC3R WT and MC3R KO mice might indicate the need of these mice to break down fatty acids to provide for the increased energy needs of other tissues brought on by e xercise (Figures 4 60 and 4 61). Entry and release of glucose into the cell is controlled by the concentration of intracellular glucose compared to extracellular glucose levels. Phosphorylation of glucose by glucokinase reduces intracellular glucose conce ntrations while dephosphorylation of glucose 6 phosphate by glucose 6 phosphatase increases intracellular glucose levels. The GCK gene is expressed at higher levels than the G6PC3 gene in the livers of male mice (Figure 4 50). Since glucose release to main tain homeostasis, activity of these proteins might be regulated at the translational or post translational levels to control glucose flow into and out of liver cells.

PAGE 369

369 Th e glycolysis and gluconeogenic pathways are opposing processes, one breaking glucose down into pyruvate, and the other synthesizing glucose from pyruvate being catalyzed by phosphofructokinase and fructose 1,6 biphosphatase respectively. FBP1 gene expressi on is much higher relative to PFKL gene expression in the liver, indicating that the gluconeogenic pathway is upregulated compared to the glycolytic maintaining appropriat e levels of blood glucose concentration, with the higher levels of FBP1 relative to PFKL indicating that at the transcriptional level, the gluconeogenesis pathway is upregulated compared to the glycolysis pathway in the liver. Glycogen synthase and glycoge n phosphorylase are responsible for the addition and removal of glucose 6 phosphate molecules from the glycogen polymer respectively. These two genes were found to be expressed in similar levels relative to each other with the largest difference being in s edentary DKO mice with PYGL being expressed approximately 3 fold higher than GYS2 (Figure 4 51). GYS2 is expressed at levels at about half of PYGL expression levels in sedentary MC4R KO and exercising MC4R KO and DKO mice (Figure 4 51). These results coupl ed with the quantitative expression of these genes (Figures 4 30 and 4 31) can be interpreted to mean that the glycogen synthesis pathway is down regulated in these mice compared to the glycogen breakdown pathway. This could result in energy being stored i n fatty acids preferentially over glycogen, which is consistent with previous reports of mice lacking the MC4R preferentially storing calories as adipose tissue. 117 Muscle Uncoupling proteins allow cel ls to bypass the ATP synthesis pathway by allowing protons to diffuse across the inner mitochondrial membrane without interacting with ATP

PAGE 370

370 synthase. Mice from the MC3R strain express UCP2 at levels approximately half of that of UCP3, while MC4R mice and DK O mice express UCP3 at higher levels than UCP2 in the muscle tissue of male mice (Figure 4 76). While both uncoupling proteins essentially perform the same task, it is interesting to note the difference seen between the MC3R and MC4R strains in relative ex pression of UCP2 and UCP3 in the muscle. Glycogen synthase 1 mRNA is expressed at lower levels than glycogen phosphorylase in male mice from both housing conditions (Figure 4 77). This difference might be explained by taking into consideration that muscle cells are more likely to have to rapidly break down glycogen stores to respond to increased energy demand due to exercise or movement than have to replenish glycogen stores quickly. Shifts in Metabolic Pathways While gene expression changed in MC3R KO mic e for AMPK and LEPR in the brain and glucokinase in the muscle, most of the differences in gene expression are seen in MC4R KO and DKO mice. The increase of SOCS3 and POMC expression of MC4R KO mice is consistent with the increased plasma leptin concentra tion associated with the genotype. The increase in POMC gene expression has been previously reported and supports the hypothesis that POMC expression would increase to counter the obese and hyperphagic phenotype of MC4R KO mice. 37 Fatty acid storage and metabolism in the liver were both affected at the transcriptio nal level in MC4R KO mice. Increases in both CPT protein genes and in hormone sensitive lipase indicate a shift to increase oxidation in sedentary MC4R KO mice. Access to voluntary exercise equipment prevented the increases in expression for these genes, returning expression of CPT1A, CPT2, and LIPE to levels not significantly different from

PAGE 371

371 MC4R WT mice. Glucose storage and synthesis were also affected in the liver of MC4R KO mice. Expression of the glycogen phosphorylase and f ructose 1,6 biphosphatase genes were both upregulated in the livers of MC4R KO mice in conventional cages compared to similarly housed MC4R WT mice. These increases taken together could indicate an increase in both gluconeogenesis and glycogen breakdown to produce glucose 6 phosphate which can be dephosphorylated and released into the blood stream. No differences in these pathways were seen in exercising MC4R KO mice compared to MC4R WT mice, indicating that voluntary exercise had an effect on the expressio n of these genes. All differences in hypothalamic gene expression seen between DKO and MC3R WT and MC4R WT mice were only observed in mice allowed to exercise. Increases in CART and POMC coupled with decreases in NPY expression in DKO mice compared to WT m ice should theoretically result in a decrease in food intake, however by the end of the experiment when the samples for these gene expression studies were taken, food intake in exercising DKO had reached levels equal to those of DKO mice in conventional ca ges (Figure 3 77). This increase in food intake does correspond to increases in AGRP and HCRT gene expression, both of which have been linked to increases in food intake. 12,135 Further experiments will need to be completed in order to better understand the effects of exercise on the expression of centrally expressed feeding pathway peptides, and the effects they have in mice lacking melanocortin receptors which are central to multiple feeding pathways. The metabolic pathways involving glucose transport, storage, and use as well as fatty acid synthe sis, storage, and use were all altered in DKO mice. Expression of glucokinase mRNA, the protein

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372 that phosphorylates glucose after uptake into the cell, was upregulated in sedentary DKO mice indicating an increase in hepatic glucose uptake capacity. Express ion of G6PC3 and SLC2A2 (GLUT2) were upregulated in exercising DKO mice compared to WT mice, signifying a potential increase in liver cell capacity to release glucose to satisfy increased peripheral organ need due to exercise. Genes involved in both glycol ysis and gluconeogenesis were upregulated in sedentary DKO mice compared to WT controls, with FBP1 being expressed at higher levels than PFKL suggesting a dominance of the gluconeogenesis over the glycolysis pathway (Figure 4 53). It is possible that this is a result of increase use of fats as source of energy in DKO mice instead of carbohydrates which would require further experiments involving calorimetric cages to either support or refute this hypothesis. An increase in PYGL expression coupled with the d ecrease in GYS2 expression suggests a decrease in energy storage as glycogen in the liver of sedentary DKO mice. This combined with the upregulation of fatty acid synthase and DGAT2 mRNA levels support the hypothesis that hepatocytes are capable of increas ing fatty acid and triglyceride production. Increased energy storage in the form over glycogen is also consistent with the high adiposity seen in DKO mice (Figure 3 20). Integrating the results of the central and peripheral gene expression experiments pres ented in Chapter 4 is a difficult task. A particular challenge is distinguishing between changes in gene expression due to melanocortin receptor inactivation, and changes in gene expression due to changes in metabolic state that are the result of melanocor tin receptor inactivation. In order to distinguish between these two effects future experiments including obese genetically WT mice (DIO model) and lean KO mice

PAGE 373

373 (pair fed KO mice) could be used to tease apart subtle differences in gene expression caused by these interrelated influences. Part of the challenge of reconciling central and peripheral changes in gene expression is due to the fact that intermediate steps cannot all be measured at the gene expression level. Song et al. have demonstrated that the M C4R is expressed in sympathetic outflow neurons that innervated both white adipose and brown adipose tissue depots, meaning central melanocortin signaling can directly influence peripheral metabolism. 124,125 Additionally, the central melanocortin system has been shown to have some control over insulin secretion by the pancreas. 120 Leptin has been reported to affect many of the genes studied including POMC, AGRP, SOCS3, and PRKAA1 (AMPK). 41,121,122,137,138,140 Increased IL 6 have been tentatively linked exercise to the improved ce ntral insulin and leptin sensitivity. 59 Insulin, leptin, and IL 6 are secreted factors that would have to be measured in the plasma in order to accurately know circulating concentrations. Finally, some of the genes studied, notably PRKAA1 (of the AMPK protein), are required to undergo post translational modification in order to be active. 100,140 This would indicate that experiments that solely use gene expression are insufficient to completely understand energy homeostasis in the body. Strain Differen ces Between MC3R and MC4R Breeding Colony Mice Further supporting the phenotypic data presented in Chapter 3, differences in gene expression between MC3R WT and MC4R WT mice indicate a difference between these strains exists. Relative expression values wer e used to identify differences between MC3R WT and MC4R WT since these values were not normalized to one of the WT groups. Relative expression levels of the MC3R and MC4R genes were significantly different in the hypothalamus of MC3R WT and MC4R WT mice. T hough

PAGE 374

374 significant, the differences in the relative expression levels of MC3R and MC4R were small between the genotypes, but still might result in subtle changes to the central melanocortin signaling pathway. These differences could be studied using selecti ve MC3R or MC4R selective agonists and antagonists and measuring the different responses of the two strains. Differences in liver gene expression also existed between the MC3R WT and MC4R WT mice. Glucokinase relative to glucose 6 phophatase was expressed in much higher levels in the livers of MC4R WT mice than in MC3R WT mice, suggesting a difference in the process than controls glucose flux into and out of the hepatocytes. Though small, there was a significant difference between MC3R WT and MC4R WT strain s in LIPE gene expression relative to DGAT1. This could indicate a difference in fatty acid storage between the two strains of WT mice. A significant difference in the relative expression of the UCP2 to the UCP3 gene was seen between exercising MC3R WT and MC4R WT mice. There also appears to be a trend that MC4R mice have a higher ratio of UCP2 to UCP3 in both housing conditions with DKO mice having what appears to be an intermediate expression ratio between the MC3R and MC4R strains. Concluding Remarks Alt ered gene expression as the result of the metabolic disorders of mice lacking either the MC3R or MC4R has been established in the literature. 36 39,139 Hypothalamic gene expression has been shown to be affected by exercise. 37,129,130,142 The e xperiments presented and discussed in Chapter 4 studied the effects of both genotype and access to running wheels for exercise on the expression of not only hypothalamic

PAGE 375

375 gene expression, but gene expression in peripheral organs involved in exercise or ener gy homeostasis. It is difficult to draw any hard conclusions from gene expression data since regulation of the studied pathways can also occur at the translation and post translational levels. However, significant differences in gene expression levels were found in all organs studied related to the management of energy balance supporting the hypothesis that voluntary exercise causes changes in expression of genes involved in energy homeostasis. While these experiments did not determine if changes in gene ex pression were the direct result of exercise or were responses to changes in metabolism because of exercise, they provide novel data contributing to the field of obesity and exercise research.

PAGE 376

376 Figure 4 1. Potential gene expression results in experiments for the identification of potential drug targets.

PAGE 377

377 Figure 4 2. Hypothalamic expression of the AGRP gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 3. Hypothalamic expression of the PRKAA1 (AMPK) gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 378

378 Figure 4 4. Effect of voluntary exercise on the hypothalamic expression of the PRKAA1 (AMPK) gene. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 5. Hypothalamic expression of the CART gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 379

379 Figure 4 6. Hypothalamic expression of the CPT2 gene in sedentary or exe rcising male mice. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 7. Hypothalamic expression of the GCK gene in sedentary or exercising male mice. No significant differen ces due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 380

380 Figure 4 8. Hypothalamic expression of the HCRT gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype and housing (P< 0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 9. Effect of voluntary exercise on the hypothalamic expression of the HCRT1 gene. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 381

381 Figur e 4 10. Hypothalamic expression of the INSR gene in sedentary or exercising male mice. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 11. Hypothalamic expression of the L EPR gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 382

382 Figure 4 12. Hypothalamic expression of the MC3R gene in sedentary or exercising male mice. Two way ANOVA si gnificance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 13. Hypothalamic expression of the MC3R gene in sedentary or exercising male mice excluding mice lacking the MC3R. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 383

383 Figure 4 14. Hypothalamic expression of the MC4R gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ** *, P<0.001) Figure 4 15. Hypothalamic expression of the MC4R gene in sedentary or exercising male mice excluding mice lacking the MC4R. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.0 01)

PAGE 384

384 Figure 4 16. Hypothalamic expression of the POMC gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 17. Hypothalamic expression of the NPY gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 385

385 Figure 4 18. Hypothalamic expression of the NPY1R gene in sedentary or exercising male mice. Two way ANOVA s ignificance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 19. Hypothalamic expression of the SOCS3 gene in sedentary or exercising male mice. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 386

386 Figure 4 20. Effect of voluntary exercise on the hypothalamic expression of the SOCS3 gene. Two way ANOVA significance achieved for genotype, housing, and the interaction betw een genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 21. Hypothalamic expression of the UCP2 gene in sedentary or exercising male mice. No significant differences due to genotype or housing were found by two way ANOVA. (*, P <0.05; **, P<0.01; ***, P<0.001)

PAGE 387

387 Figure 4 22. Relative expression of the MC3R gene to the MC4R gene in the hypothalamus. Two way ANOVA significance achieved for genotype (P<0.001) (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 23. Relative expression of the AGRP gene to the POMC gene in the hypothalamus. Two way ANOVA significance achieved for genotype (P<0.001) (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 388

388 Figure 4 24. Liver expression of the PRKAA1 (AMPK) gene in sedentary and exercising male mice. No si gnificant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 25. Liver expression of the CPT1A gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype ( P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 389

389 Figure 4 26. Effect of voluntary exercise on the expression of the CPT1A gene in male mice. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 27. L iver expression of the CPT2 gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 390

390 Figure 4 28. Effect of voluntary exercise on the expression of the CPT2 gene in mal e mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 29. Liver expression of the DGAT1 gene in sedentary and exercising male mice. No significant differences due to genotype or housing were f ound by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 391

391 Figure 4 30. Liver expression of the DGAT2 gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.0 01) Figure 4 31. Effect of voluntary exercise on the expression of the DGAT2 gene in male mice. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 392

392 Figure 4 32. Liver expression of the FASN gen e in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 33. Liver expression of the FASN gene in sedentary and exercising male mice excluding the DKO genotype. T wo way ANOVA significance achieved for housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 393

393 Figure 4 34. Effect of voluntary exercise on the expression of the FASN gene in male mice excluding the DKO genotype. Two way ANOVA significance achieved for housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 35. Liver expression of the FBP1 gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 394

394 Figure 4 36. Liver expression of the GCK gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 37. Effect of voluntary exercise on the expression of the GCK gene in male mice. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 395

395 Figure 4 38. Liver expression of the G6PC3 gene in sedentary and exercising male mice. Two way ANOVA significance achieve d for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 39. Liver expression of the SLC2A2 (GLUT2) gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.0 01)

PAGE 396

396 Figure 4 40. Effect of voluntary exercise on the expression of the SLC2A2 (GLUT2) gene in male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 41. Liver expression of the PYGL gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 397

397 Figure 4 42. Liver expression of the GYS2 gene in sedentary and exercising male mice. Two way ANOVA signi ficance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 43. Liver expression of the LIPE gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 398

398 Figure 4 44. Effect of voluntary exercise on the expression of the LIPE gene in male mice. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 45. Liver expression of the INSR gene in se dentary and exercising male mice. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 399

399 Figure 4 46. Liver expression of the LEPR gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 47. Effect of voluntary exercise on the expression of the LEPR gene in male mice. Two way ANOVA significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P< 0.01; ***, P<0.001)

PAGE 400

400 Figure 4 48. Liver expression of the PFKL gene in sedentary and exercising male mice. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 49. Relative expression of the GCK gen e to the G6PC3 gene in the livers of male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 401

401 Figure 4 50. Relative expression of the GYS2 gene to the PYGL gene in the livers of male mice. Two way ANOV A significance achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 51. Effect of voluntary exercise on the relative expression of the GYS2 gene to the PYGL gene in the livers of male mice. Two way ANOVA significanc e achieved for genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 402

402 Figure 4 52. Relative expression of the FBP1 gene to the PFKL gene in the livers of male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; ** P<0.01; ***, P<0.001) Figure 4 53. Effect of voluntary exercise on the relative expression of the FBP1 gene to the PFKL gene in the livers of male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 403

403 Figure 4 54. Relative expression of the DGAT1 gene to the DGAT2 gene in the livers of male mice. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 55. Effect of voluntary exercise on the relative expression of the DGAT1 gene to the DGAT2 gene in the livers of male mice. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 404

404 Figure 4 56. Relative expression of the LIPE gene to the DGAT1 gene in the liv ers of male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 57. Effect of voluntary exercise on the relative expression of the LIPE gene to the DGAT1 gene in the livers of male mice. Two w ay ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 405

405 Figure 4 58. Relative expression of the LIPE gene to the DGAT2 gene in the livers of male mice. Two way ANOVA significance achieved for genotype (P<0.01). (*, P <0.05; **, P<0.01; ***, P<0.001) Figure 4 59. Relative expression of the FASN gene to the CPT1A gene in the livers of male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 406

406 Figure 4 60. Relative e xpression of the FASN gene to the CPT2 gene in the livers of male mice. Two way ANOVA significance achieved for genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 61. Skeletal muscle expression of the PRKAA1 (AMPK) gene in mal e mice in conventional and running wheel cages. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 407

407 Figure 4 62. Skeletal muscle expression of the CPT1B gene in male mice in convention al and running wheel cages. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 63. Skeletal muscle expression of the CPT2 gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 408

408 Figure 4 64. Skeletal muscle expression of the GCK gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved fo r genotype and housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 65. Skeletal muscle expression of the GCK gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype and housing (P<0.01). ( *, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 409

4 09 Figure 4 66. Skeletal muscle expression of the SLC2A4 (GLUT4) gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001 ) Figure 4 67. Skeletal muscle expression of the PYGM gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

PAGE 410

410 Figure 4 68. Skeletal muscle expression of the GYS1 gene in male mice in conventional and running wheel cages. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 69. Skeletal muscle expression of the IL 6 gene in ma le mice in conventional and running wheel cages. No significant differences due to genotype or housing were found by two way ANOVA. (*, P<0.05; **, P<0.01; ***, P<0.001)

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411 Figure 4 70. Skeletal muscle expression of the PFKM gene in male mice in convention al and running wheel cages. Two way ANOVA significance achieved for housing (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 71. Skeletal muscle expression of the UCP2 gene in male mice in conventional and running wheel cages. Two way ANOVA sig nificance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

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412 Figure 4 72. Skeletal muscle expression of the UCP2 gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 73. Skeletal muscle expression of the UCP3 gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001)

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413 Figure 4 74. Skeletal muscle expression of the UCP3 gene in male mice in conventional and running wheel cages. Two way ANOVA significance achieved for genotype (P<0.01). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 75. Relative expression of the UCP2 gene to the UCP3 gene in the skeletal muscle tissue of male mice. Two way ANOVA significance achieved for genotype (P<0.001). (*, P<0.05; **, P<0.01; ***, P<0.001)

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414 Figure 4 76. Relative expression of the GYS1 gene to the PYGM gene in the skeletal muscle tissue of male mice. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001) Figure 4 77. Relative expression of the GYS1 gene to the PYGM gene in the skeletal muscle tissue of male mice. Two way ANOVA significance achieved for genotype, housing, and the interaction between genotype and housing (P<0.05). (*, P<0.05; **, P<0.01; ***, P<0.001)

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415 CHAPTER 5 EFFECT OF GENDER ON THE MITIGATING EFFEC TS OF VOLUNTARY EXER CISE ON PREVENTION OF THE OBESE PHENOTYPE OF T HE MELANOCORTIN 4 RECEPTOR MICE Breeding and genotyping of the experimental mice was done by Amy M. Andreasen, Laurie M. Koerper, and Erin B. Bruce. Mice cages were changed and cleaned by Amy M. Andreasen, Kimb erly R. Haskell, Laurie M. Koerper, and Erin B. Bruce. Measurements and blood draws were done by Amy M. Andreasen, Laurie M. Koerper, Erin B. Bruce, and Jay Schaub. Running wheel activity data was compiled by Sarah B. Carey and Jay Schaub. All plasma hormo ne assays were run by Jay Schaub. Sacrifice and dissection of organs was performed by Dr. Zhimin Xiang, Dr. Sally Litherland, and Jay Schaub. RNA extraction, cDNA synthesis of hypothalamus was done by Dr. Zhimin Xiang and Jay Schaub. RT PCR was done by Dr. Zhimin Xiang and Jay Schaub. Introductory Remarks With the rise in the number of overweight and obese individuals weight management has emerged as a critical field in healthcare. Obesity has been identified as a significant risk factor for many other dise ases including type II diabetes, hypertension, stroke, asthma, depression, and certain types of cancers. 3,143 In addition to being a detriment to health, obesity is also a financial burden. In 2009 it was estimated that yearly healthcare costs associa ted with obesity were $147 Billion in the United States. 4 Exercise has been shown to be capable of increasing energy expenditure, thereby causing weight loss in both animal and human subjects. 37,64,144 As discussed in previously, both genetic and environmental factors have been identified that contribute to the onset of obes ity. One monogenetic cause of obesity

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416 identified in both human and animal subjects are mutations resulting in a loss of function of the MC4R. 10,45,47,48 It has been shown previously that male MC4R KO mice given access to voluntary exercise equipment, in the form of running wheel apparatuses, experience a significant delay in the onset of the obese phenotype. 37,64 Exercise has also been shown to be an effective means of weight loss in huma n patients. 144 In general, male mice are larger than f emale mice in regards to body size in both WT and mutant strains. 10,36,145 Additionally, male and female animals have different responses to high fat diets. 36,146 Both male and female MC4R KO mice exhibit the hyperphagic and obese phenotype compared to WT littermates. 10 To test the hypothesis that voluntary exercise will prevent the onset of the obese phenotype in female MC4R KO mice, similar to the effect seen in male MC4R KO mice, male and female MC4R KO and WT control mic e were allowed to exercise for a period of seven weeks. Herein the effects of exercise on male and female MC4R KO mice are described. In order to expand upon the data presented in Chapter 3 where the effects of voluntary exercise on the phenotypes of male MC3R, MC4R, and DKO mice were studied, experiments were designed to study the effects of voluntary exercise on male and female MC4R KO mice. The MC4R KO genotype was selected for analysis because of the robust obese phenotype seen in the male MC4R KO mice compared to the MC3R KO mice and the relative ease of generating enough mice for a suitably sized experimental group compared to the DKO mice. Methods and Results Brief Overview of Experiment Male and female mice from the heterozygous MC4R breeding scheme identified as either WT or KO born within a one week time period were used for the experiment

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417 with between five and eleven mice in each group. At the beginning of the sixth week of age animals of each sex and genotype were split into two housing groups and placed in either conventional or running wheel cages. Body weight, body length, and food weight were measured twice each week. Food intake was determined by taking the difference between food left in the cage and food previously added to the cage. Quantit ative MRI scans were performed once a week at a time point between body weight measurements. Blood was drawn three times a week for blood glucose, cholesterol, and circulating hormone concentration determination. At sacrifice the brains were placed in RNAL ater (Ambion) until the hypothalamus was dissected out and RNA extraction and cDNA synthesis could occur. Hypothalamic gene expression was determined using commercially available Taqman gene probes (ABI). Running wheel activity was monitored by computer s oftware that registered and recorded magnetic switch closures once a minute. Rotations of the wheel were converted to distance or used as an indicator of physical activity. Statistical Analysis Data was analyzed using repeated measures analysis of varianc e (ANOVA) followed by post hoc tests as appropriate. In order to determine when differences between groups emerged, two way ANOVA tests were performed for each time point. Variables were analyzed by gender and genotype for male and female mice in the conve ntionally housed groups. Mice were also analyzed by genotype and housing within the genders. If statistical significance was found to exist between groups in the ANOVA, a Bonferroni post test was used to further compare genotype and gender or genotype and housing as appropriate. To test if voluntary exercise was able to prevent changes in the phenotypes of the KO mice from WT levels, t tests were performed

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418 between the sedentary WT mice and exercising KO mice. Significance was assumed for P values < 0.05. T o evaluate the effects of gender on energy balance pathway gene expression in both WT and KO mice, hypothalamic gene expression for mice in conventional cages was measured and normalized to levels of the sedentary male WT mice (Figure 5 8). Statistical ana ) by two way ANOVA with respects to genotype and gender. Body Size and Composition The MC4R KO mice are known to be obese with increased linear growth compared to WT littermates. 10,37,64 Addit ionally, fat mass is reported to be increased in both male and female MC4R KO mice compared to WT mice. 36 39, 147 Previous research by this laboratory has shown that voluntary exercise is able to delay the onset of the obese phenotype in male MC4R KO mice. 37,64 To test the hypothesis that voluntary exercise will prevent the onset of obesity, increased linear growth, and increased fat mass of mice lacking MC4R functionality, body weight, linear growth, and body composition were measured for male and female mice in both standard cages and cages equipped with running wheels for voluntary exercise. Body weight, body length, and body composition were m easured for all mice in each group (n=5 11). Body weights After repeated measure ANOVA, gender, genotype, and housing were found to have a significant effect on body weight (P<0.01) (Figure 5 1). An interaction between genotype and housing (P<0.01) and a s ignificant interaction between gender, genotype, and housing (P<0.05) were also found to exist (Figure 5 1). Age also had a significant effect on body weight (P<0.001) (Figure 5 1).

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419 Two way ANOVA by gender and genotype revealed a significant difference due to gender in mice in the conventionally housed group at five weeks of age (P<0.01) with the males weighing more than the females (Figure 5 1). This difference between male and female mice remained at P<0.001 for the remainder of the experiment (Figure 5 1 ). There was also a significant effect on body weight due to genotype (P<0.01) beginning at week five that persisted until the conclusion of the experiment with KO mice weighing more than WT mice (P<0.001) (Figure 5 1). Significant differences existed betw een the male and female mice at each time point for both WT and KO groups with male mice consistently weighing more than their female counterparts for both genotypes (P<0.05). At five weeks of age there was no significant difference in body weight between the male mice groups (Figure 5 1). Beginning at six weeks of age, there was a significant genotype effect with male KO mice weighing more than male WT mice (P<0.05). There was a significant difference in body weights between male KO conventional and male KO running wheel mice that started at six weeks of age and lasted until the conclusion of the experiment (P<0.01). At five weeks of age there was already a difference in body weight between the female WT and female KO mice (P<0.01) that remained for the du ration of the experiment (P<0.001) (Figure 5 1). At no point in time was there a significant difference between the female KO conventional and female KO running wheel groups. No significant difference between the male KO running wheel and male WT conventi onal mice body weights was seen until 11 weeks of age (sixth week of treatment) when a statistical difference emerged (P<0.05). A significant difference between female WT conventional and female KO running wheel began at seven weeks

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420 of age (second week of treatment) and remained for the entirety of the experiment (P<0.05). Fat masses Repeated measures ANOVA found that genotype, housing and age had significant effects on the amount of fat mass (P<0.01), while gender did not (P=0.055). There was also an inte raction between gender and housing (P<0.01). Beginning at five weeks of age male and female sedentary KO mice possessed a statistically greater fat mass than WT mice as shown in Figure 5 2 (P<0.05). Beginning at eight weeks of age (third week of treatment ) there was a statistical difference between male KO and female KO mice in conventional cages, with the males having the greater fat mass (P<0.05). For male mice there was a difference in fat mass already evident at five weeks of age due to genotype (P<0. 01), with KO mice having a greater fat mass than WT mice. Beginning at seven weeks of age (second week of treatment) a difference in fat mass from housing was seen (P<0.001), with male mice in conventional cages possessing more fat than those males in runn ing wheel cages. There was a significant difference between sedentary male KO mice and male KO mice in the running wheel cages beginning at six weeks of age (P<0.01). A significant genotype effect was present for the amount of fat mass in female mice at fi ve weeks of age (P<0.05) with female KO mice possessing more fat mass than female WT mice. There was a statistical difference in fat mass between female sedentary and exercised groups beginning at seven weeks of age (second week of treatment) that lasted f or the entirety of the experiment (P<0.05). A significant difference between the amount of fat mass in the female KO mice in conventional versus running wheel cages began at eight weeks of age (P<0.05),

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421 but disappeared at 12 weeks of age, with the mice in the running wheel cages having a reduced amount of fat mass. No difference in fat mass was observed between KO male mice in running wheels housing and male WT mice in conventional cages until 11 weeks of age (sixth week of treatment) at which point the ex ercising KO animals had a greater fat mass than the sedentary WT animals (P<0.05). At five weeks of age there was a significant difference in fat mass between sedentary female WT mice and female KO mice in running wheel cages (P<0.05), but the difference d isappeared by six weeks of age and did not reappear. Lean masses Repeated measures ANOVA revealed that age, gender, and genotype all had significant effects on the lean mass of the mice (P<0.001). Housing did not have a statistically relevant effects on le an mass. At five weeks of age there was already a difference in amount of lean mass between male and female sedentary mice as shown in Figure 5 3 (P<0.001). Additionally, there was also a genotype difference between conventionally caged mice with KO mice h aving greater lean mass than WT mice (P<0.05). A statistical difference between conventional male and female WT mice was seen beginning at five weeks of age (P<0.05), as well as a significant difference seen between the sedentary male and female KO mice (P <0.01) with the males possessing a greater lean mass than the females for both genotypes. No difference in lean mass was seen between male mice at five weeks of age. However, at six weeks both genotype and housing had significant effects on lean mass wit h KO mice having greater lean mass than WT mice and conventionally housed mice

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422 having more lean mass than mice housed in running wheel cages (P<0.05). A significant difference in amount of lean mass was observed between sedentary male KO mice and male KO m ice in cages equipped with running wheels, with the exercising mice having a lower lean mass than those in the conventional cages (P<0.01). Significant differences in lean mass were evident in female mice as early as five weeks of age with female KO mice h aving greater lean masses than female WT mice (P<0.01). At no point in time during the experiment was there a difference in lean mass due to housing for the female mice. No difference in lean mass was found between male KO mice in running wheels cages and their sedentary WT litter mates. A statistical difference was seen between female WT in conventional caging and female KO mice in cages equipped with running wheels beginning at seven weeks of age (second week of treatment) (P<0.01). Body lengths Repeated measures ANOVA found that both gender and genotype had significant effects on body length (P<0.01), while housing did not. Age did have a significant effect on body length (P<0.001) (Figure 5 4). A significant difference in nose to anus body length due to genotype and gender existed at five weeks of age and persisted through the experiment with male mice longer than female mice and the WT mice being shorter than KO mice (P<0.05) (Figure 5 4). No statistical difference in body length was seen between male mice until 11 weeks of age (sixth week of treatment) when a difference was seen between male WT mice and male KO mice (P<0.05), with male KO mice being longer than their WT littermates, lasting the duration of the experiment Figure 5 4. There was a signifi cant

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423 difference in body length between male sedentary KO mice and KO mice with access to the exercise equipment starting at 11 weeks of age (P<0.05). A difference in body length due to genotype (P<0.05) between female KO and WT mice existed from five weeks of age until the end of the experiment, with KO mice possessing the longer body lengths (Figure 5 4). No significant difference was observed in female mouse body length due to housing. No differences in male KO running wheel and male WT conventional mous e body lengths were observed. A significant difference between female WT conventional and female KO running wheel emerged and persisted at nine weeks of age (fourth week of treatment) with the female KO running wheel mice being longer than the female WT se dentary mice (P<0.05). Food Intake Age, gender, genotype, and housing were all found to have statistically significant effects on food intake after repeated measures ANOVA (P<0.01). Significant interactions also existed between gender and housing, genotype and housing, and between gender, genotype and housing (P<0.05). A gender effect on food intake is seen at six weeks of age with male mice consuming significantly more food than female mice (P<0.001). Beginning at seven weeks of age a significant differen ce caused by genotype was seen with the KO mice eating more than their WT littermates (P<0.001). A statistical difference in food intake was observed between male and female KO mice was at six weeks of age (P<0.01), and between male and female WT mice begi nning at seven weeks of age (P<0.05). Similar to previously reported studies 64 and 37 sedentary male KO mice consumed more food than conventionally housed WT mice at most time points, while

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424 male mice in running wheel cages ate comparable amounts reg ardless of genotype (data not shown). For female mice significant differences in food intake were first seen at seven weeks of age (second week of treatment) due to both genotype and housing (P<0.05), with the KO mice eating more than the WT mice and the s edentary mice eating less than the exercising mice (Figure 5 5). Female WT mice in conventional caging ate significantly less than female WT mice in running wheel cages beginning at seven weeks of age (P<0.05). Female KO mice in running wheel cages ate sig nificantly more than sedentary female KO mice at nine weeks of age (fourth week of treatment) (P<0.05). Female KO mice in conventional cages ate significantly more than female WT littermates also in conventional cages starting at 7 weeks of age (P<0.001), but this difference disappeared at 11 weeks of age. Insulin Repeated measures ANOVA found that housing, gender, genotype, and age had statistically significant effects on circulating plasma insulin concentrations (P<0.05). Interactions between gender and h ousing, genotype and housing, and gender, genotype, and housing were observed (P<0.05). A difference between circulating insulin in male and female conventionally housed mice emerged at six weeks of age with male mice having higher values of circulating in sulin compared to female mice (P<0.01) (Figure 5 6). Conventionally housed KO mice also had a greater insulin concentration than WT littermates beginning at seven weeks of age (P<0.05). A statistically significant difference in plasma insulin was seen be tween sedentary and exercised male mice at six weeks of age (first week of treatment) with the male mice in the running wheel cages having lower circulating insulin concentrations

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425 than the conventionally housed male mice.(P<0.05). A genotype difference eme rges at seven weeks of age that lasted until the conclusion of the experiment with the male WT mice having lower insulin values than male KO mice (P<0.05). Plasma insulin concentrations of male KO mice allowed to exercise were significantly lower than sede ntary male KO mice beginning with the first week of treatment (six weeks of age) (P<0.05). A difference in circulating insulin levels due to genotype was seen in female mice with KO mice having a higher circulating insulin concentration starting with the s econd week of treatment and lasting until the end of the experiment (P<0.05), excluding the tenth week of age when no difference was seen. No housing effect on insulin concentration was observed at any time point for female mice. There were no significant differences in plasma insulin level between KO mice in running wheel cages or WT mice in conventional cages for either male or female mice, except during the final week of the experiment (12 weeks of age), where a difference was seen between the groups fo r both male and female mice (P<0.05). Leptin Repeated measures ANOVA revealed that age, genotype, and housing had significant effects on plasma leptin levels (P<0.001), while gender did not (P=0.069). Interactions were seen between gender and housing, geno type and housing, and gender, genotype, and housing (P<0.05). A difference in leptin levels between conventionally housed KO and WT mice was evident as early as five weeks of age and lasted the entire experiment with KO mice having the greater plasma lepti n concentrations (P<0.05) (Figure 5 6). Sedentary male mice had higher leptin levels than conventionally housed female mice at six, seven, ten, eleven, and twelve weeks of age (P<0.05).

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426 No difference in plasma leptin concentration was observed between mal e mice at five weeks of age. A genotype effect emerged at six weeks of age with male KO mice having higher circulating concentrations of leptin than male WT mice (P<0.05). Housing played a significant role with male mice starting at the first week of the e xperiment (six weeks of age) and lasting until the end of the experiment (P<0.05), excluding the fourth week of treatment (nine weeks of age), with conventionally housed male mice having higher leptin concentrations than exercising mice. Male KO mice in ru nning wheel cages had a significantly reduced plasma leptin concentration compared to the male KO mice in the conventional cages beginning with the first week of treatment (six weeks of age) (P<0.05). The plasma leptin concentration of female mice was affe cted by genotype with KO mice having greater concentrations than WT mice at all time points except six weeks of age (P<0.05). Housing had a significant affect on leptin levels with female mice in running wheel cages having lower leptin levels than sedentar y littermates at seven, eleven, and twelve weeks of age (second, sixth, and seventh weeks of treatment respectively) (P<0.05). Leptin levels for male KO mice in running wheel cages compared to conventionally housed male WT mice were not significantly diff erent until the last week of the experiment (twelve weeks of age) (P<0.05). There was no difference in leptin levels between sedentary female WT mice and female KO mice in running wheel cages at any point in time. Hypothalamic Melanocortin Gene Expression Hypothalamic neurons expressing melanocortin receptors are part of a complex network of neurotransmitters and receptors, which under normal conditions regulate energy homeostasis and food intake by integrating and signaling downstream to

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427 mediate physiolo gical changes. Inactivation of the MC4R results in changes in the signaling pathways involved in energy homeostasis in the brain. 10,37,38 To determine the effect of gender, genotype, and exercise on hypothalamic energy homeostasis pathways, hypothalamic gene expression was measured by quantitative real time PCR Specific gene targets involved in the melanocortin and energy homeostasis pathways were selected as described previously. 37 A significant increase in brain derived neurotrophic factor (BDNF) expression is seen in female mice compared to male mice (P<0.001). Significant decreases we re seen in CART and HCRT expression in female mice compared to male mice (P<0.001), with a significant increase in CART expression seen in KO animals compared to WT animals (P<0.01). A significant difference due to genotype was observed between alues of female WT and KO mice (P=0.01). A significant increase in AGRP expression was seen in female mice compared to male mice (P<0.001). There is also significant difference in the amount of AGRP expressed between genotypes, with a significant decrease in AGRP expression observed in KO mice compared to WT mice (P<0.001). POMC gene expression was significantly increased in KO mice compared to WT mice (P<0.001), however n o difference in expression level was observed between defensin like ligands have been reported to possess the ability to bind to the melanocortin receptors in vitro. 148 A significant increase in DEFB1 expression is observed in female mice compared to male mice (P<0.001) with no genotype effect seen. No sign ificant differences in hypothalamic gene expression levels

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428 due to gender or genotype were found for the MC3R, NPY, NPY1R, or PTEN genes when analyzing male and female mice in conventional cages. To determine the effect of voluntary exercise on energy homeo stasis pathway gene expression, hypothalamic gene expression for male (Figure 5 9A) and female (Figure 5 9B) WT and KO mice in both conventional and cages equipped with running wheels was analyzed, with gene expression normalized to the WT sedentary mice f or each gen (2 ) by two way ANOVA using genotype and housing. Access to running wheels for voluntary exercise resulted in a significant decrease in CART expression in both male and fema le mice (P<0.05), while genotype only had a significant effect in female mice with CART expression higher in female KO mice than their WT littermates (P<0.01). While voluntary exercise failed to cause any changes in NPY expression in female mice, there was significant genotype effect resulting in a decrease in NPY expression in male KO mice compared to male WT mice (P<0.01). Post test revealed a significant difference in NPY expression between the sedentary WT and KO male mice (P<0.05) No significant chan ges in AGRP expression due to exercise were seen in female mice, however there was a significant increase in expression in male mice allowed access to running wheels (P<0.05). Significant differences in AGRP expression existed between the genotypes in male mice for both housing groups (P<0.01). Voluntary exercise resulted in an increase in AGRP gene expression in male MC4R KO mice compared to sedentary male KO littermates (P<0.01). There was a significant housing effect seen in the male mice with POMC expre ssion levels lower in mice housed in running wheel cages compared to those in

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429 conventional housing (P<0.05). Further analysis revealed significant differences in male mice due to genotype in both the sedentary and voluntary exercise groups with KOs having a higher level of POMC expression than WT littermates in both housing conditions (P<0.05). Voluntary exercise resulted in a decrease in POMC gene expression in male MC4R KO mice compared to sedentary male KO littermates (P<0.01).There were no significant d ifferences in hypothalamic DEFB1 expression between in male mice, but exercising female mice had decreased DEFB1 expression compared to sedentary female littermates (P<0.01). Genotype and voluntary exercise did not have a significant effect on hypothalamic expression of BDNF, HCRT, MC3R, NPY1R, or PTEN when gene expression was analyzed with respect to housing and genotype by gender. Running Wheel Activity Exercise activity, measured as rotations of the running wheel apparatus, was monitored for all animals in running wheel cages with activity recorded by computer once a minute. To determine if any of the groups were hypoactive or if differences between groups was due to different levels of activity, running wheel activity, reported in average number of runni ng wheel turns per dark cycle, was measured. No significant difference in activity during the dark cycle between any of the groups housed in running wheel cages regardless of genotype or gender as shown in Figure 5 10. To determine if increased voluntary e xercise confers extra protection from the obese phenotype for MC4R KO mice, the final body weights or final fat masses for male MC4R KO mice housed in running wheel cages were plotted against the total number of wheel rotations for that mouse for the entir e experiment and a linear regression was

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430 performed to determine the slopes of the resulting lines as seen in Figure 5 11. The slopes of both lines were found not found to be significantly different from zero. Discussion Obesity is an overwhelming health care issue affecting millions of people worldwide and is a significant risk factor for many other diseases including type 2 diabetes, hypertension, and stroke. 3,143 Exercise is an accepted method of controlling body weight and reducing fat mass in bot h human and animal models. 37,62,64,144 It is also accepted that male and female mice have different growth rates and different responses to metabolic challenges. 10,36,145,146 The effects of voluntary exercise have been studied in animal models of both genetic and diet induced obesity 37,64,71,129,149 155 In Chapter 5 the effects of gender on the obesity associated with MC4R dysfunction and the results of volunta ry exercise in mice were presented. Previously it has been reported that voluntary exercise by running wheel was able to delay the onset of the obese phenotype of male MC4R KO mice as well as alter hypothalamic expression of genes important to energy home ostasis. 37,64 Similar to previousl y reported data, a marked delay in the onset of the obese phenotype of the male MC4R KO mice given access to running wheels was observed, as evidenced by lower total body weights, fat masses, and circulating factors compared to the sedentary male MC4R KO mice. As seen previously, voluntary exercise was also able to prevent the increased linear growth in male MC4R KO using both nasal anal length and lean mass as indicators. 37,64 Sedentary female MC4R KO mice experienced many of the same indicators of obesity compared to female WT litter ma tes, however the obese phenotype in female MC4R KO mice is not as severe as that seen in male MC4R KO mice. Sedentary

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431 female MC4R KO mice had increased values for body weight, body length, fat mass, lean mass, and plasma insulin, leptin, and cholesterol co mpared to conventionally housed female WT mice. Voluntary exercise was not able to prevent increases in body weight, linear growth, or lean mass in female KO mice. Circulating insulin concentrations were significantly lower after exercise except at 11 and 12 weeks of age respectively. The lack of the pronounced obese phenotype in female MC4R KO mice might be explained by the lack of hyperphagia. Female KO mice in conventional cages ate significantly more food than female WT littermates, however this differe nce disappeared at 11 weeks of age. It is therefore possible that the lack of hyperphagia is why the female KO mice did not have the same marked obese phenotype as the male KO mice, which has been linked to the hyperphagic phenotype. 38 Marie et al. demonstrated that the while increased body weight of male MC4R KO mice can be prevented by food restriction, there is still an increase i n adipose tissue compared to WT mice. 117 It was also reported that food restriction was not able to reduce body weight to WT levels in female MC4R KO mice. 117 This suggests that, while a metabolic syndrome clearly exists, male MC4R KO mice are a model of hyperphagia induced obesity and that female mice lacking the MC4R are resistant to interventions to prevent weight gain. 117 It has been reported that susceptibility or resistance to diet induced obesity (DIO) are heritraits based on studies involving selectively bred Sprague Dawley rats. 156 Taken together, these results allow f or the possibility that female mice from our colony might have gained a resistance to the hyperphagia induced obesity, especially considering the mixed background of the founder mice, offering an explanation for lower than expected food intake and body wei ghts. Additionally, the plots of body weight, body

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432 length, and lean mass over time had a different pattern in female mice compared to male mice. In the graphs of these variables for male mice the exercising MC4R KO mice are grouped with the WT mice groups, while for the plots of the female mice are grouped separately by genotype. This suggests that over a longer period of time, especially if hyperphagia resumes, that genotype might be a more important factor in influencing these variables for female mice th an housing condition. Zachwieja et al. studied the effects of voluntary exercise on adipose tissue and circulating factors in rats prone and resistant to diet induced obesity. 152 It was reported that access to runn ing wheels for voluntary exercise reduces fat mass and lowers circulating leptin by decreasing leptin mRNA expression in adipocytes and improve insulin sensitivity in rats sensitive to diet induced obesity. Fat mass and plasma leptin concentrations are ele vated in male MC4R KO mice compared to male WT mice and responded to exercise in a similar fashion as previous reported, with voluntary exercise delaying the onset of the obese phenotype. 37 Sedentary female MC4R KO mice had less body fat and circulating leptin than conventionally caged male MC4R KO for most of the experimental time points. Vo luntary exercise was able to prevent the increase in both fat mass and plasma leptin in female MC4R KO mice in the time course of the experiment as opposed to delaying it as was seen in male mice. Grove et al. studied the effects of high fat diet on the sexual dimorphisms in adipose tissue size and gene expression in male, female, and ovariectomized female mice. 146 They concluded that there is an inherent difference in gene expression between male and female mice, including that female mice might have lower level s of insulin resistance because of increased expression of genes in the insulin signaling

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433 cascade. 146 This study demonstrates that there exists a sexual dimorphism in expression levels of some genes involved in energy homeostasis and also offers an explanation for why male animals are more prone to insulin resistance than female animals. Examining levels of hypothalamic gene expression in sedentary WT and MC4R KO genotypes reveals similarities and differences between male and female mice. Female mice had lower CAR T and HCRT gene expression levels and higher BDNF gene expression levels than male mice. When comparing both male and female mice, KO mice had a lower AGRP expression level than WT littermates. Additionally, female mice (regardless of genotype) had a highe r level of AGRP expressed in the hypothalamus compared to male mice. An increase in hypothalamic POMC expression was seen in both male and female KO mice compared to WT littermates. No gender effect was seen for POMC levels, though male mice ate significan tly more than female mice at starting at six weeks of age. With a traditional understanding of the melanocortin system, an increase in antagonist concentration (AGRP) and no change in agonist concentration (POMC), should result in food intake and orexigeni c processes. In an attempt to explain the discrepancy in food intake and endogenous ligand expression levels other potential melanocortin ligands were investigated. defensin 1 gene (DEFB1) results in different coat colorations and patterns in dogs and has also been shown to be able to bind to the MC4R. 148 Expression of the DEFB1 gene was upregulated in female mice compared to male mice, though not enough to appear d efensin binding at the MC4R and more information will be required in regards to mode of action and binding

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434 site before further conclusions regarding its role in feeding behavior and energy homeostasis can be made. Voluntary exercise has been reported to af fect hypothalamic gene expression in male WT and MC4R KO mice. 37 Access to running wheel s for exercise was able to partially correct male MC4R KO expression levels of AGRP and POMC, with expression levels in exercised mice significantly different than their sedentary counterparts (P<0.01). Gene expression in female KO mice did not have the sa me response to voluntary exercise as was seen in male KO mice. While exercise was able to partially correct hypothalamic AGRP and POMC expression levels in male KO mice, this was not seen for the exercised female MC4R KOs. Expression of DEFB1 was significa ntly lower defensin 1 expression might be involved with energy homeostasis regulation. Expression levels of NPY in male mice did not match with earlier published data, but ca n be explained by differences in time of sacrifice. 37 Male mice in this experiment were sacrificed between 0900 and 1200, while mice in our previously published experiment were sacrificed between 1230 and 1700. 37 It has been reported that amount of NPY immunoreactivity in the hypothalamus changes with time of day, with peaks in NPY levels at the transition between dark and light periods. 157,158 These data allows for the possibility that a difference in time of sacrifice is responsible for differences in NPY expression between this and previous experim ents. 37 The fact that over the course of the experiment that there was no significant d ifference in total amount of exercise between either male or female mice or the genotypes means that any benefit gained by the voluntary exercise would have been

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435 gained by all groups. When final body weight or final fat mass was plotted against total numbe r of running wheel rotations the slope of the line fitted to the data did not have a slope significantly different from zero (Figure 5 11). This data suggests that for male MC4R KO mice there is no added benefit to an increased amount of exercise. This is contrary to the findings shown in Chapter 3 (Figures 3 150 and 3 151), however since the data presented in Figure 5 11 is from a larger group of mice, it can be assumed that the effect seen in Chapter 3 was in part due to small group size. One of the pote ntial explanations for differences in melanocortin is the role of the estradiol in feeding behavior. 159 Gao et al. reported that administration of E2 causes an increase in cFos expression in POMC neurons indicating an increase in gene transcription. 160 This coupled with the fact that female MC4R KO mice respond differently to MC4R inactivation and voluntary exercise suggest that gender has a significant effect on energy balance and the changes broug ht around by voluntary exercise.

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436 Figure 5 1. Weekly average body weights for male (A) and female (B) MC4R mice in conventional and running wheel cages. Repeated measures ANOVA determined that age, genotype, housing, and gender all had significant effe cts on body weight during the experimental timeline (P<0.01).

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437 Figure 5 2. Fat mass was measured by quantitative MRI for male (A) and female (B) MC4R mice in conventional and running wheel cages. Repeated measures ANOVA determined that age, genotype, and housing all had significant effects on fat mass during the experimental timeline (P<0.01).

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438 Figure 5 3. Lean Mass measured by quantitative MRI for male (A) and female (B) MC4R mice. Repeated measures ANOVA determined that age, genotype, and gender all h ad significant effects on fat mass during the experimental timeline (P<0.001).

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439 Figure 5 4. Linear growth was measured weekly by monitoring nasal anal length for male and female MC4R mice in sedentary and exercise treatment groups. Repeated measures ANOV A determined that age, genotype, and gender all had significant effects on fat mass during the experimental timeline (P<0.01).

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440 Figure 5 5. Female MC4R mouse food intake in conventional and running wheel cages. Repeated measures ANOVA determined that age, genotype, housing, and gender all had significant effects on food intake during the experimental timeline (P<0.01).

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441 Figure 5 6. Mean plasma insulin concentration for male (A) and female (B) MC4R mice in conventional and running wheel cages. Repeated me asures ANOVA determined that age, genotype, housing, and gender all had significant effects on plasma insulin concentrations during the experimental timeline (P<0.05).

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442 Figure 5 7. Average circulating leptin concentration for male (A) and female (B) MC4 R mice in conventional and running wheel cages. Repeated measures ANOVA determined that age, genotype, and housing all had significant effects on fat mass during the experimental timeline (P<0.001).

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443 Figure 5 8. Hypothalamic gene expression of male and f emale MC4R mice in conventional cages normalized to male WT mouse group. A significant gender effect is indicated by (A), while a significant genotype effect is indicated by (B).

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444 Figure 5 9. Hypothalamic gene expression for male and female MC4R mice. H ypothalamic gene expression for male mice (A) was normalized to male WT Conv group gene expression levels. Hypothalamic gene expression for female mice (B) was normalized to female WT Conv group gene expression levels in conventional and running wheel cage s. A significant genotype effect is indicated by (G), while a significant housing effect is indicated by (H).

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445 Figure 5 10. Average number of running wheel turns per dark cycle for male and female MC4R mice housed in running wheel cages. No significant differences due to genotype or housing were found by repeated measures ANOVA.

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446 Figure 5 11. Correlations of body weights and fat masses to total amount of exercise performed. Linear regression plots of body weights versus running wheel activity (A) and f at mass versus running wheel activity (B) for male MC4R KO mice in running wheel cages.

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447 CHAPTER 6 CONCLUSION Weight gain and obesity are the result of an imbalance in energy intake and expenditure. The melanocortin system has been identified as having a m ajor role in food intake and energy homeostasis through many experiments involving feeding studies, transgenic and KO mice, and genome sequencing in obese human patients. 8 10,12,23,29,34 39,44 48,63,64,80 Generation of MC3R, MC4R, and double receptor KO mice have provided animal models for st udying the obesity and metabolic syndromes similar to the phenotypes seen in human patients. 8 10,45 48 Voluntary exercise has been shown to be an effective method for delaying the onset of the obese phenotype of male MC4R KO mice, as well as resulting in changes in the expression levels of genes involved in energy homeostasis in the hypothalamus. 37,63,64 This work presented and discussed the results from experiments in Chapter 3, examining the effects of voluntary exercise on the phenotypes in MC3R KO, MC4R KO, and DKO mice along with the appropriate control groups. Mice lacking the MC3R housed in conventional cages were not phenotypically or physiologically different from MC3R WT mice other than having elevated plasma insulin levels (Figure 6 1). Allowing the MC3R KO mice to exercise on running wheels for eight weeks prevented the increase in circulating insulin, resulting in plasma insulin c oncentrations that were not statistically different than MC3R WT mice in running wheel cages (Figure 6 1). Though conventionally MC3R KO mice did not have significantly higher adiposities than MC3R WT mice as previously reported, voluntary exercise did res ult in a significant decrease in fat mass in MC3R KO mice. 8,9 The difference in circulating plasma leptin seen between exercising and sedentary MC3R KO mice (Figure 3 137) mirrors the decrease

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448 seen in fat mass. These findings indicating that voluntary exercise might be beneficial to individuals with relatively normal phenotypes. The MC4R KO mice in conventional cages had increased body weights, fat masses, lean masses, body lengths, and plasma concentrations of both leptin and insulin (Figure 6 2). 10,37 As described previously, when allowed to exercise, MC4R KO mice had values for body weights, fat masses, body lengths, and plasma insulin and leptin conc entrations that were not significantly different from MC4R WT mice (Figure 6 2). 37,64 Voluntary exercise was unable to prevent increases in lean mass in MC4R KO mice compared to MC4R WT mice also given access to running wheels in the home cage environment (Figur e 6 2). Increased voluntary exercise, as measured by total number of turns of the running wheel, resulted in greater decreases in fat mass in a small group of male MC4R KO mice (Figure 3 151), but not when a larger group was analyzed (Figure 5 11). Convent ionally housed DKO mice possessed increased body weights, fat masses, lean masses, body lengths, food consumption, and plasma insulin and leptin concentrations as summarized in Figure 6 3. 9 Eight weeks of volunt ary exercise on running wheels did not prevent the obese phenotype or metabolic disorder seen in the DKO mice with body weights, fat masses, food intake, and plasma leptin concentrations significantly higher than those of the WT mice (Figure 6 3). It is, h owever, important to note, that while voluntary exercise did not prevent the onset of the obese phenotype, it did result in a significant reduction of both body weight and fat mass in DKO mice compared to sedentary DKO mice (Figures 3 19 and 3 38). This wo uld indicate that while voluntary exercise on running wheels was not able to fully prevent the increase in body weight and adiposity, there was still some benefit to the DKO mice achieved through exercise. Male DKO mice ran

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449 significantly less than MC3R WT mice during the final week of the experiment (Figure 6 3), though it is not possible to say whether this was the result of the genetic deletion of the MC3R and MC4R or the increased body weights of these mice resulting in decreased mobility. This could bet ter be addressed though the use of a system that could monitor ambulatory activity to determine if the DKO mice are less mobile or simply exercising less than the MC3R WT group. As discussed in Chapter 3, voluntary exercise resulted in lower fat masses in male MC3R KO, MC4R KO, and DKO mice at 13 weeks of age compared to conventionally housed mice of the same genotypes after just eight weeks of access to running wheel equipment (Figure 3 38). By using the general model presented in Figure 1 1 it is possibl e to make assumptions about the reason that increases in adiposity and total body weight were not seen in the MC4R KO mice. Simplifying the balance between energy input and energy expenditure to only consider food intake and voluntary exercise as sources allows for analysis of parameters that were measured in these experiments. Food intake for MC4R KO mice in running wheel cages was equal to or greater than mice in conventional cages, meaning that a decrease in energy intake was not responsible for the cha nges in body size and composition. This leaves the alternative solution that the prevention of the increase in fat mass and body weight was the result of increased energy expenditure by voluntary exercise on running wheels. Based on the design of this expe riment, it is not possible to identify the mechanism that resulted in different adiposities in conventionally housed MC4R KO mice compared to MC4R KO mice allowed to exercise. It is possible that the increase in energy expenditure resulted in fewer calori es that needed to be stored as lipids in adipose

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450 tissue. The lack of a need for extensive storage of excess calories in white adipose tissue might also reduce proliferation of adipocytes, further contributing the difference in adipose tissue mass seen betw een the exercising and sedentary KO mice groups. In addition to voluntary exercise having significant effects on the phenotypes of MC3R KO, MC4R KO, and DKO mice, it also resulted in changes in expression levels of genes involved in homeostatic processes i n the hypothalamus. Taqman RT PCR assay did not find any significant differences in hypothalamic gene expression for sedentary male MC3R KO mice compared to MC3R WT mice in conventional cages (Figure 6 4). Decreases in LEPR and PRKAA1 gene expression were seen in the hypothalamus of male MC3R KO mice in running wheel cages compared to exercising MC3R WT mice (Figure 6 4). The POMC gene was upregulated in male MC4R KO mice compared to MC4R WT mice in both conventional and running wheel cages (Figure 6 5). T he SOCS3 gene was upregulated in sedentary MC4R KO mice, but was not significantly different from MC4R WT levels when the MC4R KO mice were allowed to exercise (Figure 6 5). No changes in hypothalamic gene expression were seen in sedentary DKO mice compare d to the MC3R WT and MC4R WT mice also in conventional cages (Figure 6 6). Being allowed to exercise on running wheels caused significant changes to expression of many genes in the hypothalamus of male DKO mice. The AGRP, CART, HCRT, POMC, and SOCS3 genes were all upregulated in exercising DKO mice compared to at least one of the WT mouse groups (Figure 6 6). Even though the effects of AGRP and POMC peptides oppose one another, because there are no MC3R or MC4R in the hypothalamus of the DKO mice, it is unl ikely that

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451 these peptides had an effect on food intake. The NPY gene was down regulated in exercising male DKO mice compared to both WT mouse groups (Figure 6 6). In addition to the hypothalamus, expression levels of genes involved in energy homeostasis we re also measured in the liver. No changes in hepatic gene expression were seen in male MC3R KO mice in either conventional or running wheel cages compared to MC3R WT mice. Shifts and changes in gene expression in the liver pathways were seen for MC4R KO mi ce compared to MC4R WT mice. Gene expression for GLUT2, PYGL, FBP1, CPT1, CPT2, and LIPE were all upregulated in the livers of sedentary male MC4R KO mice compared to MC4R WT mice (Figure 6 8). The MC4R KO mice allowed to exercise for eight weeks on runnin g wheels did not have increases in expression for these genes compared to MC4R WT mice (Figure 6 8). Voluntary exercise resulted in the down regulation of the PFKL and FASN genes in the livers of MC4R KO mice (Figure 6 8). There were also shifts in the rat io of gene expression of some of the homeostatic pathways in the livers of male MC4R KO mice. There was a shift towards genes in the pathways for glucose release with a relative increase in G6PC3 compared to GCK in MC4R KO mice compared to MC4R WT mice for both conventional and running wheel cages (Figure 6 8). There were also changes in gene expression suggesting shifts towards glycogen breakdown and triglyceride break down in sedentary MC4R KO mice compared to MC4R WT mice (Figure 6 8). Finally, there was also an upregulation of the gluconeogenesis pathway relative to the glycolysis pathway in MC4R KO mice compared to MC4R WT mice for both conventional and running wheel housed mice (Figure 6 8). Taken together, these changes suggest an increase in the live

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452 and triglycerides), and increase hepatic glucose production and output through increases in gluconeogenesis and glucose 6 phophate dephosphorylation (Figure 6 8). Changes in hepatic gene expression were the most extensive in male DKO mice compared to either WT group (Figure 6 9). Increases in GCK, PFKL, CPT1, and DGAT2 were seen in sedentary DKO mice, but not exercising DKO mice, compared to either MC3R WT or MC4R WT mice (Figure 6 9). A decrease in GYS2 liv er expression was seen in sedentary, but not exercising, DKO mice compared to WT controls (Figure 6 9). The PYGL, FASN, CPT2, and LIPE genes were all upregulated in the livers of male DKO mice compared to either MC3R WT or MC4R WT mice in both conventiona l and running wheel cages (Figure 6 9). Finally, changes in gene expression suggesting a shift were seen in some metabolic pathways in the livers of DKO mice including a shift towards increased glycogen breakdown ability, a shift towards increased gluconeo genic potential, and a shift towards fatty acid synthesis (Figure 6 9). Overall, there appears to be a trend of an increase glycogen breakdown and fatty acid synthesis in the livers of male DKO mice, suggesting that these mice might preferentially store ex cess calories as fat rather than glycogen. Finally, skeletal (gastrocnemius) muscle was analyzed to determine the effects of genotype and housing on gene expression in a tissue involved with movement (critical for voluntary exercise). An upregulation in G CK was seen in conventionally house MC3R KO mice compared to similarly housed MC3R WT mice (Figure 6 10). No differences in skeletal muscle gene expression were seen between MC4R KO and MC4R WT in either housing condition with the exception of a shift towa rds an increase in the glycogen synthesis pathway in MC4R KO mice in RW cages (Figure 6 11). The

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453 trend of an increase in glycogen synthesis potential was also seen in the muscle of male DKO mice compared to WT mice in both running wheel and conventional ca ges (Figure 6 12). Upon further examination, it becomes evident that many more changes in gene expression levels occurred in the livers than in the skeletal muscle of the experimental mice. This relationship is most easily seen when looking at the gene ex pression results for the DKO mice (Figures 6 9 and 6 12). The large number of changes in gene homeostasis. It was surprising that more differences were not seen in ske letal muscle, but this might be explained by the fact that any changes in gene expression were acute (occurring during or immediately after a bout of exercise), and that by the time sacrifice and dissection occurred, gene expression in the gastrocnemius ha d returned to normal levels. It was also apparent that fewer changes in peripheral gene expression were observed in the male MC3R KO mice compared to either the MC4R KO or DKO mice. This could be because of the relatively normal phenotype of the MC3R KO mi ce compared to MC3R WT mice. The MC3R KO mice used as part of the gene expression study had similar body weights (Figure 3 10) and fat masses that were not significantly different from MC3R WT mice (Figure 3 29). It is possible that increasing the number o f MC3R mice in each group would reveal small, yet significant changes, however the lack of significant metabolic changes in MC3R KO mice is a logical explanation for the reduced number of changes at the gene expression level. Another tissue that would be interesting to study the gene expression profile to supplement the data collected in the experiments described in this work is white

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454 adipose tissue. Based on gene expression data from the liver and muscle, it would be expected that few changes in gene expr ession would be seen in the white adipose tissue of MC3R KO mice due to the subtly of the metabolic disorder they experience. Looking at the phenotypes of MC4R KO mice in conventional and running wheel cages, voluntary exercise prevents the onset of the ob ese phenotype and its associated increase of adiposity, keeping white adipose tissue mass at levels similar to WT mice. Therefore, it would be expected that gene expression in adipose tissue of exercising MC4R KO mice would be very similar to gene expressi on in exercising MC4R WT mice. It would be expected to see an increase in the fatty acid storage potential of the adipocytes with increases predicted for the FASN, DGAT1, and DGAT2 genes for the MC4R KO and DKO mice in conventional cages. It is also possib le that in the increased adiposity of the sedentary MC4R KO mice hypoxic zones occur, resulting in macrophage infiltration, causing the release of cytokines TNF 6. Both of these cytokines have been shown to be able to alter the insulin signaling pa thway. 59,161 Since the adipose tissue mass remains relatively small in exercising MC4R KO mice, it is unlikely that hypoxic zones would form, preventing large scale macrophage infiltration and release of pro inflammatory cytokines. Generally, one would expect an upregulation of fatty acid synthesis and storage genes to acc ommodate the excess calories available to the MC4R KO mice. It is also likely that an upregulation in adipocyte proliferation factors would be seen in sedentary MC4R KO mice to increase adipocyte number to accommodate increased fatty acid storage needs. It would be expected that sedentary DKO mice would have similar gene expression pattern in their white adipose tissue to MC4R KO mice also in conventional cages, since DKO mice also have greatly

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455 increased adipose tissue mass. It is difficult to predict the g ene expression profile of white adipose tissue for exercising DKO mice considering they still have increase fat mass compared to both WT groups in RW cages (Figure 3 29), yet have a decreased adiposity compared to sedentary DKO mice as shown in Figure 3 38 The decreased fat mass implies that exercising DKO mice definitely benefit from voluntary running on wheels, however, the exercising DKO mice possessed fat masses that appear to be near to sedentary MC4R KO levels. It would not be surprising if DKO mice in RW cages had increased expression of genes involved in fatty acid synthesis and storage since they do have an increase in fat mass. It is also possible that exercising DKO mice would have hypoxic areas in their adipose tissue mass (unless exercise incre ased blood supply through the white adipose tissue), which could result in increases in pro inflammatory cytokine gene expression might be seen. The effect of gender on the phenotype of MC4R KO mice and on the effects of voluntary exercise on MC4R KO mice were studied and presented in Chapter 5. Gender had a significant effect on body weight, lean mass, body length, food intake, and plasma insulin concentrations in MC4R KO and MC4R WT mice. The lack of hyperphagic behavior was likely the reason for the dimi nished obese phenotype of the female MC4R KO mice. Conventionally housed female mice with or without mutations resulting the deletion of the MC4R, also had different hypothalamic gene expression profiles than sedentary male mice (Figure 5 8). Future experi ments investigating the phenotypes of the central melanocortin KO mice will be able to include additional techniques to study the effects of voluntary exercise. Incorporation of calorimetric cages from TSE systems will allow for indirect

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456 calorimetry measur ements of experimental mice. Indirect calorimetry will allow for the determination of whether the mice are preferentially using fat or carbohydrates as energy sources. Treadmills might be used as alternative methods for exercising the central melanocortin receptor KO mice, allowing for a more accurate measurement of exercise duration and intensity to better understand the minimum amount of exercise required for these mice to benefit from exercise. Use of treadmills would also allow for the investigation of the differences between chronic and acute exercise, particularly on the concentrations of circulating factors (i.e. IL 6) thought to be involved in the propagation of the effects of exercise. More advanced methods, such as whole body glucose or insulin cla mps, for testing glucose and insulin homeostasis and signaling could be used to better understand the effects of voluntary exercise on these processes in central melanocortin receptor knockout mice. Fasting could be done prior to blood draws for experiment al mice to provide for more accurate measurements. Previous studies that used MC4R KO mice as models of obesity have varied in the use of a fast prior to blood draws, with Huszar and Haskell Luevano not fasting mice prior to blood draws, but both Albarado and Sutton fasting mice before blood draw. 10,36,37,39 In 2010, the NIH Mouse Metabolic Phenotyping Center Consortium published a review article providing suggested guidelines for future experiments studying glucose metabolism in experimental mic e. 162 Among the discussed material, a short fast (five to six hours) was suggested to provide a baseline metabolic state between the mice. 162 Of course fasting also effects the melanocortin signaling pathway, meaning it might be necessary to choose between which parameters are examined in a particular experiment. 122,136 Another change that would improve the experimental design would be to draw blood

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457 during a smaller window of time. In the experiments presented in Chapters 3 and 5, blood was drawn towards the end of the light cycle. However, s ince some experiments involved large number of animals, in order to complete all bloods draws prior to lights out the daily measurements were started four to five hours before the beginning of the dark cycle. It would be best to try and concentrate all blo od draws to a smaller time point, even if it means doing fewer mice per day and doing blood draws each day of the week. Similarly, scheduling of the sacrifice and tissue collection could also be improved by working in the hour before lights out and spreadi ng the final measurements and sacrificing over several days. Finally, handling the mice prior to the start of experiments would also decrease animal stress resulting from being handled, in theory reducing the contribution of stress to fluctuations in blood glucose levels. The results presented in this work support the hypothesis that voluntary exercise is capable of delaying the onset of obesity in mice lacking functional central melanocortin receptors. Additionally, evidence was found to support the hypoth esis that genotype and voluntary exercise influence both peripheral and central expression of genes involved in energy homeostasis at the transcriptional level. Advances in research examining the melanocortin pathway has led to a better understanding of th has been linked to excitation of parasympathetic neurons, connecting central melanocortin signaling to changes in peripheral tissues. 163 Evidence that brain derived neurotrophic factor (BDNF) is part of the downstream melanocortin signaling pathway, responsible for modulation of food intake, has contributed to a better understanding of how melanocortin signaling affects changes in food intake. 164,165 As technology and

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458 understanding of the melanocortin system signaling pathways expand, advances in the field of voluntary exercise will be able to be made using central melanocortin receptor KO mice as models.

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459 Figure 6 1. Figure summarizing the effects of genotype and exercise on MC3R KO mi ce compared at 13 weeks of age (after 8 weeks of exercise) to MC3R WT mice.

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460 Figure 6 2. Figure summarizing the effects of genotype and exercise on MC4R KO mice compared at 13 weeks of age (after 8 weeks of exercise) to MC4R WT mice.

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461 Figure 6 3. Fig ure summarizing the effects of genotype and exercise on DKO mice compared at 13 weeks of age (after 8 weeks of exercise) to either MC3R WT or MC4R WT mice.

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462 Figure 6 4. Figure summarizing the effects of genotype and exercise on hypothalamic gene express ion in male MC3R KO mice.

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463 Figure 6 5. Figure summarizing the effects of genotype and exercise on hypothalamic gene expression in male MC4R KO mice.

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464 Figure 6 6. Figure summarizing the effects of genotype and exercise on hypothalamic gene expression in male DKO mice.

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465 Figure 6 7. Figure summarizing the effects of genotype and exercise on hepatic gene expression in male MC3R KO mice.

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466 Figure 6 8. Figure summarizing the effects of genotype and exercise on hepatic gene expression in male MC4R KO mice.

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467 Figure 6 9. Figure summarizing the effects of genotype and exercise on hepatic gene expression in male DKO mice.

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468 Figure 6 10. Figure summarizing skeletal muscle gene expression in male MC3R KO mice.

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469 Figure 6 11. Figure summarizing skeleta l muscle gene expression in male MC4R KO mice.

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470 Figure 6 12. Figure summarizing skeletal muscle gene expression in male DKO mice.

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471 APPENDIX: ADDITIONAL INFORMATION ABOUT EX PERIMENTAL MICE Tables A 1 and A 2 provide additional information for the male m ice in the experiments presented in Chapters 3 and 4. A 3 presents additional information for the male and female mice from experiment RW 11 presented in Chapter 5. Tables A 3 through A 11 present the raw data from the mice presented in Chapters 3 and 4.

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472 Table A 1. Details for male mice that were part of the experiments presented in Chapters 3 and 4. Tag Number Genotype Housing Gender Experiment Date of Birth Date of Death Final Body Weight 11316 MC3R WT Conventional Male MC3RKO RW 1 09/18/2007 12/17/200 7 22.10 11328 MC3R WT Conventional Male MC3RKO RW 1 09/13/2007 12/17/2007 21.17 11334 MC3R WT Conventional Male MC3RKO RW 1 12/21/2007 04/01/2008 21.54 11336 MC3R WT Conventional Male MC3RKO RW 1 12/23/2007 04/01/2008 21.66 11293 MC3R WT Running Wheel Male MC3RKO RW 1 12/23/2007 04/01/2008 21.65 11306 MC3R WT Running Wheel Male MC3RKO RW 1 05/20/2009 08/29/2009 23.36 11321 MC3R WT Running Wheel Male MC3RKO RW 1 05/20/2009 08/29/2009 22.89 11332 MC3R WT Running Wheel Male MC3RKO RW 1 05/20/2009 08/29/ 2009 21.08 11340 MC3R WT Running Wheel Male MC3RKO RW 1 05/21/2009 08/29/2009 18.05 8305 MC4R WT Conventional Male RW 11 09/17/2007 12/17/2007 26.37 8437 MC4R WT Conventional Male RW 11 09/17/2007 12/17/2007 20.22 8764 MC4R WT Conventional Male RW 12 0 9/24/2007 12/17/2007 23.90 8795 MC4R WT Conventional Male RW 12 12/25/2007 04/01/2008 21.90 8816 MC4R WT Conventional Male RW 12 12/25/2007 04/01/2008 21.34 8766 MC4R WT Running Wheel Male RW 12 05/21/2009 08/29/2009 23.10 8777 MC4R WT Running Wheel Ma le RW 12 05/18/2009 08/29/2009 23.70 8813 MC4R WT Running Wheel Male RW 12 05/20/2009 08/29/2009 22.95 8841 MC4R WT Running Wheel Male RW 12 05/20/2009 08/29/2009 21.66

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473 Table A 1. Continued. Tag Number Genotype Housing Gender Experiment Date of Birth Date of Death Final Body Weight 11286 MC3R KO Conventional Male MC3RKO RW 1 05/20/2009 08/30/2009 20.75 11294 MC3R KO Conventional Male MC3RKO RW 1 04/04/2008 07/12/2008 19.39 11297 MC3R KO Conventional Male MC3RKO RW 1 04/02/2008 07/12/2008 26.62 1130 9 MC3R KO Conventional Male MC3RKO RW 1 04/02/2008 07/12/2008 23.28 11333 MC3R KO Conventional Male MC3RKO RW 1 04/23/2008 08/04/2008 21.60 11278 MC3R KO Running Wheel Male MC3RKO RW 1 04/23/2008 08/04/2008 19.09 11290 MC3R KO Running Wheel Male MC3RKO RW 1 12/21/2007 04/01/2008 20.10 11292 MC3R KO Running Wheel Male MC3RKO RW 1 12/22/2007 04/01/2008 19.73 11303 MC3R KO Running Wheel Male MC3RKO RW 1 12/23/2007 04/01/2008 18.98 11335 MC3R KO Running Wheel Male MC3RKO RW 1 12/23/2007 04/01/2008 19.64 8263 MC4R KO Conventional Male RW 11 05/18/2009 08/29/2009 37.08 8297 MC4R KO Conventional Male RW 11 05/18/2009 08/29/2009 32.66 8407 MC4R KO Conventional Male RW 11 05/21/2009 08/29/2009 39.47 8877 MC4R KO Conventional Male RW 12 05/20/2009 08/29/2009 34.80 8882 MC4R KO Conventional Male RW 12 05/20/2009 08/29/2009 37.54 8189 MC4R KO Running Wheel Male RW 11 09/14/2007 12/17/2007 26.01 8754 MC4R KO Running Wheel Male RW 12 12/21/2007 04/01/2008 25.37 8847 MC4R KO Running Wheel Male RW 12 12/23/2007 04/01/2008 25.60 8854 MC4R KO Running Wheel Male RW 12 12/23/2007 04/01/2008 26.86 8887 MC4R KO Running Wheel Male RW 12 12/25/2007 04/01/2008 27.75

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474 Table A 1. Continued. Tag Number Genotype Housing Gender Experiment Date of Birth Date of Death Final Body Weight 9320 DKO Conventional Male DKO RW 2 05/17/2009 08/30/2009 43.19 9335 DKO Conventional Male DKO RW 2 05/21/2009 08/30/2009 43.16 9337 DKO Conventional Male DKO RW 2 05/18/2009 08/30/2009 45.42 9566 DKO Conventional Male RW 13 05/18/2009 08/ 30/2009 46.18 9568 DKO Conventional Male RW 13 05/21/2009 08/30/2009 42.43 9569 DKO Running Wheel Male RW 13 04/23/2008 08/04/2008 37.90 9329 DKO Running Wheel Male DKO RW 2 04/05/2008 07/12/2008 26.22 9330 DKO Running Wheel Male DKO RW 2 04/05/2008 07 /12/2008 31.61 9563 DKO Running Wheel Male RW 13 04/23/2008 08/04/2008 28.32 9571 DKO Running Wheel Male RW 13 04/28/2008 08/04/2008 33.00

PAGE 475

475 Table A 2. Details for male MC3R mice that were part of the expanded statistical analysis presented in Chapter 3 Tag Number Genotype Housing Gender Experiment Date of Birth Date of Death Final Body Weight 11286 MC3R KO Conventional Male MC3RKO RW 1 5/21/2009 8/29/2009 20.75 11294 MC3R KO Conventional Male MC3RKO RW 1 5/18/2009 8/29/2009 19.39 11295 MC3R KO Conve ntional Male MC3RKO RW 1 5/18/2009 8/29/2009 18.94 11297 MC3R KO Conventional Male MC3RKO RW 1 5/20/2009 8/29/2009 26.62 11309 MC3R KO Conventional Male MC3RKO RW 1 5/20/2009 8/29/2009 23.28 11312 MC3R KO Conventional Male MC3RKO RW 1 5/20/2009 8/29/200 9 22.45 11313 MC3R KO Conventional Male MC3RKO RW 1 5/20/2009 8/30/2009 21.68 11317 MC3R KO Conventional Male MC3RKO RW 1 5/20/2009 8/30/2009 19.37 11320 MC3R KO Conventional Male MC3RKO RW 1 5/21/2009 8/30/2009 20.72 11324 MC3R KO Conventional Male MC 3RKO RW 1 5/21/2009 8/30/2009 19.97 11333 MC3R KO Conventional Male MC3RKO RW 1 5/20/2009 8/30/2009 21.60 11341 MC3R KO Conventional Male MC3RKO RW 1 5/20/2009 8/30/2009 21.22 11342 MC3R KO Conventional Male MC3RKO RW 1 5/21/2009 8/30/2009 20.60 11278 MC3R KO Running Wheel Male MC3RKO RW 1 5/17/2009 8/30/2009 19.09 11285 MC3R KO Running Wheel Male MC3RKO RW 1 5/21/2009 8/30/2009 21.45 11290 MC3R KO Running Wheel Male MC3RKO RW 1 5/21/2009 8/30/2009 20.10 11292 MC3R KO Running Wheel Male MC3RKO RW 1 5 /18/2009 8/30/2009 19.73 11301 MC3R KO Running Wheel Male MC3RKO RW 1 5/18/2009 8/30/2009 20.62 11303 MC3R KO Running Wheel Male MC3RKO RW 1 5/18/2009 8/30/2009 18.98 11308 MC3R KO Running Wheel Male MC3RKO RW 1 5/20/2009 8/30/2009 18.54 11310 MC3R KO Running Wheel Male MC3RKO RW 1 5/20/2009 8/30/2009 19.63 11315 MC3R KO Running Wheel Male MC3RKO RW 1 5/20/2009 8/30/2009 19.99 11322 MC3R KO Running Wheel Male MC3RKO RW 1 5/21/2009 8/30/2009 20.18 11335 MC3R KO Running Wheel Male MC3RKO RW 1 5/21/2009 8/30/2009 19.64 11338 MC3R KO Running Wheel Male MC3RKO RW 1 5/21/2009 8/30/2009 22.67

PAGE 476

476 Table A 2. Continued. Tag Number Genotype Housing Gender Experiment Date of Birth Date of Death Final Body Weight 11274 MC3R WT Conventional Male MC3RKO RW 1 5/17/2 009 8/29/2009 22.89 11276 MC3R WT Conventional Male MC3RKO RW 1 5/17/2009 8/29/2009 19.91 11316 MC3R WT Conventional Male MC3RKO RW 1 5/20/2009 8/29/2009 22.10 11327 MC3R WT Conventional Male MC3RKO RW 1 5/20/2009 8/29/2009 25.03 11328 MC3R WT Conventi onal Male MC3RKO RW 1 5/20/2009 8/29/2009 21.17 11334 MC3R WT Conventional Male MC3RKO RW 1 5/20/2009 8/29/2009 21.54 11336 MC3R WT Conventional Male MC3RKO RW 1 5/21/2009 8/29/2009 21.66 11279 MC3R WT Running Wheel Male MC3RKO RW 1 5/17/2009 8/29/2009 18.91 11282 MC3R WT Running Wheel Male MC3RKO RW 1 5/18/2009 8/29/2009 24.27 11293 MC3R WT Running Wheel Male MC3RKO RW 1 5/18/2009 8/29/2009 21.65 11306 MC3R WT Running Wheel Male MC3RKO RW 1 5/18/2009 8/29/2009 23.36 11321 MC3R WT Running Wheel Male MC3RKO RW 1 5/21/2009 8/29/2009 22.89 11332 MC3R WT Running Wheel Male MC3RKO RW 1 5/20/2009 8/29/2009 21.08 11340 MC3R WT Running Wheel Male MC3RKO RW 1 5/20/2009 8/29/2009 18.05 11344 MC3R WT Running Wheel Male MC3RKO RW 1 5/21/2009 8/29/2009 17.17

PAGE 477

477 Table A 3. Details for male and female MC4R mice from RW 11 presented in Chapter 5. Tag Number Genotype Housing Gender Date of Birth Date of Death Final Body Weight 8263 MC4R KO Group Conventional Male 9/17/2007 12/17/2007 37.08 8264 MC4R KO Group Conve ntional Male 9/17/2007 12/17/2007 36.78 8272 MC4R KO Group Conventional Male 9/17/2007 12/17/2007 33.69 8273 MC4R KO Group Conventional Male 9/17/2007 12/17/2007 33.70 8295 MC4R KO Group Conventional Male 9/17/2007 12/17/2007 36.91 8297 MC4R KO Group C onventional Male 9/17/2007 12/17/2007 32.66 8403 MC4R KO Group Conventional Male 9/24/2007 12/17/2007 37.38 8407 MC4R KO Group Conventional Male 9/24/2007 12/17/2007 39.47 8189 MC4R KO Running Wheel Male 9/14/2007 12/17/2007 26.01 8351 MC4R KO Running Wheel Male 9/17/2007 12/17/2007 28.48 8278 MC4R KO Running Wheel Male 9/17/2007 12/17/2007 28.80 8303 MC4R KO Running Wheel Male 9/18/2007 12/17/2007 36.01 8274 MC4R KO Running Wheel Male 9/17/2007 12/17/2007 26.10 8359 MC4R KO Running Wheel Male 9/20/ 2007 12/17/2007 23.77 8449 MC4R KO Running Wheel Male 9/25/2007 12/17/2007 20.34 8461 MC4R KO Running Wheel Male 9/14/2007 12/17/2007 34.91 8468 MC4R KO Running Wheel Male 9/14/2007 12/17/2007 25.06 8304 MC4R WT Group Conventional Male 9/18/2007 12/17/ 2007 26.62 8305 MC4R WT Group Conventional Male 9/18/2007 12/17/2007 26.37 8436 MC4R WT Group Conventional Male 9/13/2007 12/17/2007 22.29 8437 MC4R WT Group Conventional Male 9/13/2007 12/17/2007 20.22 8443 MC4R WT Group Conventional Male 9/14/2007 12 /17/2007 20.82 8466 MC4R WT Group Conventional Male 9/14/2007 12/17/2007 21.88 8323 MC4R WT Group Conventional Male 9/17/2007 12/17/2007 22.41 8324 MC4R WT Group Conventional Male 9/17/2007 12/17/2007 20.44

PAGE 478

478 Table A 3. Continued. Tag Number Genotype H ousing Gender Date of Birth Date of Death Final Body Weight 8319 MC4R WT Running Wheel Male 9/17/2007 12/17/2007 20.60 8344 MC4R WT Running Wheel Male 9/17/2007 12/17/2007 23.04 8353 MC4R WT Running Wheel Male 9/17/2007 12/17/2007 26.81 8277 MC4R WT Ru nning Wheel Male 9/17/2007 12/17/2007 22.87 8460 MC4R WT Running Wheel Male 9/14/2007 12/17/2007 26.08 8469 MC4R WT Running Wheel Male 9/17/2007 12/17/2007 21.92 8179 MC4R KO Group Conventional Female 9/14/2007 12/17/2007 23.15 8181 MC4R KO Group Conve ntional Female 9/14/2007 12/17/2007 23.99 8280 MC4R KO Group Conventional Female 9/17/2007 12/17/2007 24.10 8282 MC4R KO Group Conventional Female 9/17/2007 12/17/2007 17.61 8291 MC4R KO Group Conventional Female 9/15/2007 12/17/2007 26.50 8292 MC4R KO Group Conventional Female 9/15/2007 12/17/2007 24.63 8356 MC4R KO Group Conventional Female 9/20/2007 12/17/2007 23.72 8357 MC4R KO Group Conventional Female 9/20/2007 12/17/2007 29.30 8268 MC4R KO Running Wheel Female 9/17/2007 12/17/2007 26.35 8289 MC4R KO Running Wheel Female 9/15/2007 12/17/2007 21.37 8299 MC4R KO Running Wheel Female 9/17/2007 12/17/2007 25.12 8314 MC4R KO Running Wheel Female 9/17/2007 12/17/2007 34.38 8327 MC4R KO Running Wheel Female 9/17/2007 12/17/2007 22.61 8341 MC4R KO Running Wheel Female 9/17/2007 12/17/2007 21.71 8395 MC4R KO Running Wheel Female 9/21/2007 12/17/2007 18.23 8409 MC4R KO Running Wheel Female 9/24/2007 12/17/2007 4.23 8439 MC4R KO Running Wheel Female 9/14/2007 12/17/2007 20.07 8455 MC4R KO Running W heel Female 9/25/2007 12/17/2007 22.99 8462 MC4R KO Running Wheel Female 9/13/2007 12/17/2007 17.81

PAGE 479

479 Table A 3. Continued. Tag Number Genotype Housing Gender Date of Birth Date of Death Final Body Weight 8194 MC4R WT Group Conventional Female 9/14/2007 12/17/2007 16.32 8195 MC4R WT Group Conventional Female 9/14/2007 12/17/2007 20.38 8339 MC4R WT Group Conventional Female 9/17/2007 12/17/2007 17.48 8340 MC4R WT Group Conventional Female 9/17/2007 12/17/2007 19.15 8396 MC4R WT Group Conventional Fema le 9/21/2007 12/17/2007 17.68 8397 MC4R WT Group Conventional Female 9/21/2007 12/17/2007 18.00 8366 MC4R WT Running Wheel Female 9/20/2007 12/17/2007 16.76 8193 MC4R WT Running Wheel Female 9/14/2007 12/17/2007 17.76 8197 MC4R WT Running Wheel Female 9/14/2007 12/17/2007 18.02 8275 MC4R WT Running Wheel Female 9/17/2007 12/17/2007 17.09 8279 MC4R WT Running Wheel Female 9/17/2007 12/17/2007 19.92 8290 MC4R WT Running Wheel Female 9/15/2007 12/17/2007 20.70 8300 MC4R WT Running Wheel Female 9/17/200 7 12/17/2007 18.73 8316 MC4R WT Running Wheel Female 9/17/2007 12/17/2007 18.07 8329 MC4R WT Running Wheel Female 9/17/2007 12/17/2007 17.49 8463 MC4R WT Running Wheel Female 9/13/2007 12/17/2007 18.75

PAGE 480

480 Table A 4. Body weights (in grams) of male mice p resented in Chapters 3 and 4. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC3R WT Conv 17.60.3 17.10.2 17.60.3 18.30.2 18.80.2 19.60.2 20.00.2 20.70.3 21.20.3 11316 17.59 17.15 17.8 18.43 18.9 19.66 20.18 20.8 21.24 11328 17.33 16.61 16.79 17.57 18.12 19.05 19.35 19.89 20.53 11334 17.18 17.15 17.99 18.54 19.02 19.89 20.17 21.29 21.83 11336 18.36 17.62 18 18.49 19.08 19.8 20.32 20.95 21.24 MC3R WT RW 18.30.5 18.40.8 48.70.9 19.31.0 19.50.9 20.00.9 20.40.9 20.80.9 21.00.9 11293 18 .83 18.88 19.18 19.98 20.14 20.65 21.15 21.04 21.49 11306 18.6 18.85 19.79 20.77 20.87 21.69 21.68 22.58 22.56 11321 19.23 20.18 20.31 20.98 21.09 21.43 21.83 22.38 22.42 11332 18.58 18.39 18.75 19.2 19.37 19.66 20.07 20.72 21 11340 16.16 15.59 15.4 15 .56 16.17 16.81 17.13 17.31 17.6 MC3R KO Conv 17.70.7 17.20.8 18.10.7 18.80.8 19.31.0 19.71.1 20.41.1 21.11.2 21.91.2 11286 15.69 15.86 16.91 17.79 18.53 19.06 19.48 19.75 20.53 11294 16.48 14.92 16.4 17.08 17.22 17.25 17.8 18.42 19. 27 11297 19.61 19.33 20.09 21.48 22.33 23.65 24.2 25.2 26.02 11309 19.12 18.69 19.27 19.76 20.64 20.8 21.54 22.21 22.81 11333 17.59 17.35 17.73 17.71 17.64 17.96 19.01 20.1 20.87 MC3R KO RW 17.80.4 17.50.3 17.50.3 17.70.3 18.00.3 18.30 .3 18.50.3 18.90.2 19.20.2 11278 18.43 17.34 16.96 17.33 18.0 18.15 18.4 18.9 18.89 11290 18.68 18.77 18.53 18.79 19.11 19.45 19.43 19.54 19.7 11292 17.1 16.91 17.06 17.45 17.63 17.98 18.63 18.84 19.23 11303 17.95 17.2 17.11 16.84 17.46 17.71 17.7 1 8.25 18.63 11335 16.9 17.04 17.85 17.97 17.86 18.32 18.64 19.21 19.55

PAGE 481

481 Table A 4. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC4R WT Conv 20.11.0 20.71.2 20.71.1 21.11.2 21.51.2 21.91.1 22.11.1 22.11.0 22.10.7 8305 23.42 25.01 24.74 24. 84 25.59 25.37 25.61 25.55 8437 18.27 19.24 19.01 18.94 19.56 19.63 19.58 19.76 8764 20.88 21.84 21.75 22.76 22.87 23.63 24.06 23.28 23.49 8795 18.24 18.68 19.04 19.37 19.96 20.73 20.99 21.43 21.63 8816 19.46 18.61 19.03 19.64 19.44 19.97 20.2 20.6 6 21.07 MC4R WT RW 20.20.6 19.80.5 20.60.4 21.00.4 21.40.6 21.60.5 21.80.4 21.90.4 22.40.4 8766 20.29 20.33 20.82 21.31 22.22 21.78 21.91 22.11 22.66 8777 21.37 20.6 21.32 21.7 22.36 22.71 22.67 22.75 23.14 8813 20.68 19.92 20.72 21 .31 21.24 21.44 21.69 21.89 22.5 8841 18.56 18.48 19.44 19.87 19.88 20.46 20.75 20.95 21.38 MC4R KO Conv 21.50.7 23.41.0 25.21.0 27.51.0 29.71.0 31.41.2 33.40.2 34.81.2 35.11.6 8263 22.9 25.81 27.74 29.79 31.87 33.75 35.04 36.46 8 297 23.21 24.78 25.82 27.49 29.27 29.91 31.9 32.08 8407 20.87 24.1 26.84 29.48 32.31 34.86 37.02 38.11 8877 20.65 21.26 22.41 24.9 27.12 28.92 30.79 32.37 33.57 8882 19.77 20.84 23.1 25.69 28.12 29.8 32.08 34.93 36.67 MC4R KO RW 21.10.4 20.90.6 21.60.5 22.50.4 23.00.3 23.80.5 24.30.4 25.20.5 25.80.5 8189 20.86 19.98 20.44 21.3 22.16 23.25 24.32 25.32 8754 21.6 21.97 22.41 22.72 22.51 22.84 23.77 24.1 24.73 8847 21.73 21.35 22.11 22.94 22.9 23.16 23.6 24.48 25.29 8854 19.55 18.92 20.39 21.98 23.63 24.33 24.27 24.85 25.86 8887 21.84 22.3 22.44 23.34 24.01 25.26 25.7 27.03 27.22

PAGE 482

482 Table A 4. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 DKO Conv 20.61.3 23.51.4 28.11.3 32.31.3 35.31.0 38.70.8 40.60.9 42.50.4 44.00. 7 9320 20.72 23.16 26.07 30.79 34.52 38.54 38.16 41.57 43.14 9335 16.72 19.45 25.37 29.79 33.79 38.16 41.29 42.44 43.22 9337 23.4 27.05 31.44 35.02 37.89 41.36 43.04 43.59 45.48 9566 23.235 26.055 31.19 36.095 37.435 39.46 41.32 42.86 45.72 9568 18.84 21.855 26.465 29.95 32.965 36.2 39.24 41.945 42.515 DKO RW 17.81.4 18.92.0 20.62.1 23.02.3 25.02.3 26.92.4 28.32.2 29.92.0 31.41.9 9569 22.325 25.825 28.165 31.465 32.965 35.105 35.83 36.26 37.75 9329 17.36 17.45 17.88 19.04 20.52 2 1.52 22.88 24.73 26.21 9330 19.31 20.22 22.24 23.96 26.36 27.73 28.38 30.23 31.35 9563 16.4 17.305 18.85 20.19 20.795 22.88 25.345 27.025 28.96 9571 13.785 13.87 16.025 20.49 24.33 27.445 29 31.43 32.765

PAGE 483

483 Table A 5. Fat masses (in grams) of male mice presented in Chapters 3 and 4. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC3R WT Conv 1.60.1 1.30.0 2.30.2 0.40.2 1.40.3 1.50.3 2.00.4 2.10.4 2.30.3 11316 1.75 1.38 2.82 1.88 2.16 1.66 2.78 2.81 2.96 11328 1.38 1.19 1.96 1.13 1.08 2.21 0.98 1.26 2.0 9 11334 1.69 1.35 2.09 1.37 1.06 1.14 2.16 2.57 2.48 11336 1.47 1.2 2.47 1.06 1.39 1.14 2.05 1.8 1.79 MC3R WT RW 1.40.3 1.60.3 2.00.2 1.10.3 1.40.2 1.50.2 1.10.2 1.10.1 1.50.1 11293 2.09 2.13 2.61 1.3 1.35 1.93 1.36 1.32 1.61 11306 2.15 1.57 1.86 0.95 0.86 1.02 0.58 0.9 1.55 11321 0.52 1.12 1.38 0.18 1.46 1.44 1.33 0.69 1.35 11332 1.35 2.3 1.96 1.79 1.86 1.8 1.41 1.5 1.81 11340 1.06 0.75 1.94 1.27 1.45 1.45 0.91 0.94 1.16 MC3R KO Conv 2.20.3 2.00.1 3.000.4 2.50.2 2.90.3 3.10.4 3.40.5 3.60.5 4.10.6 11286 1.91 1.92 1.62 1.71 2.4 2.49 2.2 2.26 2.86 11294 1.92 1.91 2.76 2.22 2.53 2.36 2.49 3.15 3.11 11297 2.0 1.97 3.49 3.17 3.81 4.48 5.22 5.38 6.39 11309 3.37 2.51 3.9 2.76 3.25 3.6 3.87 3.4 4.29 11333 1.97 1. 91 3.3 2.61 2.34 2.53 3.01 3.63 3.98 MC3R KO RW 1.50.3 1.80.1 1.90.1 1.40.2 1.00.1 1.60.2 1.30.2 1.10.1 1.50.1 11278 2.07 2.19 1.93 1.33 1.22 1.81 1.71 1.45 1.53 11290 1.36 1.61 1.89 1.21 0.8 1.32 1.11 0.83 1.23 11292 2.71 1.77 2.25 1.93 0.82 2.15 1.43 1.11 1.76 11303 1.39 1.81 1.82 0.92 1.01 1.15 0.66 0.96 1.32 11335 1.66 1.57 1.51 1.81 0.95 1.42 1.46 1.21 1.53

PAGE 484

484 Table A 5. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC4R WT Conv 1.90.0 1.60.3 1.90.3 1.90.2 2.00.3 2.20 .3 2.00.2 2.10.2 2.10.1 8305 1.98 2.3 2.79 2.61 2.81 2.84 2.37 2.27 8437 0.74 1.24 1.28 1.38 1.44 1.32 1.32 8764 1.965 2 2.06 2.155 2.365 2.75 2.665 2.66 2.395 8795 1.895 1.595 1.955 1.84 1.685 1.87 1.715 2.11 1.955 8816 1.91 1.58 1.595 1.775 1.75 1.935 1.725 2.1 2.02 MC4R WT RW 1.60.1 1.60.1 1.50.2 1.90.2 2.10.2 2.00.2 2.00.2 1.80.1 1.80.1 8766 1.19 1.445 1.445 1.915 2.425 2.205 2.395 2.015 1.895 8777 1.805 1.87 1.475 1.735 1.875 2.05 1.955 1.9 1.89 8813 1.69 1.645 1.95 2.5 2.385 2.19 2.04 1.85 2.02 8841 1.625 1.285 1.21 1.44 1.545 1.425 1.505 1.505 1.575 MC4R KO Conv 2.60.1 2.90.2 4.40.4 6.10.6 7.70.7 9.30.8 10.50.7 11.60.9 12.51.1 8263 2.88 3.89 5.71 7.87 9.73 11.08 11.97 12.98 8297 2.32 2.43 3.71 5.07 6.4 7.33 8.6 8.87 8407 2.75 5.12 6.9 8.7 11.17 12.4 13.56 8877 2.645 2.755 3.57 4.735 6.05 8.22 9.395 10.46 11.39 8882 2.365 2.87 4.07 5.805 7.415 8.57 10.27 12.26 13.61 MC4R KO RW 2.20.2 1.70.1 2.00.2 2.30.3 2.70.4 2.90 .4 3.10.3 3.50.4 3.40.3 8189 2.6 1.52 1.92 2.33 2.37 2.99 3.65 4.15 8754 2.64 1.82 1.965 1.675 1.895 1.995 2.3 2.45 2.89 8847 2.035 1.75 1.635 1.885 2.705 2.245 2.47 2.785 2.825 8854 1.97 1.65 2.57 3.255 4.055 4.335 3.96 3.73 4.045 8887 1.79 1.88 5 2.02 2.34 2.435 3.1 3.34 4.25 4.025

PAGE 485

485 Table A 5. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 DKO Conv 3.20.5 5.20.9 8.91.1 12.61.1 15.11.0 17.50.7 19.10.6 20.40.6 21.10.7 9320 3.53 4.07 6.52 11.26 13.62 16.56 17.22 18.65 19.59 9335 1.57 2.99 6.85 10.16 13.19 16.1 18.23 19.65 19.88 9337 3.69 7.76 11.66 14.71 16.62 19.44 20.26 21.18 22.09 9566 4.59 6.95 11.35 15.98 18 19 20.59 21.93 23.26 9568 2.4 4.23 8.07 10.97 13.88 16.5 18.97 20.53 20.86 DKO RW 1.90.5 2.20.7 3.51.0 4.7 1.3 6.21.4 7.61.5 8.41.5 9.71.3 10.81.2 9569 3.72 4.895 7.3 9.3 10.91 12.31 12.95 13.24 13.82 9329 1.23 1.5 2.31 2.58 3.27 4.77 5.44 6.49 7.29 9330 2.23 2.52 3.98 5.43 7.2 8.3 9.27 10.22 11.33 9563 0.95 1.265 1.75 2.41 3.36 3.79 5.0 6.99 8.92 95 71 1.24 0.71 2.035 3.68 6.06 8.79 9.46 11.44 12.77

PAGE 486

486 Table A 6. Lean masses (in grams) of male mice presented in Chapters 3 and 4. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC3R WT Conv 13.30.1 13.00.2 13.20.3 14.10.2 14.70.3 15.50.4 14.90.3 15.40.3 15 .90.3 11316 13.27 13.12 12.72 13.71 13.93 15.59 14.27 14.94 15.56 11328 13.1 12.36 12.77 13.89 14.66 14.49 15.65 15.78 15.91 11334 13.21 13.04 13.73 14.27 15.3 16.4 14.7 15.09 15.59 11336 13.64 13.39 13.45 14.55 14.83 15.5 15.16 15.95 16.73 MC3R WT RW 14.10.6 13.90.5 15.01.0 15.40.9 15.50.9 15.90.9 16.30.7 16.50.8 16.60.8 11293 14 13.92 14.45 15.71 16.21 15.85 16.55 16.58 16.68 11306 13.47 14.42 16.62 16.64 17.39 18.19 18.25 18.31 18.37 11321 16.37 15.47 17.27 17.57 16.58 16.96 17.03 17.74 17.82 11332 14.0 13.28 15.22 14.73 14.72 15.58 15.98 16.02 16.13 11340 13.0 12.38 11.58 12.31 12.54 12.88 13.82 13.7 13.93 MC3R KO Conv 13.00.6 12.80.5 13.30.4 13.40.5 13.80.7 14.30.6 14.30.6 14.80.7 15.40.6 11286 11.53 11.55 13.83 13.08 13.49 13.94 14.65 14.9 15.47 11294 12.23 12.14 12.25 12.41 12.17 13.15 12.49 12.64 13.7 11297 14.81 14.61 14.62 15.03 15.93 16.17 16.07 16.6 17.17 11309 13.38 13.17 13.5 14.39 14.84 15.02 14.75 15.66 16.22 11333 13.14 12.3 12.43 12.36 12.74 13.33 13.66 14.03 14.51 MC3R KO RW 13.30.6 12.80.3 13.80.4 13.40.4 14.40.3 14.30.3 14.60.2 15.00.3 15.10.3 11278 13.14 11.82 13.1 13.42 13.79 14.11 14.17 14.44 14.8 11290 14.61 13.81 14.81 14.61 15.46 15.55 15.53 15.91 16.18 11292 11.39 12.32 12.79 12.33 14.23 13.58 14.46 14.95 14.66 11303 14.12 13.13 13.54 13.31 13.88 13.9 14.27 14.45 14.49 11335 13.38 13.08 14.99 13.6 14.43 14.55 14.34 15.2 15.23

PAGE 487

487 Table A 6. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC4R WT Conv 1 6.61.0 15.50.7 15.60.6 16.00.7 16.30.7 16.30.8 16.70.7 16.90.8 16.60.5 8305 19.12 17.75 17.64 18.09 18.77 18.37 18.76 19.46 8437 14.44 14.54 14.53 14.86 14.64 14.88 15.38 8764 16.945 16.145 16.455 17.15 17.22 17.735 17.805 17.76 17.55 879 5 14.635 14.585 14.67 15.115 15.575 15.805 16.485 16.285 16.44 8816 15.52 14.405 14.66 15.35 15.22 14.805 15.34 15.445 15.875 MC4R WT RW 15.90.3 15.30.4 15.70.3 15.90.3 16.20.4 16.20.3 16.60.2 16.30.02 16.60.1 8766 15.8 15.91 15.925 16.56 16.795 16.365 17.065 16.665 16.685 8777 16.345 15.78 16.305 16.225 16.855 17.075 16.725 16.77 16.92 8813 16.415 15.15 15.56 15.805 15.7 15.635 16.285 15.875 16.475 8841 14.96 14.3 14.995 15.07 15.425 15.87 16.15 15.99 16.325 MC4R KO Co nv 16.41.1 17.10.6 17.60.6 17.90.5 18.60.5 18.90.4 19.20.4 19.70.5 19.00.4 8263 17.72 18.41 18.53 18.98 18.94 19.69 19.96 20.51 8297 18.66 18.4 18.45 18.47 18.98 18.97 19.29 19.62 8407 17.4 18.57 18.94 20.17 20.13 20.28 20.93 8877 14.76 5 16.035 16.55 16.785 17.53 17.985 18.195 18.375 18.645 8882 14.47 15.24 15.685 16.545 17.16 17.95 18.42 19.15 19.36 MC4R KO RW 16.30.3 15.90.6 16.70.5 17.00.4 17.20.4 17.70.4 18.10.3 18.10.2 18.50.3 8189 16.4 14.82 15.39 16.08 16.03 16.56 17.21 17.87 8754 16.565 17.305 17.84 18.115 18.005 18.36 18.415 18.69 18.555 8847 16.7 16.655 17.095 16.9 17.295 17.545 18.105 18.035 18.58 8854 15.19 14.295 15.585 15.955 16.52 17.335 17.675 17.375 17.82 8887 16.58 16.42 17.4 17.795 17.915 18 .66 19.2 18.61 19.055

PAGE 488

488 Table A 6. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 DKO Conv 14.30.4 15.10.5 15.80.4 16.50.4 16.90.3 17.70.4 18.10.3 18.80.3 19.40.3 9320 14.74 15.62 16.21 16.56 16.82 17.91 17.88 19.39 20.13 9335 12.97 13.51 15. 06 16.51 17.26 18.25 18.94 18.93 19.43 9337 14.29 16.29 16.79 17.73 17.84 18.67 18.95 19.23 20.14 9566 15.28 15.39 16.145 16.11 16.68 16.94 17.54 17.83 18.76 9568 14.2 14.47 14.84 15.49 15.92 16.54 17.42 18.44 18.56 DKO RW 13.60.8 13.81.2 14.50.9 15.40.7 15.70.8 16.00.7 16.70.7 17.10.6 17.20.6 9569 15.6 17.36 17.35 18.26 18.51 18.71 19.21 19.23 19.55 9329 13.99 13.49 12.82 14.26 14.27 14.18 15.11 15.6 15.8 9330 14.59 15.43 15.57 15.33 16.05 16.06 16.45 17.18 16.73 9563 12.72 12.6 95 14.575 15.14 14.5 15.35 16.25 16.5 16.64 9571 11.29 10.12 12.215 14.13 15.06 15.92 16.48 16.92 17.02

PAGE 489

489 Table A 7. Body lengths (in millimeters) of male mice presented in Chapters 3 and 4. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC3R WT Conv 78.30.6 78.1 1.4 78.90.3 80.80.5 80.70.2 80.80.4 82.60.6 83.50.5 83.50.6 11316 77.15 74.8 78.7 79.2 81.0 81.85 82.6 82.6 82.5 11328 79.35 78.4 78.35 80.9 80.95 82.5 84.1 83.85 83.65 11334 77.2 77.4 78.7 81.55 79.95 80.45 81.45 82.75 82.75 11336 79.4 81.65 7 9.85 81.3 80.8 82.05 82.1 84.7 85.1 MC3R WT RW 79.5039 80.50.6 81.41.0 81.90.7 82.80.9 83.50.9 84.20.6 85.00.9 85.60.5 11293 81.8 81.55 83.35 82.35 82.85 83.65 85.1 85.4 85.55 11306 80.55 81.2 83.0 83.15 84.45 85.3 85.45 86.5 86.55 11321 78.35 80.45 80.85 81.75 83.1 84.8 84.15 84.5 85.9 11332 80.15 80.85 81.85 82.7 83.85 83.2 84.25 86.5 86.3 11340 76.45 78.25 77.7 79.15 79.55 80.3 81.95 81.9 83.7 MC3R KO Conv 78.41.1 78.91.0 79.41.0 80.51.3 81.21.0 81.31.3 82.01. 3 83.41.3 84.21.1 11286 76.35 77.65 79.45 80.05 80.9 81.8 81.55 83.95 83.85 11294 76.5 77.15 77.15 77.2 78.45 78.6 78.9 80.1 81.6 11297 81.15 81.75 81.55 84.2 82.7 84.6 85.15 86.95 87.05 11309 80.95 80.7 81.4 82.65 84.15 83.75 84.75 84.95 86.35 1133 3 76.8 77.3 77.2 78.5 79.7 77.85 79.5 80.8 81.85 MC3R KO RW 78.60.7 78.70.8 79.30.5 79.90.4 80.40.3 81.10.5 81.70.5 82.90.4 82.70.2 11278 78.9 75.6 79.6 80.05 80.0 80.65 80.65 83.15 82.35 11290 79.7 80.3 80.35 81.25 80.8 82.15 82.55 83.3 82.6 11292 80.2 79.3 78.75 79.65 80.0 82.35 82.25 83.5 82.95 11303 76.45 78.45 77.35 78.45 79.65 79.65 80.2 81.2 81.95 11335 77.65 79.7 80.1 79.9 81.45 80.7 82.6 83.4 83.4

PAGE 490

490 Table A 7. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC4R WT Conv 77.01.7 87.01.6 87.71.4 89.51.4 89.51.3 89.61.4 90.51.2 90.01.1 88.11.0 8305 93.2 92.7 92.9 94.4 94.5 95 94.6 94.5 8437 87.5 86.5 86.8 90.6 88.0 89.5 87.9 89.0 8764 85.7 87.6 87.3 88.65 89.2 88.75 91.45 89.5 89.7 8795 83.5 84.8 86.6 87.1 8 8.55 87.5 90.35 88.05 86.4 8816 84.9 83.4 84.8 86.95 87.1 87.35 88.3 89 88.05 MC4R WT RW 86.20.9 85.90.4 86.70.3 88.00.4 88.60.6 89.00.5 89.50.3 89.50.9 89.20.4 8766 87.5 86.05 86.5 87.45 89.9 89.7 90.0 90.6 89.3 8777 86.45 86.65 86 .95 87.65 88.5 90.05 90.0 91.25 88.1 8813 87.2 86.1 87.45 89.25 89.0 88.45 89.2 87.5 89.95 8841 83.55 84.85 85.95 87.55 87.05 87.95 88.85 88.5 89.55 MC4R KO Conv 87.61.9 88.91.3 89.91.3 90.91.0 92.60.9 93.21.0 94.40.9 94.90.6 94.30.3 8263 90.5 91.8 92.6 92.2 94.7 95.8 96.1 96.5 8297 91.9 91.8 91.8 92.5 93.9 92.9 94.8 94.6 8407 88.5 88.8 91.6 92.8 93.6 94.8 96.4 96 8877 85.25 85.6 86.65 87.45 89.6 90.1 90.6 90.9 90.7 8882 85.45 86.15 87.6 88.95 90.55 91.9 92.35 93.9 94.55 MC4R KO RW 85.40.6 85.70.6 86.90.7 87.20.4 88.20.5 88.20.5 89.90.4 89.90.7 89.90.6 8189 86.5 85.1 85.6 87.5 87.3 87.5 88.7 88.2 8754 85.75 87.55 89.15 88.15 89.9 88.95 90.4 91.55 90.9 8847 85.95 85.0 86.6 86.6 87.3 87.9 89.6 89.4 89 .05 8854 83.15 84.55 85.5 85.95 87.5 87.15 89.85 88.6 88.75 8887 85.45 86.45 87.65 87.6 88.8 89.65 91.0 91.95 91.0

PAGE 491

491 Table A 7. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 DKO Conv 82.61.0 85.30.9 86.50.7 87.21.0 88.40.9 88.50.9 88.30.5 89.0 0.5 89.50.7 9320 81.9 84.65 86.9 86.35 88.5 90.05 87.45 87.4 90.3 9335 80.15 84.8 87.05 88.45 90.2 89.35 88.5 90.05 91.1 9337 84.85 88.0 88.25 90.5 90.3 90.6 90.2 90.0 90.45 9566 84.95 86.35 86.15 85.65 87.3 86.75 87.85 88.6 88.7 9568 81.15 82.5 84. 05 85.05 85.45 85.95 87.5 89.1 87 DKO RW 80.31.5 82.72.2 83.22.1 83.81.7 85.61.8 86.21.4 87.11.7 87.71.4 88.41.7 9569 83.7 88.8 89.5 89.65 92.2 91.75 93.6 92.95 94.6 9329 80.65 81.95 82.7 81.7 81.8 84.4 83.15 84.3 85.5 9330 80.9 84. 45 84.15 85.7 86.5 86.3 87.0 87.6 88.25 9563 80.3 83.25 83.25 82.3 84.9 84.2 86.15 86.05 88.6 9571 74.6 75.05 76.35 79.75 82.7 84.35 85.8 87.65 84.9

PAGE 492

492 Table A 8. Average daily food intake (in grams) of male mice presented in Chapters 3 and 4. Age (Week s) 5 6 7 8 9 10 11 12 13 MC3R WT Conv 2.70.1 3.30.2 3.20.2 2.80.2 3.50.3 3.40.2 3.50.3 3.80.1 3.40.2 11316 2.63 3.15 2.92 2.49 2.98 2.99 3.07 3.45 3.04 11328 2.46 3.04 3.01 2.89 3.21 3.35 3.49 3.9 3.27 11334 2.72 3.25 3.12 2.4 3.31 3.31 3.22 4.03 3.46 11336 2.93 3.72 3.79 3.33 4.42 3.91 4.36 3.78 3.98 MC3R WT RW 2.80.2 3.50.2 3.40.1 3.70.2 3.80.2 3.90.2 1.00.2 3.90.1 4.30.2 11293 2.76 3.31 3.47 3.61 3.91 4.02 4.04 3.7 4.02 11306 2.89 3.21 3.32 3.78 3.91 4.23 4.06 4.21 4.27 11321 3.51 4.15 3.97 4.46 4.53 4.38 4.51 3.47 4.88 11332 2.61 3.37 3.34 3.5 3.15 3.22 3.51 3.73 4.11 11340 2.34 3.22 3.13 3.11 3.53 3.78 3.92 4.17 4.05 MC3R KO Conv 2.80.2 3.10.1 3.20.1 3.40.1 3.50.1 3.50.1 3.50.1 3.60.1 3.70.0 11286 2.74 3.00 3.09 3.29 3.26 3.31 3.46 3.44 3.59 11294 2.24 2.86 2.9 3.29 3.40 3.26 3.30 3.77 3.75 11297 3.29 3.17 3.28 3.60 3.78 3.66 3.56 3.51 3.78 11309 3.09 3.27 3.19 3.31 3.47 3.41 3.45 3.52 3.73 11333 2.78 3.16 3.38 3.44 3.74 3.74 3.89 3.57 3 .87 MC3R KO RW 2.60.1 3.00.1 3.20.1 3.60.1 3.90.1 3.90.2 3.90.2 3.90.1 4.00.2 11278 2.65 3.02 3.3 3.47 3.83 3.48 3.42 4.15 3.63 11290 2.99 3.45 3.31 3.49 3.98 4.06 3.97 3.84 4.03 11292 2.33 2.85 2.98 3.4 3.61 3.51 3.58 3.60 3.81 11 303 2.56 2.71 3.08 4.02 4.26 4.30 4.33 3.97 4.21 11335 2.51 2.80 3.43 3.76 3.92 4.17 4.11 3.92 4.56

PAGE 493

493 Table A 8. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC4R WT Conv 3.20.1 3.50.2 3.10.1 3.00.1 3.00.1 2.90.1 2.90.1 2.90.2 3.10.1 8305 4.46 3.5 3.22 3.06 3.22 3.06 3.21 8437 3.27 2.98 2.67 2.7 2.59 2.55 2.59 8764 3.24 3.34 2.86 2.89 2.79 2.74 2.78 2.59 2.91 8795 3.43 3.47 3.32 3.32 3.33 3.11 3.19 3.36 3.34 8816 3.01 3.02 2.99 3.1 2.93 2.97 2.98 2.91 3.07 MC4R WT RW 3.50.2 3.40.2 3.70.2 3.50.2 3.50.2 3.40.1 3.40.1 3.50.1 3.60.1 8766 3.62 3.55 3.57 3.55 3.66 3.47 3.33 3.47 3.54 8777 3.82 3.9 4.07 3.88 3.94 3.77 3.56 3.7 3.71 8813 3.30 3.2 3.7 3.37 3.23 3.31 3.36 3.34 3.56 8841 3.09 3.03 3.26 3.17 3.23 3.23 3.37 3.37 3.46 MC4R KO Conv 3.00.2 3.60.2 3.80.2 3.90.2 3.80.2 3.70.2 3.80.1 3.70.1 3.70.1 8263 4.07 4.11 4.15 3.99 3.86 3.46 3.61 8297 3.77 4.22 3.92 3.8 3.34 3.82 3.52 8407 3.62 4.11 4.27 4.28 4.4 4.08 4.0 8877 2.81 3.1 3.13 3.15 3.54 3.45 3.66 3.6 3.63 8882 3.23 3.21 3.29 3.81 3.35 3.32 3.62 3.85 3.85 MC4R KO RW 2.90.0 3.30.1 3.80.1 3.80.1 3.80.1 3.80.1 3.70.1 3.90.1 4.00.1 8189 3.04 3.68 3.66 3.65 3.68 3.69 3.62 8754 2.97 3.82 4.32 4.27 4 .24 4.14 4.12 4.22 4.34 8847 2.81 3.26 3.65 3.77 3.61 3.59 3.43 3.78 3.8 8854 2.92 3.03 3.51 3.51 3.68 3.66 3.42 3.7 3.93 8887 2.77 3.43 3.62 3.78 3.61 3.73 3.83 3.99 3.77

PAGE 494

49 4 Table A 8. Continued. Age (Weeks) 5 6 7 8 9 10 11 12 13 DKO Conv 2.80.4 4 .70.3 5.20.2 5.10.1 4.90.2 5.00.2 4.70.2 4.50.1 4.50.1 9320 2.13 4.19 4.56 5.25 4.74 5.06 3.98 4.58 4.24 9335 2.15 4.28 5.28 5.23 5.37 5.44 5.3 4.74 4.52 9337 4.23 5.78 5.33 5.09 5.29 5.27 4.87 4.29 4.97 9566 3.07 4.82 5.75 5.2 4.33 4.45 4.28 4 .31 4.62 9568 2.35 4.41 5.21 4.6 5.0 4.8 4.89 4.68 4.27 DKO RW 2.70.3 3.60.5 3.80.4 4.10.3 4.40.4 4.50.4 4.30.2 4.50.2 4.50.2 9569 3.64 5.38 5.23 5.33 5.32 5.43 4.83 4.68 5.21 9329 2.18 2.97 2.79 3.26 3.31 3.32 3.51 3.8 4.01 9330 2 .69 3.59 3.98 4.02 4.65 4.21 4.08 4.43 4.3 9563 2.95 3.71 3.94 3.96 3.74 4.25 4.58 4.65 4.67 9571 2.04 2.5 3.17 4.09 4.82 5.04 4.53 4.86 4.39

PAGE 495

495 Table A 9. Average daily number of running wheel rotations during the dark cycle of male mice presented in Cha pters 3 and 4. Age (Weeks) 6 7 8 9 10 11 12 13 MC3R WT RW 2136614 39411080 40531028 54831132 5820752 6218829 6629615 6860585 11293 1547 3567 5685 6339 4980 6225 6657 6819 11306 2869 4363 6415 6948 6517 6813 4654 5114 11321 3454 6157 3185 468 7 6734 7125 7081 6658 11332 2787 5594 586 1485 3316 3072 6291 6914 11340 18 19 4393 7956 7551 7854 8460 8795 MC3R KO RW 2260885 39961451 6010786 6234859 56611004 5232888 5764938 5755707 11278 960 568 7204 5350 3362 2698 5230 5449 112 90 0 387 6844 7314 4355 3633 6952 6253 11292 1973 5873 6778 8604 4499 5766 6747 6588 11303 3342 6061 6305 6344 8534 6880 2337 3187 11335 5022 7089 2919 3556 7552 7181 7553 7297

PAGE 496

496 Table A 9. Continued. 6 7 8 9 10 11 12 13 MC4R WT RW 2214697 19597 16 1967513 2979654 3310847 3133643 3321438 2721392 8766 1225 871 1079 1182 1435 1578 3389 2503 8777 3832 4066 3266 3428 2319 2740 2234 1717 8813 2909 1523 2304 4282 4676 4604 4377 3177 8841 889 1372 1215 3022 4810 3610 3282 3483 MC4R K O Conv 3241885 44651300 46391190 47901046 4317890 4517785 46991096 5194835 8263 1976.4 2860 3294 2756 1617 1946 1555 8297 6045 8971 8587 7305 5553 5404 8306 7478 8407 4557 5813 6153 7378 6668 6476 5323 5177 8877 1376 2140 2296 3271 4621 5103 3897 3529 8882 2253 2538 2863 3237 3126 3653 4410 4588 DKO RW 22461259 1609534 2646821 2004883 2299776 2174763 2129695 2091673 9569 4131 2235 2314 0 0 0 0 0 9329 257 314 829 272 862 888 1410 1465 9330 363 1339 3782 3987 3584 3217 303 8 2891 9563 6295 3327 5182 4124 3664 4174 4074 4001 9571 182 827 1120 1633 3380 2589 2122 2096

PAGE 497

497 Table A 10. Average weekly plasma insulin concentration (pM) for male MC3R mice presented in Chapters 3 and 4. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC3R WT Conv 25050 23682 257100 18359 16221 14522 24859 26246 25846 11316 315.198 452.66 556.93 355.01 204.67 207.5 417.465 302.787 348.6463 11328 344.661 173.7 135.4 92.284 190.25 116.931 216.663 124.962 278.4735 11334 213.818 153.18 175.32 144.53 12 5.19 146.342 215.938 321.645 274.622 11336 126.127 164.07 159.43 140.14 128.87 109.858 142.321 300.087 129.0558 MC3R WT RW 21416 16223 13610 14321 18752 18142 15943 12114 15425 11293 228.034 88.181 108.81 109.88 87.719 122.525 92.662 5 127.601 190.6383 11306 187.505 207.09 168.7 133.28 132.9 95.027 66.5305 74.2998 226.1645 11321 167.398 204.83 122.56 93.16 155.45 328.486 196.886 145.48 90.61425 11332 245.123 177.57 141.73 209.87 171.44 219.674 306.526 152.746 152.2918 11340 244.2 1 31.7 140.11 168.15 386.76 139.791 130.698 102.515 109.513 MC3R KO Conv 22725 23635 23865 28899 23443 19446 20826 29680 457103 11286 262.332 142.67 116.12 152.83 150.35 101.673 128.985 146.948 272.7563 11294 179.78 196.05 134.89 170.8 6 117.18 157.544 179.813 222.669 259.3093 11297 293.535 207.53 280.51 263.63 259.06 257.028 247.379 591.061 501.1455 11309 160.71 305.42 473.52 173.36 323.59 341.854 281.5 183.743 425.8418 11333 237.088 326.84 186.75 677.9 318.02 111.38 200.971 336.301 823.7965 MC3R KO RW 32332 19635 21555 20241 17342 15218 23451 16223 14629 11278 245.364 127.95 171.46 117.61 80.743 175.28 99.4645 125.838 87.99575 11290 327.309 169.91 121.37 145.23 103.94 95.103 142.291 140.144 102.3685 11292 436. 907 313.31 177.34 349.29 177.34 202.944 382.699 158.345 107.2013 11303 317.421 235.96 432.49 220.89 320.43 138.623 278.053 250.477 196.9355 11335 286.54 131.39 170.41 174.94 184.61 149.638 268.103 133.241 234.698

PAGE 498

498 Table A 11. Average weekly plasma lept in concentration (pM) for male MC3R mice presented in Chapters 3 and 4. Age (Weeks) 5 6 7 8 9 10 11 12 13 MC3R WT Conv 5934 8322 6230 10135 597 9325 8024 13639 11644 11316 158.311 146.59 150.33 170.77 75.305 152.459 126.843 217.015 133.7805 11328 11.0655 52.483 41.658 42.104 50.4025 20.117 54.627 11334 49.765 80.067 20.94 74.68 60.921 118.493 108.203 186.585 182.5788 11336 15.9175 52.841 34.802 58.051 57.371 51.056 65.0195 84.403 30.536 MC3R WT RW 3216 5613 4411 4113 46 18 3510 202 395 235 11293 15.5435 64.89 61.951 46.658 12.066 38.294 23.887 37.9573 19.82467 11306 96.489 50.875 29.046 6.2 24.057 20.0555 12.5265 27.1415 17.0885 11321 10.568 37.508 15.863 17.084 23.765 13.959 21.025 31.5523 14.83725 11332 10.5185 101.46 33.867 80.569 107.61 27.499 22.2765 52.7908 11340 25.5325 27.011 77.773 56.773 62.093 73.1465 21.082 46.8678 38.53275 MC3R KO Conv 3110 11224 12631 10926 8717 15527 16540 18550 18242 11286 7.8355 56.941 96.056 93.55 48.833 8 5.182 72.177 112.895 165.861 11294 49.383 77.064 94.83 36.224 61.573 96.7855 86.7185 120.278 195.548 11297 9.6735 99.479 179.64 114.12 82.344 213.624 289.104 320.14 176.5373 11309 54.2855 133.08 46.315 102.14 94.464 170.245 193.48 80.7105 55.7745 11333 31.4795 192.35 214.3 196.53 145.67 210.546 184.884 291.577 316.948 MC3R KO RW 2910 8815 8018 5413 285 417 375 466 298 11278 55.2645 126.32 141.83 101.23 35.62 44.616 38.153 34.1305 49.69075 11290 33.872 32.867 92.769 31.902 19.112 3 5.1695 38.014 41.994 19.11675 11292 11.115 96.108 69.16 39.529 22.115 59.3355 52.5475 61.1888 28.9775 11303 97.206 39.089 35.039 22.544 20.824 34.7228 41.337 11335 15.7925 88.055 58.575 59.904 42.992 26.2115 37.421 57.254 6.2

PAGE 499

499 Table A 12. Hypotha lamic gene expression presented as group averages of fold change (from conventionally housed WT group). Gene Housing MC3R WT MC3R KO MC4R WT MC4R KO DKO AGRP Conventional 1.30.4 1.40.1 1.00.2 0.60.0 2.10.9 Running Wheel 2.10.3 1.90.4 0.10.1 0 .70.0 3.41.1 PRKAA1 Conventional 1.00.1 0.90.0 1.00.0 1.10.1 1.10.0 Running Wheel 1.20.1 0.90.0 1.20.0 1.10.0 1.10.1 CART Conventional 1.10.2 1.10.1 1.00.1 1.40.0 1.30.1 Running Wheel 1.00.1 1.20.2 0.90.1 1.0 0.1 1.30.1 CPT2 Conventional 1.10.2 1.00.1 1.00.1 1.20.1 1.10.0 Running Wheel 1.20.1 0.90.1 1.20.1 1.10.0 1.20.1 GCK Conventional 1.20.3 1.10.1 1.00.1 0.90.0 1.00.0 Running Wheel 0.10.2 1.10.1 0.90.0 1.00.1 1.00.1 HCRT Conventional 1.20.3 1.10.2 1.00.1 0.90.0 1.30.2 Running Wheel 1.50.2 1.80.2 1.00.1 1.10.1 2.00.3 INSR Conventional 1.00.2 1.10.1 1.00.0 1.00.0 1.10.1 Running Wheel 0.90.1 1.10.1 1.00.1 1.00.0 1.0 0.1 LEPR Conventional 1.00.1 0.60.1 1.00.0 1.10.1 1.20.2 Running Wheel 1.30.2 0.70.1 1.10.0 1.10.0 1.30.2 MC3R Conventional 1.10.2 0.00.0 1.10.0 1.20.0 0.00.0 Running Wheel 1.20.1 0.00.0 1.10.1 1.10.0 0.00.0 MC4R Conventional 1.00.1 1.10.1 1.00.0 0.00.0 0.00.0 Running Wheel 1.10.1 1.20.2 1.20.1 0.00.0 0.00.0

PAGE 500

500 Table A 12. Continued. Gene Housing MC3R WT MC3R KO MC4R WT MC4R KO DKO POMC Conventional 1.10.2 0.60.0 1.00.1 2.00.5 0.3 0.2 Running Wheel 0.60.1 0.60.2 0.90.1 1.80.1 1.90.3 NPY Conventional 1.00.2 1.40.1 1.00.1 1.10.1 0.80.1 Running Wheel 1.40.1 1.80.3 1.40.1 1.20.2 0.90.1 NPY1R Conventional 1.10.3 0.60.1 1.00.1 1.30.2 1.00.1 Running Wheel 0.70.1 0.70.1 1.40.1 1.20.1 1.00.1 SOCS3 Conventional 1.00.1 0.70.1 1.00.1 1.90.1 1.10.1 Running Wheel 0.60.2 0.60.0 1.40.2 1.20.2 1.00.1 UCP2 Conventional 1.20.3 1.40.1 1.00.1 1.20.2 1.20.1 Running Wheel 1.30.1 1.30.2 1.10.0 1.10.0 1.20.1

PAGE 501

501 Table A 13. Liver gene expression presented as group averages of fold change (from conventionally housed WT group). Gene Housing MC3R WT MC3R KO MC4R WT MC4R KO DKO PRKAA1 Conventional 1.00.0 1.10.1 1.00.1 1.20.1 1.30.2 Running Wheel 1.10.1 1.20.0 1.00.1 1.20.1 1.20.1 CPT1A Conventional 1.00.0 1.20.1 1.10.2 1.80.2 1.80.2 Running Wheel 1.20.1 1.30.1 1.00.2 1.10.1 1.10.1 CPT2 Conventional 1.00.1 1.2 0.0 1.00.1 1.50.2 1.60.1 Running Wheel 1.00.0 0.10.1 1.40.1 1.10.1 1.40.2 DGAT1 Conventional 1.00.0 1.00.0 1.00.1 1.10.1 1.10.1 Running Wheel 1.00.0 1.00.0 1.20.1 1.10.0 1.20.1 DGAT2 Conventional 1.00.0 1.10 .1 1.10.2 1.30.1 1.40.1 Running Wheel 1.00.1 1.00.1 1.00.1 0.80.0 1.30.2 FASN Conventional 1.20.4 1.00.1 1.00.1 1.20.2 4.30.5 Running Wheel 0.50.1 0.60.0 1.30.2 0.70.1 2.81.4 FBP1 Conventional 1.00.1 1.20.1 1 .00.1 1.50.2 2.00.1 Running Wheel 1.20.0 1.20.1 1.20.1 1.10.1 1.60.2 G6PC3 Conventional 1.00.0 1.10.1 1.00.1 1.30.1 1.30.1 Running Wheel 1.00.0 1.10.1 1.20.1 0.90.1 1.30.2 GCK Conventional 1.20.3 1.70.1 1.00 .0 0.90.1 1.70.2 Running Wheel 0.60.2 0.90.2 1.30.2 0.70.1 1.10.3 SLC2A2 Conventional 1.00.1 0.90.1 1.00.1 1.80.2 1.30.1 Running Wheel 0.90.0 0.90.0 1.10.1 1.30.1 1.30.2

PAGE 502

502 Table A 13. Continued. Gene Housing MC3R WT MC3 R KO MC4R WT MC4R KO DKO PYGL Conventional 1.00.1 1.10.1 1.00.1 1.40.1 1.70.1 Running Wheel 0.80.0 0.90.0 1.10.1 1.20.1 1.40.2 GYS2 Conventional 1.00.1 0.90.1 1.00.1 0.80.1 0.60.0 Running Wheel 1.10.1 1.10.1 1.00.1 0.7 0.1 0.80.1 LIPE Conventional 1.00.1 1.10.1 1.00.1 1.90.2 1.60.1 Running Wheel 1.10.1 1.10.1 1.40.1 1.00.1 1.50.2 PFKL Conventional 1.00.1 1.10.0 1.00.1 1.10.1 1.40.0 Running Wheel 1.00.0 1.00.0 1.40.1 1.00.1 1.20.2

PAGE 503

503 Table A 14. Skeletal muscle (gastrocnemius) gene expression presented as group averages of fold change (from conventionally housed WT group). Gene Housing MC3R WT MC3R KO MC4R WT MC4R KO DKO PRKAA1 Conventional 1.00.0 0.90.0 1.00.1 1.00 .1 1.20.1 Running Wheel 1.20.1 1.10.1 1.00.1 1.00.0 1.20.1 CPT1B Conventional 1.00.1 1.00.0 1.00.1 0.90.1 1.00.1 Running Wheel 1.30.1 1.40.1 0.90.2 0.80.2 1.20.3 CPT2 Conventional 1.00.1 1.00.1 1.00.1 1.00.2 1.20.2 Running Wheel 1.60.1 1.40.1 1.20.2 1.10.1 1.50.3 GCK Conventional 1.00.1 2.00.3 1.10.2 0.80.1 0.80.1 Running Wheel 0.60.2 0.80.2 0.80.2 0.40.1 0.70.2 SLC2A4 Conventional 1.00.1 1.10.1 1.00.1 0.80.2 0.7 0.1 Running Wheel 1.00.1 1.20.2 0.70.1 0.60.2 0.80.1 PYGM Conventional 1.00.1 0.80.0 1.00.1 0.90.2 0.70.1 Running Wheel 0.70.0 0.70.1 0.80.2 0.60.1 0.70.1 GYS1 Conventional 1.00.0 1.00.0 1.00.1 1.10.2 1.10.2 Running Wheel 1.20.1 1.20.1 0.80.2 0.80.1 1.30.3 IL 6 Conventional 1.00.1 0.90.0 1.00.1 0.80.2 1.00.1 Running Wheel 0.90.1 1.10.0 0.70.1 0.80.0 1.20.2 PFKM Conventional 1.00.0 1.00.1 1.00.2 0.90.2 0.80.1 Running Wheel 0.70.1 0.80.1 0.70.1 0.60.1 0.80.1 UCP2 Conventional 1.00.1 1.80.1 1.20.3 1.00.4 2.00.4 Running Wheel 2.40.2 2.20.4 0.80.2 1.20.2 2.40.7

PAGE 504

504 Table A 14. Continued. Gene Housing MC3R WT MC3R KO MC4R WT MC4R KO DKO UCP3 Conventional 1.00.2 1.30.1 1.00.1 0.90.1 1.40.2 Running Wheel 1.90.2 1.90.3 0.80.3 0.60.1 1.20.3

PAGE 505

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520 BIOGRAPHICAL SKETCH Jay was born and raised in Southwest Florida. Jay graduated with honors from the University of Florida with a degree in chemistry in 2005. After working as laboratory manager for Dr. Carrie Haskell Luevano for two years, he joined the graduate research program in the Department of Medicinal C hemistry, College of Pharmacy at the University of Florida and later transferred to the Department of Pharmacodynamics. He worked under the supervision of Dr. Carrie Haskell Luevano studying the effects of voluntary running wheel exercise on obese mouse mo dels.