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1 COMPLICATIONS OF LEPTIN AND SEROTONIN AGONIST THERAPY IN REGULATION OF FOOD INTAKE AND THE PROMISE OF WHEEL RUNNING INTERVENTION IN RATS By KEVIN YANNICK EMANUEL STREHLER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 Kevin Yannick Emanuel Strehler
3 To my family, my colleagues an d those in pursuit of knowledge
4 ACKNOWLEDGEMENTS It would not be possible for me to have reached this point without the continual support of those whom have made my success a part of their own interests. First and foremost I wish to thank my parents, Em anuel and Marie Antoinette, their support of my pursuits in all facets of life has been unequivocal. No words are sufficient for my gratitude. I would also like to thank my extended family for not only their direct support but also for setting an example o f what can be achieved. Next I would like to thank those that have guided me through my graduate studies. My mentor, Dr. Philip Scarpace, has provided an environment with enough structure that I could succeed, yet enough flexibility to follow my own ideas. I am grateful to feel my best interests were always paramount in the decisions we made together. I must thank all the individuals, current and former, in the Scarpace and Tmer Labs for their technical assistance with experiments. Specifically, I would li ke to thank Michael Matheny for keeping the lab functioning and willingly working in the same lab space. Also my committee members: Dr. Christy Carter, Dr. Michael King, Dr. Neil Rowland and Dr. Peter Sayeski, provided me with helpful critiques and suggest ions, as well as training me in specialized techniques and analysis. I also wish to thank the support staff in the Department of Pharmacology and Therapeutics for assisting me in navigating through the bureaucratic tasks and allowing me to focus on my stud ies. Finally I would like to thank my friends and colleagues. They have always been there providing support in either verbal or occasionally fluid form. Sharing each others failures, and more importantly our successes, ha s made the past years immensely mo re enjoyable. Thank You.
5 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................ ................................ ............................... 4 L IST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 13 ABSTRACT ................................ ................................ ................................ ................... 16 CHAPTER 1 BACKGROUND ................................ ................................ ................................ .. 18 1.1 Introduction ................................ ................................ ................................ ....... 18 1.2 Obesity and Overweight ................................ ................................ .................... 18 1.2.1 Definition ................................ ................................ ................................ 18 1.2.2 Consequences ................................ ................................ ....................... 19 1.2.3 Causes ................................ ................................ ................................ .. 20 1.2. 4 Treatments ................................ ................................ ............................ 21 1.3 Physiological Regulation of Feeding ................................ ................................ 22 1.3.1 Gastrointestinal ................................ ................................ ...................... 22 1.3.2 Central Nervous System ................................ ................................ ........ 23 1.4 Leptin and Leptin Resistance ................................ ................................ ............ 24 1.4.1 Discovery ................................ ................................ ............................... 24 1.4.2 Leptin Action ................................ ................................ .......................... 24 1.4.3 Leptin Receptor Expression ................................ ................................ ... 26 1.4.4 Leptin Resistance ................................ ................................ .................. 27 1.5 Serotonin Pathways and Feeding ................................ ................................ ..... 28 1.6 Rat Models and Obesity ................................ ................................ .................... 29 2 DOSE RESPONSE TO ADENO ASSOCIATED VIRAL DELIVERY OF LEPTIN IN FISCHER 344 X BROWN NORWAY RATS ................................ ....................... 31 2.1 Introduction ................................ ................................ ................................ ....... 31 2.2 Results ................................ ................................ ................................ .............. 33 2.2.1 Two Month Adeno Associated Viral Leptin Treatment ........................... 33 184.108.40.206 AAV1 leptin delivery via 3rd ventricle causes dos e dependent body weight change ................................ ................ 33 220.127.116.11 Rats treated with increasing doses of leptin display greater but transient decreases in food consumption ............................ 35 18.104.22.168 Leptin treatment induces changes to body composition ........... 36 22.214.171.124 Leptin treatment reduces iBAT size and changes relative UCP1 levels ................................ ................................ .............. 38 126.96.36.199 Leptin elevates metabolic rate ................................ .................. 38
6 188.8.131.52 Increasing doses of leptin desensitize leptin receptors in the mediobasal hypothalamus ................................ ........................ 39 2.2.2 Six Month Adeno Associated Viral Leptin Treatment ............................. 39 184.108.40.206 AAV1 leptin delivery via 3 rd ventricle causes dose dependent body weight cha nge; high dose of leptin results in earlier onset and more rapid weight regain ................................ .......... 39 220.127.116.11 Rats treated with increasing doses of leptin display greater but transient decreases in food consumpti on; the highest dose results in long term increased chow intake ...................... 41 18.104.22.168 Changes to body composition over time are dependent on the dose of leptin treatment ................................ ...................... 42 22.214.171.124 Leptin treatment changes relative UCP1 levels at six months in iBAT but does not change the size of depot .......................... 44 126.96.36.199 Dose of leptin may alter metaboli c substrate preference long term without change to overall metabolism ....................... 45 188.8.131.52 Chronic leptin treatment diminishes the ability of acute leptin to stimulate STAT3 phosphorylation in select ce ntral nervous system areas ................................ ................................ ............ 45 2.3 Materials and Methods ................................ ................................ ...................... 46 2.3.1 Animals ................................ ................................ ................................ .. 46 2.3.2 Adeno Associated Viral Vectors ................................ ............................ 47 2.3.3 Surgery ................................ ................................ ................................ .. 47 2.3.4 Food Intake & Body Weight Assessment ................................ ............... 48 2.3.5 Body Composition ................................ ................................ .................. 48 2.3.6 Respiratory Measurements ................................ ................................ .... 48 2.3.7 Acute Lepti n Signaling ................................ ................................ ........... 49 2.3.8 Interscapular Brown Adipose Tissue Assessment ................................ 49 2.3.9 Evaluation of Signal Transducer and Activator of Tra nscription 3 Phosphorylation ................................ ................................ ..................... 50 2.3.10 Statistics ................................ ................................ ................................ 51 2.4 Discussion ................................ ................................ ................................ ........ 52 3 ALTERNATIVE THERAPEUTIC OPTIONS TO ALTER FOOD CONSUMPTION AND PHYSIOLOGICAL OUTCOMES ................................ ................................ ..... 78 3.1 Introduction ................................ ................................ ................................ ....... 78 3.1.1 Serot onin Agonists and Feeding ................................ ............................ 78 3.1.2 Wheel Running and Food Intake ................................ ........................... 80 3.2 Results ................................ ................................ ................................ .............. 82 3.2.1 Central Nervous System Treatment with ( ) Trans PAT ........................ 82 184.108.40.206 Long term infusion of ( ) trans PAT into ventricles of Sprague Dawley rats produces increased weight gai n ............. 82 220.127.116.11 Prolonged infusion of ( ) trans PAT does not reduce chow nor high fat diet intake ................................ .............................. 82 18.104.22.168 ( ) Trans PAT does not change body composition ................... 83 3.2.2 Acute Treatment with the Selective Serotonin 2C Receptor Agonist WAY 161503 in the Central Nervous System ................................ ........ 83
7 22.214.171.124 Overnight fasting induces a re feeding response in Sprague Dawley rats ................................ ................................ ............... 83 126.96.36.199 Acute lateral ventricle delivery of vehicle does not change re feeding following overnig ht fast ................................ ................. 84 188.8.131.52 Lateral ventricle delivery of 5 g WAY 161503 does not change chow intake after overnight fast ................................ .... 85 184.108.40.206 Lep tin given acutely to lateral ventricle reduces 24 hour food intake in Sprague Dawley rats ................................ .................. 85 220.127.116.11 Decrease in food intake was observed after overnight fasting following acute administration of a 40 g dose of WAY 161503 ................................ ................................ ...................... 86 3.2.3 Peripheral Injection of WAY 161503 and ( ) Trans PAT ........................ 87 18.104.22.168 WAY 161503 treatment giv en peripherally is able to decrease chow food intake ................................ ....................... 87 22.214.171.124 No reduction in chow intake during re feeding is observed when rats are treated with ( ) trans PAT ................................ ... 87 3.2.4 Voluntary Wheel Running and Diet Preferences in Sprague Dawley Rats ................................ ................................ ................................ ....... 88 126.96.36.199 Wheel running alters the proportion of diet consumed in the chow vers us high fat diet choice paradigm ............................... 88 188.8.131.52 Diet choice with one week wheel running intervention .............. 88 184.108.40.206 Rats maintained on h igh fat diet, introduced to running wheels and then exposed to dietary choice .............................. 89 220.127.116.11 Wheel running activity in CWC and HWC experiments ............. 90 3.2.5 Central Nervous System Administration of Leptin Antagonist Increases Food Intake but Does Not Prevent Reduced Intake Attributable to Voluntary Wheel Running ................................ ............... 9 1 18.104.22.168 Incr ease in chow intake following voluntary wheel running is not prevented by leptin antagonist ................................ ............ 91 22.214.171.124 Leptin antagonist increases high fat diet consumption, but does not prevent wheel running induced reduction of intake .... 92 126.96.36.199 Leptin antagonist treatment did not reduce wheel running activity, however leptin antagonist dramatically increased body weight ................................ ................................ ............... 92 3.3 Materials and Methods ................................ ................................ ...................... 93 3.3.1 General ................................ ................................ ................................ .. 93 188.8.131.52 Animals ................................ ................................ ..................... 93 184.108.40.206 Statistics ................................ ................................ ................... 93 3.3.2 Long Term Infusion of ( ) Trans PAT ................................ ..................... 94 220.127.116.11 Surgery ................................ ................................ ..................... 94 18.104.22.168 Treatments ................................ ................................ ................ 95 22.214.171.124 Food consumption ................................ ................................ .... 95 126.96.36.199 Body composition ................................ ................................ ..... 95 3.3.3 Acute CNS Treatment Experiments Involving WAY 161503, Leptin, and ( ) Trans PAT ................................ ................................ .................. 96 188.8.131.52 Surgery ................................ ................................ ..................... 96
8 184.108.40.206 Treatments ................................ ................................ ................ 96 220.127.116.11 Diets ................................ ................................ .......................... 97 3.3.4 Peripheral Injection of WAY 161503 and ( ) Trans PAT ........................ 97 18.104.22.168 Animals ................................ ................................ ..................... 97 22.214.171.124 Treatments ................................ ................................ ................ 98 3.3.5 Wheel Running Diet Choice Experiments ................................ .............. 98 126.96.36.199 Experimental design of choice wheel running choice experiment (CWC) ................................ ................................ .... 98 188.8.131.52 Experimental design of high fat wheel running choice experiment (HWC) ................................ ................................ .... 99 3.3.6 Wheel Running Diet Choice with Leptin Antagonist Treatment ............. 99 184.108.40.206 Animals ................................ ................................ ..................... 99 220.127.116.11 Surgery ................................ ................................ ..................... 99 18.104.22.168 Treatment ................................ ................................ ............... 100 22.214.171.124 Diets ................................ ................................ ........................ 100 126.96.36.199 Wheel running ................................ ................................ ......... 101 188.8.131.52 Body composition ................................ ................................ ... 101 3.4 Discussion ................................ ................................ ................................ ...... 101 3.4.1 Selective Serotonin Agonists ................................ ............................... 101 3.4.2 Wheel Running ................................ ................................ .................... 104 4 BLOCKADE OF CENTRAL NERVOUS SYSTEM MELANOCORTIN 3/4 RECEPTORS IN FISHER 344 X BROWN NORWAY RATS ................................ 124 4.1 Introduction ................................ ................................ ................................ ..... 124 4.2 Results ................................ ................................ ................................ ............ 126 4.2.1 SHU9119 Treatment Increases Body Weight Gain in Presence of High Fat Diet that is Attenuated by Voluntary Wheel Running ............. 126 4.2.2 SHU9119 Increases Food Consumption and Delays Homeostatic Regulation of Caloric Consumption, While Voluntary Wheel Running Accelerates Regulation of Caloric Intake ................................ ............. 128 4.2.3 SHU9119 Reduces Wheel Running but Pair Feeding Attenuates Wheel Running Reduction ................................ ................................ ... 130 4.2.4 SHU9119 Treatment Increases Adiposity and Reduc es Lean Mass ... 131 4.2.5 SHU9119 Treatment Increases Interscapular Brown Adipose Tissue Size and Changes the Expression of Uncoupling Protein 1 ................. 132 4.3 Materials and Methods ................................ ................................ .................... 134 4.3.1 Animals ................................ ................................ ................................ 134 4.3.2 Surgery ................................ ................................ ................................ 134 4.3.3 Treatments ................................ ................................ .......................... 135 4.3.4 Diet Choice Paradigm and Pair Feeding ................................ ............. 135 4.3.5 Wheel Running ................................ ................................ .................... 135 4.3.6 Body Composition ................................ ................................ ................ 136 4.3.7 UCP1 ................................ ................................ ................................ ... 136 4.3.8 Statistics ................................ ................................ .............................. 137 4.4 Discussion ................................ ................................ ................................ ...... 137
9 5 CONCLUDING REMARKS ................................ ................................ ................... 151 LIST OF REFERENCES ................................ ................................ ............................. 157 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 175
10 LIST OF FIGURES Figure Page 2 1 Body weight of rats following 3 rd ventric le AAV1 leptin injection. ........................ 57 2 2 Change in body weight of rats respective to date of injections. .......................... 58 2 3 Food intake of rats trea ted with varying doses of AAV1 leptin. ........................... 59 2 4 Cumulative food intake of rats treated with varying doses of AAV1 leptin. ......... 60 2 5 B ody composition changes following AAV1 leptin treatment. ............................. 61 2 6 Effect of leptin on interscapular brown adipose tissues and levels of UCP1 two months following leptin treatment.. ................................ ............................... 63 2 7 Thermogenic evaluation of animals treated with increasing doses of leptin observed at day 55. ................................ ................................ ............................ 65 2 8 Phosphorylated STAT3 protein in t he mediobasal hypothalamus two months after treatment with leptin vector following acute leptin stimulation. ................... 66 2 9 Body weight of rats injected with AAV1 leptin vector over the 6 month experi mental period.. ................................ ................................ .......................... 67 2 10 Change in body weight over 6 months in rats injected with AAV1 leptin. ........... 68 2 11 Chow food consumed follow ing treatment with AAV1 leptin. .............................. 69 2 12 Body composition of AAV1 leptin treated rats. ................................ ................... 71 2 13 Changes in interscapular brown adi pose tissue following 6 months of treatment with AAV Leptin. ................................ ................................ ................. 73 2 14 Thermogenic evaluation of animals treated with increasing doses of leptin observed at day 163. ................................ ................................ .......................... 75 2 15 Long term leptin over expression diminishes the ability of acute leptin to induce phosphorylation of STAT3 in select CNS nuclei.. ................................ .... 76 3 1 Central ner vous system delivery of ( ) trans PAT results in increased body weight gain in SD rats. ................................ ................................ ...................... 108 3 2 Food Intake of rats treated with ( ) trans PAT on chow and high fat diet. ........ 108 3 3 Change in body composition following treatment with ( ) trans PAT for two weeks. ................................ ................................ ................................ .............. 109
11 3 4 Food Restriction by overnight fasting induces a re fe eding response in SD rats. ................................ ................................ ................................ .................. 110 3 5 Acute injection of 5 l of aCSF vehicle to lateral ventricle does not change re feeding response to overnight fast in SD rats.. ................................ ................. 111 3 6 Acute injection of 5 g WAY 161505 into lateral ventricle does not change re feeding response to overnight fast. ................................ ................................ ... 112 3 7 Acute injection of 5 g of l eptin produces a significant decrease in chow intake in the 24 hour period following an overnight fast. ................................ ... 113 3 8 Acute lateral ventricle injection using a 40 g dose of WAY 161503 produces a de crease in re feeding response following overnight fast. ............................. 114 3 9 Intraperitoneal 3 mg/kg WAY 161503 inhibits chow food intake following 12 hour daytime fast. ................................ ................................ ............................. 115 3 10 Intraperitoneal injection of 10mg/kg ( ) trans PAT has no effect on food intake following a 12 hour daytime fast. ................................ ............................ 116 3 11 Voluntary wheel running changes the amount of diet consumed in two diet choice paradigm in SD rats. ................................ ................................ .............. 117 3 12 Voluntary wheel running while on HFD reduces intake and produced a shift in consumption toward chow in response to the two diet choice paradigm. ......... 118 3 13 Average wheel running of Sprague Dawley rats in CWC and HWC experiments. ................................ ................................ ................................ ..... 119 3 14 Leptin antagonist increases caloric intake but does not prevent decreased intake in response to wheel running. ................................ ................................ 120 3 15 Wheel running activity does not change with leptin antagonist treatment in SD rats. ................................ ................................ ................................ ............ 122 3 16 Change in body weight following leptin antagonist treatment. .......................... 122 3 17 Wet tissue weight of multiple adipose tissu e depots collected after two weeks of leptin antagonist treatment. ................................ ................................ .......... 123 4 1 Daily change in body weight with SHU9119 treatment during dietary choice with introduction of running wheels. ................................ ................................ .. 142 4 2 Daily intake of chow and high fat diet in aCSF and SHU9119 treated rats with or without access to running wheels.. ................................ ............................... 143
12 4 3 Aver age daily caloric intake of high fat diet in aCSF and SHU9119 treated groups at baseline (Phase A) and the one week period following the introduction of running wheels (Phase B). ................................ ........................ 144 4 4 Avera ge daily wheel running activity in SHU9119 treated diet choice paradigm. ................................ ................................ ................................ ......... 145 4 5 Change in body composition following one month of SHU9119 treatment assessed by TD NMR.. ................................ ................................ .................... 146 4 6 Cumulative wet tissue weight of selected white adipose tissue depots from rats treated for 28 days with SHU9119. ................................ ............................ 147 4 7 Interscapular brown a dipose tissue (iBAT) depot size following 28 day treatment with SHU9119. ................................ ................................ ................. 148 4 8 Uncoupling Protein 1 expression in iBAT depots. ................................ ............. 149 4 9 Effect of voluntary wheel running on UCP1 expression in iBAT depots. Expression of UCP1 with wheel running was compared within each individual treatment condition.. ................................ ................................ ......................... 150
13 L IST O F ABBREVIATIONS AAV1 Adeno A ssociate d Virus Type 1 aCSF Artificial Cerebrospinal Fluid AgRP Agouti related Peptide AMPK 5' Adenosine Monophosphate activated Protein Kinase MSH Alpha Melanocyte Stimulating Hormone ANOVA Analysis of Variance BDNF Brain derived Neurotrophic Factor BMI Body Ma ss Index C Experimental Phase: Diet Choice C1 Experimental Phase: First Diet Choice C2 Experimental Phase: Secondary Diet Choice Cal Calorie CCK Cholecystokinin CNS Central Nervous System CWC Experiment: Choice followed by Wheel Running and Choice C/WR Ex perimental Phase: Choice with Wheel Running db Diabetes Gene (Produces Leptin Receptor) DIO Diet Induced Obese DMHa Dorsomedial Hypothalamic Area DMSO Dimethyl Sulfoxide DR Dietary Resistant EWAT Epididymal White Adipose Tissue FBN Fischer 344 x Brown Norw ay Rat Strain 5 HT 5 Hydroxytryptamine (Serotonin)
14 GFP Green Fluorescent Protein GI Gastrointestinal GLP 1 Glucagon Like Peptide 1 H Experimental Phase: High Fat Diet HD High Dose (1.76x10 10 viral genomes per rat leptin) HFD High Fat Diet HWC Experiment: H igh Fat Diet followed by Wheel Running and Choice H/WR Experimental Phase: High Fat Diet with Wheel Running iBAT Interscapular Brown Adipose Tissue IP Intraperitoneal Injection JAK2 Janus Kinase 2 LD Low Dose (1.76x10 6 viral genomes per rat leptin) LH Late ral Hypothalamus MC3R Melanocortin 3 Receptor MC4R Melanocortin 4 Receptor MD Middle Dose (1.76x10 8 viral genomes per rat leptin) MPOA Medial Preoptic Area mTOR Mammalian Target of Rapamycin NPY Neuropeptide Y NTS Nucleus Tractus Solitarius o b Obesity Gene (Produces Leptin) ObRb Long form of Leptin Receptor P/F Pair Fed POMC Pro opiomelanocortin pSTAT3 Phosphorylated Signal Transducer and Activator of Transcription 3
15 PTP1B Protein Tyrosine Phosphatase 1B PWAT Perirenal White Adipose Tissue PYY Protein Tyros ine Tyrosine RTWAT Retroperitoneal White Adipose Tissue RQ Resp i ratory Quotient SD Sprague Dawley Rat Strain SED Sedentary SEM Standard Error of the Mean SHP2 Src Homology 2 Domain Containing Protein Tyrosine Phosphatase SHU9119 Ac Nle Asp His D Nal(2) Arg Trp Lys NH 2 Amide Bridge: Asp 3 Lys 8 SNK Student Newman K eu ls Post Test SOCS3 Suppressor of Cytokine Signaling 3 STAT3 Signal Transducer and Activator of Transcription 3 TD NMR Time Domain Nuclear Magnetic Resonance UCP1 Uncoupling Pr otein 1 vg viral genomes VMH Ventromedial Hypothalamus VTA Ventral Tegmental Area WAT White Adipose Tissue WAY 161503 8,9 Dichloro 2,3,4,4a tetrahydro 1H pyrazino[1,2 a]quinoxalin 5(6H) one hydrochloride WR Wheel Running ( ) Trans PAT (1R,3S) trans 1 p henyl 3 dimethylamino 1,2,3,4 tetrahydronaphthalene
16 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy COMPLICATIONS OF LEPTIN AND SEROTONIN AGONIST THERAPY IN REGULATION OF FOOD INTAKE AND THE PROMISE OF WHEEL RUNNING INTERVENTION IN RATS By Kevin Yannick Emanuel Strehler August 2013 Chair: Philip Scarpace Major: Medical Sciences Physiology and Pharmacology S taggerin g prevalence rates and link age as significant risk factors for numerous other diseases overweight and obesity are major challenges in modern healthcare. Obesity results from long term dysregulation of energy intake and reduced energy expenditure. Energy b alance is regulated by various physiological processes; the experiments within focus specifically on the role of leptin and serotonin along with the intervention of wheel running on food intake and energy expenditure. Using a viral mediated delivery system we show that rats res pond to leptin in a dose depende nt fashion and while higher doses produce greater changes with respect to reduction of food intake and body weight in the short term they also accelerate the onset of resistance to leptin in the long term. The development of specific serotonin receptor agonists has show n promise in treatment of obesity, as such we evaluated the role of a newly synthesized compound (1 R ,3 S ) trans 1 phenyl 3 dimethylamino 1,2,3,4 tetrahydronaphthalene on food intake in rats. Our results show that neither peripheral, nor central nervous system delivery was able to reduce food intake in Sprague Dawley rats. More promising, we show that vo luntary wheel running is able to reduce high fat
17 diet intake as well as increase preference for chow diet in a two diet choice paradigm. Furthermore, the wheel running effect on diet intake is not mediated by leptin, nor does it depend on the melanocortin pathway. Antagonists of both leptin and melanocortin receptors did not prevent the effectiveness of wheel running. Taken together, the results of our experiments show that while there are inherent difficulties in the treatment of obesity using current phar macologic therapies the future may hold successful therapies. Elucidating the physiological mechanisms regulating food intake by voluntary wheel running may illuminate novel therapeutic strategies to control energy balance.
18 CHAPTER 1 BACKGROUND 1.1 Introductio n In recent decades the rates of obesity and overweight have been rising at staggering levels across much of the globe and specifically within the United States. The issue of overweight and obesity is not a cosmetic one, as the risks of developing a multit ude of other diseases is strongly linked to excessive body weight. In the simplest form obesity can be defined as the resu lt of a long term imbalance where energy intake exceed s energy expenditure. The major contribution on the energy intake side is food consumption; while the major components of expenditure are basal metabolic rate, thermogenesis and activity level. Numerous physiological regulatory pathways exist in an attempt to regulate both components of energy balance. Providing effective treatment f or obesity and overweight has proven to be more complicated than anticipated. Options including surgical, pharmacological and behavioral treatments have all been shown to have a degree of effectiveness, but to date each also has its own set of shortcomings The current studies have been designed and executed in order to better understand the physiological responses and underlying mechanisms controlling feeding regulation. The results of these studies will hopefully aid in the understanding of how feeding is regulated and thereby identifying new potential therapeutic targets or alternate interventions. 1.2 Obesity and Overweight 1.2.1 Definition Overweight and obesity are characterized by increases in total body weight of individuals yet more clinically relevant than total weight is the increased adiposity.
19 Multiple methods of characterizing individuals as over weight or obese exist, ant h ropometric measures including body mass index (BMI, calculation of ratio between weight and height kg/m 2 ) waist circumference and ski n fold thickness act as estimates of body fat. Additionally more direct methods of determining body composition exist such as bioelectric impedance spectroscopy, a ir displacement plethysmography and dual energy X ray absorptiometry The aforementioned met hods each have their own advantages and limitations including cost, invasiveness and validity, reviewed in [1, 2] As such, due to its universal application, the most common measure is BMI with a score greater than 25 being considered overweight and a score of greater th an 30 being class ified as obese. H owever in evaluating treatment needs for patients it is important to assess multiple fact ors  1.2.2 Consequences Interestingly overweight individuals have been shown to have reduced mortality compared to normal weight individuals  ; th is phenomenon and open to differential interpretation. H owever it is well established that indi viduals fitting the obese classification have increased comor bidity for numerous other diseases as well as increased mortality [4 7] Beyond the negative impact on personal health, obesity also generates a substantial economic burden. Increased prevalence of obesity in the United States raised obesity associated healthcare spending from $78.5 billion in 1998 to $147 billion in 2008  Results from the 2009 2010 National Health and Nutrition Examination Survey indicated that 69.2% of the adult population in the United States was overweight (BMI > 25) with 35.9% being classified as obese (BMI > 30), and these rates continue to increase although the rate of increase has slowed over the last decade  High rates of childhood obesity and
20 individuals r eaching the obese state at an earlier age  result i n longer periods of individuals being subjected to the obese state thereby creating even higher healthcare costs. 1.2.3 Causes Continuous excessive caloric intake compared to energy expenditure results in storage of extra calories as fat. Prolonged positive en ergy imbalance eventually will produce the obese state. However the energy balance hypothesis does not fully explain the etiology of obesity, as likely it is itself a response to an initial insult  Some cases of obesity can be attributed to genetic abnormalities and their presenting syndromes, but in most cases genetics simply provide the background predisposition to the effects of the environment  Among the environmental factors one determinant is food cost. Energy dense foods cost less and are typically less nutritious [13, 14] A disproportionate number of individuals with lower socioeconomic status are obese  Secondly the nutrient composition of diets also plays a potential role [16, 17] Diets high in fat or simple carbohydrates alter the phys iological response to nutrient intake and promote or inhibit physiological regulators of feeding  Furthermore, t he availability of calories per individual has increased  and likely is one of the factors that ha s contributed to the observed increase d intake of ~570 kcal/day over the last three decades in the United States  In addition to the increased availability of calories, compounded by calorically dense foods, recent data show s that total activity levels have decreased globally and future predictions suggest further increases in sedentary behavior will be observed  The current environment is so highly obes o genic that negative physiological regulators are not eno ugh to combat the system I n turn the development of obesity results in activation of a feed forward mechanism that further
21 drive s the progression of obesity a phenomenon that has been termed by Swinburn  1.2.4 Treatments While there are a variety of treatment options for overweight and obesity each has its own limitations therefore the development of better and more effective treatments is necessitated  Treatments fall into 3 major categories: b ehavioral s urgical and pharmacological  Behavioral therapies including lifestyle interventions or cognitive behavioral therapy, provide reductions in body weight and improvements in physiological parameters, especially when combined with pharmacotherapy [24 ] However such therapies require continued consultations for weight loss to be maintained [24, 25] Surgical treatments by gast r ic bypass or banding have produced significant weight loss but due to the nature of such an invasive procedure this strategy is only advised for individuals wit h severe obesity  Whil e there have been a variety of pharmacological agents that have acted to induce weight loss these treatments have led to the development of serious side effects and resulted in a removal from the market [27, 28] R ecently two n ew treatments for obesity, Lorcaserin and P hent er mine T opiramate [29, 30] have been approved for use in addition to the only drug having been on the market previously O rlistat  The number of physiological regulators of feeding and energy expenditure, described in detail later in this c hapter, will hope fully result in new therapies or more efficacious polytherapies in the future [28, 32] While there are multiple pharmaceuticals in the pipeline, including those at various stages of clinical trials, the past history of success necessitates that further understanding into the regulation of feeding and energy expenditure be undertaken.
22 1.3 Physiological Regulation of Feeding 1.3.1 Gastrointestinal The stomach and gastrointestinal (GI) tract release a my riad of signals to regulate feeding reviewed in [33 37] The following paragraph highlights, in brief, some of the most importan t concepts. Whil e signals originate in the stomach or GI tract they involve terminal c entral nervous s ystem (CNS) areas to integrate and provide the response to such signa ls acting either to induce sati ation (termination of ongoing meal) or sat iety ( relat ion of inter meal interval). The primary signals include g astric distention sensed by stretch and tension receptors within the stomach and relayed to hindbrain by vagal afferents. C holecystokinin (CCK) is produced in the proximal intestine by I cells of th e duodenum and jejunum and acts as a short term satiation signal. Multiple cleavage products of CCK act with two types of CCK receptors, CCK1 in the gut and CCK2 receptors expressed in the brain. Intestinal signals, including CCK are responsive to macronu trient contents of diets, where as dietary composition does not appear to have an effect within the stomach. Additionally, distal intestinal L cells produce glucagon like peptide 1 (GLP 1) peptide tyrosine, tyrosine (PYY) and oxyntomodulin which all act as satiation signals and are responsive to micronutrients. While GLP 1 action is primarily through vagal afferent signaling following activation of the GLP 1 receptor, PYY satiation actions are more likely attributed to interaction with arcuate nucleus Y2 re ceptors. PYY is able to induce rapid satiation but also acts in a long term fash ion more along the lines of non GI satiety signals such as leptin, insulin and glucagon. In contrast to the aforementioned anorectic signals, ghrelin is an orex i genic signal th at is released from the stomach. Ghrelin levels peak prior to the initiation of a meal. Additional GI derived signals not covered may also play important roles but their
23 actions on satiety and satiation are not yet completely understood. As their roles ar e further elucidated additional therapeutic targets for the treatment of overweight and obesity are likely to develop. 1.3.2 C entral Nervous System Traditionally hypothalamic nuclei have been attributed to regulate higher level feeding integration; however suc h a restri cted view overlooks the impo rtance of hindbrain nuclei and CNS areas involv ed with determining the reward value of food. Several excellent reviews on the complete and complex relationships of the CNS and feeding can be referred [36, 38 42] T he following section addresses topically some of the major c oncepts; while latter sections will spotligh t specific components of selected systems. The hypothalamus is very significant in feeding regulation due to its ability to respond to peripheral circulation signals and the ability to modulate output connectivit y by both hormonal as well as sympat hetic nerve activity. Similarly the hindbrain specifically the area post rema is sensitive to peripheral circulation whereas other hindbrain nuclei are linked to the vagal afferents responsive to GI signals. Furthermore there is bidirectional neuronal connectivity between hypothalamic and hindbrain areas. It is disingenuous to disregard other aspects of feeding including olfactory visual and gustatory components of food. Beyond that midbrain reward and incentive coding is responsive to feeding and components of this system are altered within the obese state. Finally it is also important to consider CNS areas involved with the formation of learned associations and memor y as feeding is not an isolated one time event. C learly illustrated is the complex nature of feeding as disruption of any component can result in dysregulation of feeding, while on the other hand multiple inputs and redundancies
24 allow for compensation, and thus can make the dissection of a singular alte ration difficult to observe or interpret. 1.4 Leptin and Leptin Resistance 1.4.1 Discovery Parabiosis experiments in which the blood supply of two animals is surgically connected, conducted by Hervey showed marked differences in fat content and body weight between animals of a pair in which one had hypothalamic lesions  Further parabiosis experiments involving genetically deficient mice with either the ob/ob (obese) or the db/db (diabetes) mutation led to the d iscovery that there was a deficiency in the production of a factor within ob/ob mice that could be recovered by parabiosis with either a db/db or wild type partner  In 1994, using a positional cloning approach the ob gene was shown to have a 167 amino acid open reading frame containing an N terminal sign al sequence which produced a secreted protein of 16 kilodalton s  Further s tudies confirmed the initial observation of expression within adipose tissue and that the ob product is expressed in relation to the amount of adipose tissue in multiple species [45, 46] Recombinant ob gene product was shown to decrease food intake and body weight in mice, pri marily through fat loss, and was gi ven the name leptin  Shortly after the discovery of leptin its receptor was also cloned  The discovery of leptin was initially thought to be the breakthrough for obesity treatment. 1.4.2 Leptin Action Adipose tissue is an important e ndocrine organ and releases multiple endocrine signals and cytokines  Important among these is l eptin which is released into circulation in proportion to the level of adiposity [50, 51] and produces effects on feeding [47, 52, 53] reproduction [54, 55] and immunity  Leptin induces intracellular
25 signaling cascades through binding of the long form of the leptin receptor (ObRb)  one of 6 subtypes of the leptin receptor  Alternative forms of the leptin receptor include short forms as well as a secreted form, these receptors are involved with le ptin binding [58 60] and transport across the blood brain b arrier  Signaling occurs through the association of proteins interacting with phosphorylated intr acellular tyrosine residues [48, 62 64] ; these residues become phosphorylated via receptor associated jan us kinase 2 (JAK2) that can bind to Box1 and Box2 motifs within the cytoplasmic tail upon leptin receptor dimerization [65 67] Feeding is controlled s pec ifically through signal transducer and activator of transcription 3 (STAT3) at Y1185  with STAT3 translocating to the nucleus to regulate the transcription of the pro opiomelanocortin (POMC) gene  and inhibition of aguti related peptide (AgRP)  Leptin receptors can also signal through mitogen activated kinase pathways  which is thought to involve the protein tyrosine phosphatase SHP2 at Y985  Further intracellular cascades can be stimulated by the leptin receptor including phosphatidylinositide 3 kinase, allowing for activation of protein kinase B and eventual actions with mammalian target of rapamycin (mTOR), AMP activated protein kinase (AMPK) and nitric oxide among others [64, 71 73] AMPK and mTOR are important energy regulating pathways and contribute to regulation of feeding [74, 75] Inhibitio n of receptor signaling occurs through binding of the protein tyrosine phosphatase SHP2 at Y985 and can act to decrease the activity of receptor assoc iated JAK2 [76, 77] leading to decreased phosphorylation of STAT3 Furthermore STAT3 activates the negative feedback regulator suppressor of cytokine signaling 3 (SOCS3)
26 which acts to suppress JAK2 phosphorylation  Additionally the protein tyros ine phosphatase 1B (PTP1B) is also able to inhibit JAK2 adding another mechanism of leptin receptor inhibition [79, 80] While the preceding mechanisms act to inhibit leptin signaling, it can also be sensitized  1.4.3 Leptin Receptor Expression The signaling form of the leptin receptor is expressed in diverse areas of the CNS  Dense expression occurs within the mediobasal hypothalamus  specifically in the arcuate nucleus in POMC  and neuropeptide Y ( NPY ) /AgRP neurons [84 88] Leptin a ctivation of POMC neurons  leads to the production of alpha melanocyte stimulating MSH) one of multiple gene products produced from POMC MSH binds to melanocortin receptors (MCRs) p roducing an anorectic response (reviewed in  ) Conversely AgRP acts as an antagonist of MCRs thereby acting in an orexigenic fashion [90, 91] Other hypothalamic areas associated with feeding regulation expressing leptin receptors include the ventromedial hypothalamus (VMH) [83 86] dorsomedial hypothalamic area (DMHa) [83 86] and the lateral hypothalamus (LH) [83, 85] Beyond hypothalamic areas, leptin receptors have also been found in the hindbrain, importantly withi n the nucleus tractus solitarius ( NTS ) [86, 92] an area important for integration of peri pheral feeding signals and coordinating energy expenditure. Midbrain dopamine neurons of the ventral tegmental area (VTA) and substa nt ia nigra also express leptin receptors [85, 93 95] Action of leptin in the VTA has been shown to alter dopamine neuron firing  and increase levels of the enzyme involved with dopamine synthesis  providing evidence that leptin i s potentially involved with altering food reward
27 1.4.4 Leptin Resistance Resistance to leptin, as the result of changes to intracellular signaling capacity is known as cellular leptin resistance while the general term of leptin resistance can be attributed to other causes  including reduced transport across the blood brain barrier  I n certain cases such as pregnancy or animals t hat undergo seasonal regulation, it can be considered a beneficial physiologi cal process  However in the case of human obesity leptin resistance is a mal adaptation that acts to defend the higher body weight  The development of leptin resistance in common obesit y has resulted in leptin monotherapy being an ineffective method of pharmacological treatment  Centra l leptin resistance appears to be a secondary process as in the early stages animals are not responsive to peripheral injection of leptin but can still respond to CNS infusion  Prolonged stimulation of leptin receptors results in down regulation of receptors and inhibited signaling capacity yet in cases of calorie restriction and lower leptin levels recep tors are upregulated [101, 102] Furthermore, l eptin resistance does not develop equal ly in all CNS nuclei with the arcuate nucleus being most prone [103, 104] In addition to the effect of leptin inhibiting signaling through negative feedback, increased endoplasmic reticulum stress  as well as CNS inflammation have been suggested as causes  In order to overcome leptin resistance strategies to prevent inhibition of leptin receptor signaling by SOCS3 [107, 108] and PTP1B [80, 109, 110] have been proposed Sensitization of leptin receptors has been shown to occur with short bouts of exercise  voluntary wheel running [112, 1 13] and the hormone amylin  Further research into the development of
28 nuclei specific resistance and re sensitization may indeed lead to leptin being a viable therapy for overweight and obesity. 1.5 Serotonin Pathways and Feeding In the pursuit of pharmacotherapy for depressi ve and anxiety disorders, which invole pertu r bations of the sero tonin ( 5 hydroxytryptamine; 5 HT ) system, it was discovered that modulation of 5 HT changed feeding. The receptors for 5 HT are classified into 7 families containing 14 receptor subtypes  While multiple families are implicated in effects on food intake  this section will focus on the 5 HT 2 B,C and 5 HT 1 b receptor, for full review of 5 HT receptors see  The 5 HT 2 receptors are G protein receptors coupled to G q resulting in phosphoinositide hy drolysis and production of inos itol phosphates and diacylglycerol causing increases in intrac ellular calcium and activation of protein kinase C, respectively ; in contrast the 5 HT 1B receptor is a G protein receptor coupled i/o which elicits an inhibitory action of the cAMP pathway therefore resulting in neuronal hyperpolarization [115, 117] The 5 HT 1B receptor is express ed thro ugh out the CNS, but shows a high level of expression within the arcuate nucleus  Furthermore it was shown by double labeling to be expressed on NPY neurons of the arcuate nucleus [119, 120] Consistent with the mechanism of action and its expression on NPY neurons, 5HT 1B agonists decrease feeding  NPY/AgRP neurons in the arcuate nucleus also contain inhibitory projections to POMC neurons and 5 HT 1B agonists show the ability to inhibit POMC neurons within the ARC  The 5HT 2C receptor is also expressed in the arcuate nucleus, specifically on POMC neurons [120, 122] MSH is potentiated by 5 HT  further linking the effect of 5 HT to control of feeding. The effects of 5 HT or 5 HT drugs on feeding behavior requi r e melanocortin 4 receptors
29  and expre s sion of the melanocortin 4 receptor specifically on POMC neurons is enough to res cue reduced food intake in response to drugs that release 5 H T  Furthermore 5 HT elicits important effects on dopamine, the neurotransmitter involved in reward behavior, another important component of feeding behavior. Dopa minergic neurons of the substant ia nigra and VTA are innervated by 5 HT neurons ; 5 HT stimulation alters the release of dopamine in the nucleus accumb e ns and striatum by a 5 HT 2 C/2B receptor mechanism  Of additional note, 5 HT and fenfluramine ar e able to stimulate energy expe n d iture  While the anorectic effects of the 5 HT 2B / 2C receptor agonist is well established as the mechanism of action for the drug fen fluramine [127, 128] the agonist effect at the 5 HT 2B recept or leads to cardiac valvopathy resulting in the drug being withdrawn from the market  The necesity of 5 HT 2C receptor specific agonists has lead to the development of new compounds and the newly approved obesity therapeutic lorcaserin is an example. Lor caserin shows higher selectivity for 5 HT 2C receptors but posseses agonistic activity at all 5 HT 2 receptors  A potentia lly safer therapeutic ( ) trans PAT has 5 HT 2C agonist properties but inverse agonist/antagonist properties at the 5 HT 2A/2B receptors  and was shown to reduce the intake of palatable treats i n fed mice  Interactions of short term satiety actions of 5 HT and the long term actions of leptin have been reported  B ased on such observations the ex periments in the following chap ters were conducted to further understand the contributions of serot onergic and leptin mechanisms on food intake and body weight homeostatsis. 1.6 Rat Models and Obesity Although it is inherently obvious that rodents are not humans, they serve as an adequate surrogate to model the human obesity condition. Monogenic cases of obesity
30 in humans are relatively rare and therefore animal mo dels with genetic causes need to be interpreted with caution. However rats exposed to dietary obesity share many of the similar characteristics observed with human obesity ; including altered lipid and glucose profiles, increased adiposity and leptin resis tance [134, 135] and are the result of perturbations to multiple gene pathways more similar to the human condition  With regard to leptin receptors the rat and human signaling form of the leptin receptor share 83% homology  while the leptin peptide shares 82% homology (NCBI BLASTP; P41159.1 Homo sapiens & P50596.1 Rattus norvegicus ). Not to be overlooked are important differences in metabolic rates between rodents and humans and alternate feeding patter n s. Additionally complications in translation between observations in rats an d humans occurs due to the inbr ed nature of laboratory animals and the outbred characteristics of humans  Even though distinct differences occur between rats and humans the nature of similarities between the species, the ease of handling, ease of measuring food intake and lower cost of drug delivery warrant the ir use in the study of obesity.
31 CHAPTER 2 DOSE RESPONSE TO ADENO ASSOCIATED VIRAL DELIVERY OF LEPTIN IN FIS C HER 344 X BROWN NORWAY RATS 2.1 Introduction The failure of leptin as a monotherapy for common obesity may be due to ob ese individuals presenting in a hyperleptinemic state [50, 51] and the resultan t resistance to the effects of leptin tempered the expectations of its usefulness as a therapeutic agent [99, 138] Y et leptin is an effective treatment for numerous other conditions that present with hypoleptinemic states such as hypothalamic am e no r rhea [139, 140] and l ipodystrophies where it improves not only the lipid profile but also has actions on glucose regulation [141 143] Furthermore leptin therapy in rodents with type 1 and type 2 diabet es has been shown to improve both the glucose and lipid profiles [144 146] unfortunately to date these result s have not translated to humans with type 2 diabetes [147, 148] As the mechanisms contributing to leptin resistance continue to be elucidated potent ial polytherapies involving leptin and sensitizing agents may indeed provide applicable therapeutic action for common obesity. Animals deficient in PTP1B [80, 109, 110] and SOCS3 [107, 108] have enhanced sensitivity to the anorexic act ions of leptin. Addi tionally, combination therapy with leptin and amylin (or its analog pramlintide) is able to produce greater reductions in food intake and bodyweight than either compound alone attributed to the sensitizing effects of amylin on leptin a ction [114, 149 151] further demonstrating the potential for leptin as a treatment for common obesity. A ca veat to the sensitization of leptin action is that the studies were performed in the short term. A fundamental issue with leptin therapy is that leptin itsel f is able to induce leptin resistance. High fat diet (HFD) feeding results in increased adiposity a nd
32 therefore increased levels of serum leptin ; in an attempt to increase sensitivity to leptin by over expression of leptin receptors specifically on POMC neurons mic e fed HFD actually had increased food intake and gained more body weight than wild type m ice fed HFD  A cleverly designed experiment by Knight et al.  using ob/ob mice (incapable of producing functional endogenous leptin) that were infused with exogenous leptin to match basal wild type leve ls showed conclusively that hyperleptinemia is necessary for the development of leptin resistance. When the ob/ob mice with normalized basal leptin levels and wild type mice were placed on HFD for 20 weeks then subsequently tested for leptin response usin g a 450 ng/hr infusion over 12 days the wild type were fully resistant while the ob/ob mice remained responsive to leptin  Furthermore CNS infusion of leptin in rats also produces only a short term decrease in food intake and resistance in NPY neurons is observed even w hen fat mass is significantly lower in animals compared to those pair fed to leptin infusion  In young lean rats long term CNS over expression of leptin, using adeno associated virus (AAV) resulted in an attenuation of leptin action on food intake red uction after a few months, while reduced food intake in aged rats was abolished by 3 weeks  In a study with middle aged rats the loss of effect on food intake occurred at one month however body wei ght remained lower than pair fe d rats presumably due to the continued action of leptin on increasing energy expenditure  The effect of leptin on energy expenditure is lost by 225 days of treatment in rats undergoing long term over expression of leptin ; additionally, the rats are n o longer able to respond to exogenous leptin treatment even though their endogenous serum leptin levels are lower than their matched controls that have higher adiposity yet maintain exogenous leptin
33 response  The lack of effect to exogenous leptin is due to a decrease in to tal signaling capacity of the leptin receptor as basal levels of STAT3 are increased with long term leptin treatment  F urthermore the lack of response to leptin in rats pretreate d with AAV leptin over expression results in exacerbated obesity when challenged with HFD, suggesting lack of response to increased endogenous leptin  In summary, the current study was designed based upon the following four observations: 1) c urrent and potential u s es of leptin in pharmacotherapy ; 2) t he differen t temporal physiological responses attributed to leptin action described in the previous paragraph ; 3) t he ability of leptin receptors to be up or down regulated [102, 159] ; and 4) t he finding of region specific leptin resistance in the CNS  In such, the experiment was design ed to examine both short and long term leptin action s including food intake, body composition changes, energy expenditure and CNS signaling capacity, in resp onse to dose dependent AAV leptin delivery within the CNS. Based on previous knowledge we expect to induce a leptin resistant state with high dose treatment b ut hypothesize that the lower dose treatments will provide a positive physiological effect withou t inducing a leptin resistant state. 2.2 Results 2.2.1 Two Month Adeno Associated Viral Leptin Treatment 184.108.40.206 AAV1 leptin delivery via 3rd ventricle causes dose dependent body weight change Rats were administered control vector or an escalating dose of r ecombinant AAV v ector encoding leptin to the 3 rd ventricle and resultant body weight and food consumption examined. Body weight was calculated as five day averages and results
34 are shown in Figure 2 1 Two Way ANOVA on bodyweight o ver time demonstrated a significant effect of treatment (p<0.001) and interaction of treatment by time (p= 0.042). Animals receiving 1.77x10 10 vg/rat AAV1 GFP, control group (CTRL), resumed normal development and weight gain following initial surgical insu lt observed from day 0 to day 5. In the group receiving the lowest dose of leptin1.76x10 6 vg/rat, low dose (LD), body weight responded similarly to the control condition. Rats that received the 1.76x10 8 vg/rat leptin, middle dose (MD), show no apparent wei ght loss over the experimental period, but in contrast to LD leptin and control, these animals did not display the normal growth associated weight gain. Treatment with the 1.76x10 10 vg/rat of leptin, high dose (HD), led to a decline in body weight, reachin g a nadir at 25 days following surgery, whereafter body weight stabilized for the remainder of the experiment. Body weight differed significantly for HD treatment compared to day 0 from day 25 onward (p<0.05). Beginning at day 20 HD treatment was signific antly different from all other treatment groups at each time point evaluated (p<0.05 day 20; p<0.01 days 25 60). When the body weight is expressed as the difference from body weight at the ti me of vector injection ( Figure 2 2 ), t he results were similar to that for body weight over time. Statistical testing by Two Way ANOVA demonstrated significant effects of treatment, time, and interaction of treatment and time (p<0.05). SNK post hoc test revealed that time was onl y significant in comparison to baseline in the HD leptin group from day 10 onward (p<0.001). Injection of GFP control vector produced a surgical insult and was followed by a resumption of normal growth associated weight gain. No effect of LD leptin was obs erved, as weight changes were virtually identical to the changes observed in the CTRL group. Treatment with 1.76x10 8 vg/rat leptin produced a
35 statistically significant difference from control and low dose treatment from day 20 onward. The highest dose of l eptin induced the greatest decrease in body weight and resulted in a statistically significant difference from day 10 onward when compared to control and low dose; with respect to the middle dose used, the difference became statistically significant at day 15. 220.127.116.11 Rats treated with increasing doses of leptin display greater but transient decreases in food consumption Food consumption of chow diet was evaluated following treatment with rAAV control and leptin vectors and the results are expressed in Figure 2 3 Data was analyzed using Two Way ANOVA with SNK post hoc tests. Animals in the control group responded to vector injection with a post surgical decrease in chow intake yet resumed consumption of diet at baseline lev els 10 days post surgery. LD leptin treatment did not differ from controls during the two month experimental period. Food consumption in the MD leptin treated group was significantly less than in the control group for days 5 20 (p<0.05). Treatment with HD leptin demonstrated the greatest reduction in food intake, and was significantly different from control for days 5 20 (p<0.01 day 5, p<0.001 for days 10 20), from LD days 10 20 (p<0.001), and from MD for days 10 15 (p<0.01). As can be observed from Figure 2 3 these effects are transient as all groups returned to approximately baseline consumption values by day 30. Further investigation into the cumulative food consumption is presented in Figure 2 4 Thirty days after treatment, significant differences exist between groups as demonstrated in Figure 2 4 A (One Way ANOVA, p<0.001). SNK post tests revealed no significant difference betwee n LD and control group; MD was significantly different from both CTRL and LD (p=0.017 and 0.035, respectively). The HD group had consumed the
36 least diet after 30 days and was significantly different from both the control and LD groups, but not from the MD group (p<0.001, p=0.001 and 0.095, respectively). Two Way Repeated Measures ANOVA for the entire experimental period maintain the group differences throughout the 60 day expe riment, however simple analysis at endpoint day 60 ( Figure 2 4 B ) revealed no statistically significant difference by One Way ANOVA. Of principal interest is the observation that consumption in the HD leptin group after day 30 for the remainder of the experimental period remains elevated in compa rison to the other groups ( Figure 2 3 black triangles, and Figure 2 4 C ). 18.104.22.168 Leptin treatment induces changes to b od y c omposition Body composition was altered in a dose depende nt fashion with leptin treatments. Animals were analyzed for total adiposity and total lean mass using TD NMR on day 0 prior to injection with vectors, and at four additional time points throughout the experiment. Individual rats were evaluated with respec t to their own baseline value. Data were analyzed using Two Way ANOVA with Repeated Measures using SNK post hoc tests. Figure 2 5 A demonstrates that normal growth results in moderate increase of body fat accumulati on in the control group. The pattern of increased body fat is echoed in the LD treated group. Treatment with MD leptin resulted in a decrease in body fat at time point 1 (day 15 p=0.013); the initial decrease was maintained for time point 2 (day 30 p=0.016 ); however by time points 3 and 4 (day 45 and 60) body fat composition returned to approximately baseline values (p=0.068 and p=0.538, respectively). At day 60 body fat following MD leptin treatment was at a lower level than in both control and LD treated rats, however this decrease was not statistically significant with respect to either control or LD group. HD leptin treatment resulted in the greatest reduction in body fat as expected. The reduction in fat was
37 preserved through the time frame of the exper iment (p<0.001), nevertheless even in the HD group fat began to accumulate after the observed decrease at time point 1. Body fat percentage in the HD group was significantly different from all other groups at all time points (p<0.001, except from MD at tim e points 2 and 4, p=0.006 and p=0.003, respectively). Lean mass changes are generally the inverse of changes to adiposity and indeed this was observed within the experiment ( Figure 2 5 B ). With respect to lean mass in both the control and LD treated group, no significant differences were observed from baseline or between the groups at any time point. The MD treated group showed a significant increase in lean percentage from baseline at time points 1 and 2 (p<0.05). H owever, MD group did not differ from either control or LD at any time point. Similar to the body fat percentage results, the HD group was significantly different from baseline and from all other groups at all time points (p<0.001, except for MD at time poi nts 2 and 4 where p=0.009 and p=0.005 respectively). In order to substantiate the body composition data, selected adipose tissue depots were excised and weighed from each of the animals at the end point two months after treatment. Results of the pooled wet tissue weights of the depots from individual animals are presented in Figure 2 5 C One Way ANOVA revealed a significant effect of treatment (p<0.001), and SNK post test revealed that the HD treatment group was sig nificantly different from all other groups (p<0.001, except for MD where p=0.014). Inter group differences were present in the individual fat depots (epididymal, perirenal and retroperitoneal) with the exception of the control and LD treated groups (p<0.05 data not shown).
38 22.214.171.124 Leptin t reatment reduces iBAT size a nd changes relative UCP1 levels Leptin not only alters feeding and white adipose tissue depots but controls thermoregulatory pathways. Interscapular brown adipose tissue (iBAT) size changed in a dose de pendent fashion to treatment with leptin, as in the MD treated group iBAT was decreased by ~20% and HD resulted in a ~40% reduction compared to control ( Figure 2 6 A ). One Way ANOVA detected an overall effect of p<0 .01; SNK post hoc testing revealed statistically significant differences for HD treatment in comparison to LD and CTRL (p<0.05) but not compared to MD treated group. UCP1 levels were also evaluated, and as indicated in Figure 2 6 B and 2 6C an interesting divergence was observed depending on the dose of leptin used. Low dose leptin treatment reduced levels of UCP1 per microgram of iBAT protein; MD leptin treatment resulted in slight increase of UCP1 in comparison to GFP control, while UCP1 levels were elevated with HD leptin treatment although One Way ANOVA did not detect a statistically significant difference between groups (p=0.112). When levels were adjusted to the total level of iBAT protein present within the dep ot, UCP1 levels were decreased ~20%, 17% and 3% compared to CTRL in LD, MD and HD groups respectively. There was no statistically significant difference detected between any groups. These results suggest that the changes in bodyweight at two months are lik ely not due to alterations in UCP1. 126.96.36.199 Leptin elevates metaboli c rate Fifty five days after surgical delivery of rAAV vector expressing leptin rats were evaluated for changes to metabolism using indirect calorimetry. Rats were e valuated for oxygen consumptio n and carbon dioxide production the respiratory quotient (RQ) a s well as heat production was then calculated for individual animals. Leptin treatment did not greatly affect RQ values for the groups; values were in the range of 0.852 0.873,
39 with LD leptin having the lowest and HD treatment having the highest RQ value ( Figure 2 7 A ). Statistical analysis revealed no significant differences between means. In regards to heat production, treatment with leptin elevated th e rate of heat production in all groups compared to control. Interestingly observing the trends for heat production the MD treatment produced the greatest increase while the increase observed with HD leptin treatment was less than that of either MD or LD treated rats ( Figure 2 7 B ). As with the analysis of RQ values, One Way ANOVA revealed no statistically significant differences between groups for heat production The analysis was limited by the lack of power due t o the small sample size used. 188.8.131.52 Increasing doses of leptin desensitize leptin receptors in the mediobasal h ypothalamus At sacrifice animals were stimulated with an acute supra physiological dose of leptin (1 g)  in the 3 rd ventricle to examine the ability of receptors to respond to leptin by induction of STAT3 phosphorylation. Figure 2 8 shows that there was a dose dependent desen sitization that occurred in the mediobasal hypothalamus of rats treated with leptin. Phosphorylated STAT3 was reduced compared to GFP control treated rats by ~22, 35, and 45% in LD, MD and HD treated groups, respectively. One Way ANOVA resulted in an overa ll group effect with p=0.015. SNK post test indicated differences between groups were only evident between MD and control, as well as HD and control. 2.2.2 Six Month Adeno Associated Viral Leptin Treatment 184.108.40.206 AAV1 leptin delivery via 3 rd ventricle causes dose depen dent body weight c hange ; high dose of leptin results in earlier onset and more rapid weight regain Rats were administered control vector or an escalating dose of rAAV vector encoding leptin to the 3 rd ventricle on day zero. Body weight of the rats was eval uated
40 daily. Five day averages were calculated and are represented in Figure 2 9 Two W ay ANOVA on bodyweight over time demonstrated a significant effect of treatment (p<0.001) and of time (p= 0.010), with no signi ficant interaction of treatment and time. Control group animals resumed normal development and weight gain following initial surgical insult observed from day 0 to day 5. The LD leptin treated group had body weights virtually identical to that of the contr ol group throughout the experiment. The observed decrease in body weight following day 60 was likely due to the experimental interventions performed between day 55 and 60, including respiratory measurements, body composition and tail bleeding. The MD lepti n group did not resume growth related weight gain as was observed in both the control and LD leptin groups, yet weight reduction did not occur until day 45 and reached its nadir at day 80. The leptin mediated delayed onset of weight reduction in the MD tre ated group is in contrast with the HD leptin group in which accelerated weight loss was observed; the minimum weight in the HD group was reached at day 65, a fter which weight regain began. Data expressed as the change in body weight from surgical day 0 in Figure 2 10 more clearly define the differential response to doses of leptin used. Two Way ANOVA identified a significant effect of both treatment and time (p<0.001), but no interaction between treatment and time. SNK post hoc test revealed significant differences between all groups (p<0.001) with the exception of CTRL and LD (p=0.731). Control and LD groups recovered from the initial surgical insult rapidly, while the rats in groups treated with either MD or HD lep tin initially showed a decrease in body weight. Of note was that between 2.5 and 3 months post treatment both MD and HD leptin groups began to regain body weight. Comparison of the average daily weight gain from day 80
41 until the terminal data point at day 180 in the experiment indicates that daily weight gain was greatest in the HD group. Daily weight gain in HD treated animals was 43% greater than in the CTRL group; LD and MD treated animals gained 10% and 17% less weight per day than CTRL. The increased r ate of weight regain in the HD group resulted in the HD group having a slightly greater body weight at sacrifice than MD treated group, even though the initial weight loss was approximately 15 grams greater. Both MD and HD treated groups still had lower bo dyweights than CTRL and LD treated animals at 6 months. 220.127.116.11 Rats treated with increasing doses of leptin display g r eater but transient decreases in food c onsumption ; the highest dose results in long term increased chow intake Five day averages of chow food con sumption were calculated for the duration of the experiment and all groups responded to the surgical procedure by decreasing consumption and then returning to baseline consumption. Consumption fluctuated around baseline value by plus/minus 2 grams in all g roups at all data points that were measured (data not shown) Two Way ANOVA revealed a significant effect of both treatment and time (p<0.001); SNK post test re vealed in significant differences between all groups (p<0.05) with the exception of Control and L D leptin treatment (p=0.141). Leptin treated rats exhibited a dose dependent decrease in food consumption for the initial month post surgery, with HD leptin treated rats consuming the least chow ( Figure 2 11 A ). On e Way ANOVA for the total chow consumed for days 0 30 did not reach the statistically significant threshold (p=0.067), however the HD treated group consumed 72.3 grams less than CTRL. Thirty days post treatment, the effect of HD leptin treatment was to rev erse the decreased consumption ( Figure 2 11 B ), and in fact this group consumed 142.4 grams more over this period than the next highest group.
42 The dichotomy of HD leptin treatment effect on food intake is better obs erved in Figure 2 11 C and 2 11D which display the average grams of chow consumed per day for the first 30 days and for the remaining 150 days. The daily difference for HD leptin treated animals after day 30 ( Figure 2 11 D ) is ~2.2 grams greater than in the first month following treatment ( Figure 2 11 C ). When the total chow consumption for the entire duration of the experiment was eva luated, HD treated group consumed 2901 119 grams, compared to 2830 81 grams for CTRL; both LD and MD treated groups consumed less than CTRL group, 2801 89 and 2730 75 grams, respectively (Mean SEM). One Way ANOVA did not reveal a statistically si gnificant difference between groups. 18.104.22.168 Changes to body c omposition over time are dependent on the dose of leptin treatment Leptin is known to alter body composition by preferentially reducing fat mass in animals that are sensitive to leptin. In order to eval uate changes to body composition, rats were subjected to TD NMR periodically throughout the six month experimental period. Changes in percentage of fat mass and lean mass were calculated relative to baseline values obtained prior to treatment and are expre ssed in Figure 2 12 Two Way ANOVA of percentage fat mass revealed a significant effect of both treatment and time (p<0.001); SNK post test indicated differences between all groups (p=0.006 for MD and HD, p<0.001 f or other comparisons), with the exception of CTRL versus LD leptin group. The CTRL and LD leptin groups both exhibited an increase in the percentage of body fat as is expected with normal development; however the MD and HD treatment revealed different temp oral effects of treatment ( Figure 2 12 A ) The HD leptin group had the greatest magnitude decrease which occurred immediately after treatment, yet the
43 decrease in body fat percentage was lost by four months post tre atment and values were again similar to baseline levels. In the MD leptin treated animals the reduction of body fat percentage was not as great as in HD treated, but the reduction appeared more stable for the first three months post treatment in contrast t o the changes observed in the HD group. No overall difference between groups was present at the endpoint of the experiment, and all groups had increased their percentage of body fat over baseline. Lean mass changes tend to occur in opposition to changes to body fat mass; indeed such a pattern was observed over the 6 month post treatment period ( Figure 2 12 B ) HD animals demonstrated the greatest increase in lean mass percentage initially, but as fat mass percentage increased, the gain in lean mass percentage decreased. Approximately 1% increase in lean mass as a percentage of body weight compared to baseline was observed in the MD leptin treated group in the first 3 months post treatment, after which this gain was lo st. Two Way ANOVA for lean mass percentage mimicked that of fat percentage as a significant effect of both treatment and time were detected (p<0.001). All treatment groups with the exception of Control and LD were identified as being statistically differen t by SNK post test (p=0.012 for MD to HD, all other comparisons p<0.001). At sacrifice 6 months post treatment, the epididymal, perirenal and retroperitoneal adipose tissue depots were excised and weighed in order to corroborate the TD NMR body composition data. The results of the pooled weight of the respective depots are presented in Figure 2 12 C Although the changes are not drastic, even 6
44 months post treatment a dose dependent trend in the reduction of adipose tissue remained (One Way ANOVA p=0.198). 22.214.171.124 Leptin t reatment changes relative UCP1 levels at six months in iBAT but does not change the size of depot After six months of treatment with increasing doses of AAV1 leptin the iBAT was removed at sacrifice and UCP1 levels were evaluated. Six months of treatment did not alter the size of iBAT in rats as demonstrated in Figure 2 13 A The weight of iBAT in CTRL, LD and HD treated groups was virtually identical, while the MD tre ated group had a slightly lower iBAT weight; no statistical significance was detected between groups by One Way ANOVA (p=0.739). Six months of AAV leptin treatment did slightly increase UCP1 levels per microgram in both the MD and HD treated groups over bo th LD and CTRL groups ( Figure 2 13 B ). The variance in the LD group was quite large and therefore required Kruskal Wallis One Way ANOVA on Ranks to be performed, no statistical difference was observed (p=0.248); how ever it is important to note the when the median value was considered a dose dependent increase in UCP1 per microgram was observed (median of LD group greater than CTRL but less than MD group, greatest median in the HD group). Adjusting UCP1 levels to the size of the iBAT depot revealed a dose dependent increase in UCP1 per depot ( Figure 2 13 C ). As with the per microgram results, variance was large and demanded use of Kruskal Wallis One Way ANOVA on Ranks testing, w hich indicated no statistical differences between the groups (p=0.649). Interestingly UCP1 appears to be elevated in the HD treated group even though this group was in a rapid weight gain state at 6 months; additionally UCP1 is elevated compared to levels observed after 2 months of treatment.
45 126.96.36.199 Dose of leptin may alter metabolic substrate preference long term without change to overall metabolism Indirect calorimetric analysis was performed 163 days post treatment to determine oxygen consumption and carbon dio xide production. The respiratory quotient was calculated for all groups and the values were ~0.815 for Control, LD, and MD; while the RQ for HD leptin treated animals was higher at 0.841 ( Figure 2 14 A ). The differe nces between groups did not reach the statistically significant threshold that was set (One Way ANOVA p=0.063); however it should be noted that the power of the test was relatively low. More interestingly, when heat production was analyzed all leptin treat ed groups, irrespective of dose, showed a trend toward elevated heat production compared to CTRL ( Figure 2 14 B ). 188.8.131.52 Chronic leptin treatment diminishes the ability of acute leptin to stimulate STAT3 phosphorylation in select central nervous system areas Different central nervous system (CNS) nuclei may play independent roles in the diverse effects of leptin in regards to maintaining body weight homeostasis. Four CNS areas were selected to evaluate signaling capacity of leptin receptors in response to acute leptin stimulation following the long term treatment with increasing doses of leptin. Both the arcuate nucleus (ARC) and the dorsomedial medial hypothalamic area (DMHa) have relatively high level expression of leptin receptors; in each of these areas escalating doses of chronic leptin treatment demonstrated a dose dependent decrease in STAT3 phosphorylation in response to a single acute pharmacological 3 rd ventricle injection of leptin (1g) ( Figure 2 15 A & B ). The reductions in comparison to CTRL were approximately 12.5%, 25% and 35 % for LD, MD, and HD pretreated groups, respectively in the ARC and approximately 25%, 34% and 37% in the DMHa. The
46 observed decreases did not reach the statistically significant threshold when evaluated by One Way ANOVA (p=0.115 for ARC, p=0.482 for DMHa). In addition to the DMHa, the nucleus tractus solitarius (NTS) and medial preoptic area (MPOA) contain leptin receptors and have projections to the iBAT. Therefore the ability of leptin receptor signaling was also evaluated in these areas. Figure 2 15 C demonstrates that six months post treatment there appears to be no effect of any dose of leptin in altering the signaling capacity within the NTS. In contrast, within the MPOA ( Figure 2 15 D ) the HD leptin treatment reduced STAT3 phosphorylation in response to acute leptin (~30%), a magnitude similar to what was observed after HD treatment in both the ARC and DMHa. MD leptin treatment reduced phosphorylation of STAT3 by only ~10% in the MPOA compared to CTRL, while there was no difference between LD treatment and CTRL. One Way ANOVA for the NTS and MPOA did not detect stat istically significant differences. 2.3 Materials and Methods 2.3.1 Animals Six month old male Fisher 344 x Brown Norway rats were obtained from the National Institute of Aging colony. Rats were single housed in ventilated cages to allow for measurements of chow cons umption in individual rats. Animals were cared for Guide for the Care and Use of Laboratory Animals and the protocols were approved by the University of Florida Institutional Animal Care and Use Committee. Animals were assigned to one of four treatment groups (n=12/group), half of the rats from each treatment were sacrificed at 2 months while remaining rats were sacrificed at 6 months.
47 2.3.2 Adeno Associated Viral V ectors The pTR(2)ObW construct encoding leptin transgene under a chicken actin promoter linked to CMV enhancer was packaged into recombinant adeno associated virus (rAAV) serotype 1 by the Powell Gene Therapy Vector Core Lab at the University of Florida using the methods of Zolotukhin et al.  with the exception that the AAV1 helper plasmid pKRAP1A was used Dot Blot titer of rA AV1 leptin was 5.86x10 12 viral genomes (vg) per milliliter. Control vector UF11 rAAV1 encoding green fluorescent protein (GFP) was obtained from the same facility. The rAAV1 GFP titer was calculated by Dot Blot at 5.90x10 12 vg/ml. The stock rAAV1 leptin ve ctor was used as the High Dose (HD) leptin treatment. 1:100 dilutions of rAAV1 leptin were made in artificial cerebrospinal fluid (aCSF; 125mM sodium chloride, 3mM potassium chloride, 1mM disodium phosphate, 1.4mM calcium chloride, 1mM magnesium chloride, 10mM alpha D glucose, pH7.4 filter sterilized through 0.2 m nylon membrane) to obtain the Middle Dose (MD) and Low Dose (LD) treatments. 2.3.3 Surgery Anesthesia was induced using 5% isoflurane. Upon failure to present toe pinch reflex, the animal was mounted into a stereotaxic frame and anesthesia was maintained at 2% isoflurane throughout the duration of surgery. 3 l of rAAV1 vector was delivered into the third ventricle (coordinates: 1.3 mm anterior to Bregma, 0.0 mm from midline, depth of 9.6 mm ventral fr om surface of skull, at an angle of 20, [16 2] ) at a rate of 1 l/minute followed by a retention time of 2 minutes. Coordinates were verified previously in separate rats using injection of bromothymol blue dye. The use of 3 l resulted in a dose of 1.77x10 10 vg/rat for CTRL (rAAV1 GFP), 1.76x10 10 v g/rat for HD, 1.76x10 8 vg/rat for MD and 1.76x10 6 vg/rat for LD (rAAV1 leptin). Immediately preceding, and up to 48
48 hours post surgery rats were given buprenorphine analgesic in 12 hour increments; rats were also provided with a nutritiously dense post sur gical recovery gelatin cube. 2.3.4 Food Intake & Body Weight A ssessment Rat body weight was obtained at ~24 hour intervals, however for representation and clarity of data, 5 day averages were calculated. Similarly food consumption of chow diet (Harlan Standard Rodent Chow Diet 7912, 3 .1 kcal/g, 17 % kcal Fat, 25% kcal Protein ) was obtained daily for individual rats by calculating the difference in weight of chow for the 24 hour period accounting for any spillage into the cage. Diet was replaced every two weeks co inciding with cage changes. 2.3.5 Body C omposition Time Domain Nuclear Magnetic Resonance (TDNMR) was used to perform measurements of fat, lean and fluid mass. Live unanaesthetized animals were weighed and placed into sampling tubes and measured using a minispec LF90 analyzer (Bruker Optics, The Woodlands, T X ). Measurements were taken in duplicate for each animal at approximately 15 day intervals throughout the experiment. Fat and lean mass were calculated as a percentage of body weight for each individual animal at time point zero immediately prior to surgery and the resulting value was used as the baseline value for all future calculations. At sacrifice, select fat depots were collected and weighed to provide a secondary measurement for alterations in body compo sition. The epididymal (EWAT), perirenal (PWAT), and retroper itoneal white adipose tissue (RTWAT) were summated to provide values for total white adipose tissue. 2.3.6 Respiratory M easurements Oxygen consumption and carbon dioxide production were assessed using a modular animal respirometry system consisting of an Oxygen Analyzer S 3A/I and
49 Carbon Dioxide Analyzer CD 3A (AEI Technologies, Naperville, IL). The unit consists of 8 chambers allowing for assessment of 7 animals per run with 1 reference chamber. Instru ments were calibrated on a two point calibration curve using room air and a nitrogen gas standard containing 20% O 2 /4% CO 2 Flow rates for each chamber were set to 1L/minute. Prior to taking measurements a 60 minute acclimation time was used, followed by a 64 minute analysis period. Each chamber was analyzed in rotating one minute intervals and data was captured using Logger Pro 3.3 Software (Vernier, Beaverton, OR). 2.3.7 Acute Leptin S ignaling Leptin peptide (Amgen, Thousand Oaks, CA) was diluted to a concentr ation of 0.2 g/l in aCSF. Rats were anesthetized using a cocktail of ketamine and xylazine, then mounted into a stereotaxic apparatus, and the 3rd ventricle was targeted for delivery of acute peptide using the same coordinates as for initial vector treat ment (1.3 mm anterior to Bregma, 0.0 mm from midline, depth of 9.6 mm ventral from surface of skull, at an angle of 20). Acute leptin was delivered at a rate of 1 l/minute over 5 minutes, resulting in a total injection volume of 5l and a resulting dose of 1 g leptin per rat. Following leptin delivery, a 1 minute retention time was maintained prior to removal of the cannula. Rats were sacrificed 1 hour following the acute leptin injection. 2.3.8 Interscapular B rown Adipose Tissue A ssessment An incision was mad e along the dorsal midline of the rat and the interscapular brown adipose tissue (iBAT) was removed. Muscle and white adipose tissue that were attached to the iBAT was subsequently removed. The remaining iBAT tissue was weighed using an analytical scale. F or measurement of UCP1 a n approximately 20 mg sample was removed from the iBAT and homogenized in 0.300 ml buffer (10 mM Tris
50 HCl, 2% SDS pH 6.8) by sonication. Remaining cellular debris was removed by centrifugation and supernatant was transferred to fres h tubes. In order to remove lipid component, the supernatant was passed through a 0.45 m syringe filter (Whatman Inc., Florham Park, NJ). The p rotein concentration of samples was determined by detergent compatible protein assay (Bio Rad Laboratories, Herc ules, CA). 10 g of protein samples was electrophoresed on 10% Tris HCl polyacrylamide gels and subsequently transferred to nitrocellulose membranes. UCP1 expression was detected using primary antibody to UCP1 (ab10983, A bcam, Cambridge, MA) and horseradis h peroxidase conjugated secondary antibody (Cell Signalling Technology Inc., Danvers, MA). The blots were developed using Amersham ECL P rime reagent (GE Healthcare Biosciences, Piscataway, NJ) and scanned using a (Bio Rad Laboratories, Hercules, CA) Image Lab 3 .0 software (Bio Rad Laboratories, Hercules, CA) was used for densitometry analysis of bands. UCP1 expression was adjusted for the size of the iBAT depot from the individual rat sample. Res ults were expressed as the percentage of expression in the appropriate control treated rats. 2.3.9 Evaluation of S ignal T ransducer and A ctivator of T ransc ription 3 P hosphorylation In animals that were sacrificed at the two month time point the mediobasal hypotha lamus was collected by cutting medially of the piriform lobes, caudal to the optic chiasm and rostral to the cerebral crus at a depth of 2 mm. Brains from animals sacrificed at 6 months were placed on tissue slicer ( Stoelting Co. Wood Dale, IL) and two, 2 mm slices starting from the caudal end of optic chiasm extending in both directions were made. In the rostral section, the MPOA was punched bilaterally using a 1 mm micropunch ( Stoelting Co. Wood Dale, IL). In the caudal slice the DMHa was
51 punched out bi laterally and the ARC was removed using a surgical blade. For the NTS the slicer was aligned with the rostral edge of the cerebellum and then moved caudally 2 mm; a slice extending 2.5 mm caudally was taken and the NTS punched out bilaterally. Tissue was c ollected in 200 l of homogenization buffer (10 mM Tris HCl, 2% SDS, pH 6.8) for MBH, and in 50 l homogenization buffer for 6 month brain punches. Samples were briefly sonicated. Protein concentration of samples was determined by detergent compatible prot ein assay (Bio Rad Laboratories, Hercules, CA). Thirty g of protein samples w ere electrophoresed on 10% Tris HCl polyacrylamide gels and subsequently transferred to nitrocellulose membranes. Phosphorylated STAT3 was detected using primary antibody to pST AT3 ( 9131 Cell Signaling Technology Inc., Danvers, MA ) and horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology Inc., Danvers, MA). After obtaining the pSTAT3 signal, the blots were stripped and probed for total STAT3 ( 9132 Cel l Signaling Technology Inc., Danvers, MA ). B lots were developed using Amersham ECL Plus reagent (GE Healthcare Biosciences, Piscataway, NJ) and scanned using a Storm 860 Phosphorimager (GE Healthcare Biosciences, Piscataway, NJ). ImageQuant5.0 software (Mo lecular Dynamics Inc., Sunnyvale, CA) was used for densitometry analysis of bands. Phosphorylated STAT3 was normalized to total STAT3 and results are expressed as percentage of GFP control treated samples. 2.3.10 Statistics One Way ANOVA Kruskal Wallis One Way A NOVA on Ranks when necessary or Two Way ANOVA (with Repeated Measures when appropriate) were preformed using SigmaSt at v3.1 (Systat Software, Inc., San Jose, CA). Student Newman Keuls
52 (SNK) posttests were used for group comparisons where the main effect wa s found to be significant. The s tatistical signific ance threshold value was set at p<0.05. 2.4 Discussion Administration of HD leptin resulted in similar changes in magnitude and temporal response with respect to both body weight and food intake as compared t o a previous experiment employing the same dose  indicating reproducibility of responses. While t he expression of leptin within the target areas was not directly evaluated within these experiments, in the aforementioned study  leptin was detected by reverse transcription polymerase chain reaction 452 days after injection using the same injection coordinates within the hypothalamus. The evidence of expression and similar physiological responses s trongly suggests that delivery of rAAV1 leptin produced long term sustained leptin production in the current experiment. Additional evidence for the ability for rAAV to express in target cells was de monstrated by injection using a rAAV GFP vector and obser ving GFP expression by immunohistochemistry. Transduced cells were observed in the areas lining the 3 rd ventricle injection site including the bed nucleus of the anterior commi s sure, ventral preoptic area, suprachiasmatic nucleus, anterior hypothalamus, pa raventricular nucleus, DMH, and arcuate nucleus  Furthermore, the ability for rAAV leptin to produce secreted leptin was previously confirmed by observing an approximate two fold increase in cerebrospinal fluid leptin levels assessed from the spinal column following leptin treatment; no increase in serum leptin levels was observed  Detection of elevated leptin levels within the spinal fluid provides evidence that even hindbrain leptin receptors were exposed to elevated leptin levels following 3 rd ventricular treatment. Finally, basal leptin signa ling as measured by STAT3 phosphorylation is elevated two fold 152 days
53 after treatment with rAAV1 leptin, and the administration of leptin antagonist was able to reduce levels to untreated baseline values  The dose dilution technique used suggests that there is a minimum threshold response for leptin to elicit physiological effects as no differences we re observed between the LD and control vector. A general pattern of dose response was observed in the two month data with one important exception, food intake. The reduced food intake response to leptin only followed within the dose response pattern for on e month following treatment, after which HD treatment resulted in greater intake than all other groups ( Figure 2 4 A versus C ). This observation suggests that the onset of leptin resistance occurs along a distinct t imeline with the food intake being the earliest target. This observation appears to be logical considering that major sites of regulation of food intake are the POMC and AgRP/ NPY neurons of the arcuate hypothalamus, and it has been previously shown that t he arcuate nucleus is most susceptible to the development of leptin resistance  When pSTAT3 responsiveness to an acute injection of leptin was measured after 60 days, a measure of leptin receptor signaling capacity ( Figure 2 8 ), the HD trea tment exhibited the greatest reduction in STAT3 phosphorylation. Although the measurement used the whole mediobasal hypothalamus and not specifically the arcuate nucleus it is likely reflective of the action of POMC and Ag RP/NPY neurons as the predominan t leptin receptor expressing neurons within the mediobasal hypothalamus are located in the arcuate nucleus (Figure 1 in  ). Though the reduction of food intake is abolished in both the MD and HD group after the first 30 days of treatment the reduction in body weight is maintained through day 60. Presumably the maintenance of body weight reduction occurs through elevated
54 energy expenditure due to leptin The loss of the anorexic action of leptin preceding that of the leptin effect on energy expenditure has been demonstrated previously  Leptin activates the sympathetic nervous sy stem and produces action on adipose tissue  Leptin action on UCP1 activation in iBAT requires sympathetic enervation  At 2 months UCP1/g was elevated in the HD treatment group while in the MD group this effect was not observed. This observation likely explains why HD leptin body weight is lower with HD treatment than MD considering that by day 60 both these groups consumed an equal quantity of chow ( Figure 2 4 B ). In contrast to previous observations in which UCP1/g was no longer elevated at 153 days, UCP1/g remained slightly elevated in HD compared to control treatmen t after 180 days ( Figure 2 13 B ); s uggesting that leptin may indeed be able to maintain an increase to energy expenditure long term, though it should be noted the HD lep tin used in this experiment 1. 76 x10 10 vg/rat was less than the 2.5x10 11 vg/rat dose used in the previous study  Thus the observation that an even higher dose of leptin than the one used in the current experiment abolishes the effect on energy expenditure at an earlier time point furthers the theory that onset of leptin resistance is related to the amount of leptin present. Further evidence to support that the onset and sever ity of leptin resistance is related to the amount of leptin present can be observed in the 6 month leptin treatment data. Daily food consumption after the dose dependent hypophagic effect during the fi r st 30 days, is markedly elevated in HD treatment comp ared to the other groups ( Figure 2 11 D ). The elevation in food consumption markedly accelerates the weight gain in this group beginning at 3 months ( Figure 2 10 ), in fact b y 6 months the weight of HD treated rats was greater than MD eliminating the 15 gram difference between the
55 groups observed at the nadir of weight loss. Although both the MD and HD groups exhibited lower bodyweights at day 180 it is expected that the trend in weight gain would continue in the HD group had the experiment continued. Daily weight gain in HD treated animals was 43% greater than the control group whereas the LD and MD treated animals gained 10% and 17% less weight per day than controls beginning at 3 months. Moreover, the effect of leptin on body composition was short lived as even at 15 days following treatment the percentage of body fat began to increase in the HD treate d group, whereas with MD treatment the increase occurred after 30 days ( Figure 2 5 A and Figure 2 12 A ) It would be interesting to observe a HFD challenge between the groups at some point after 3 months of treatment, as presumably the state of lept in resistance induced by the varying doses would be amplified  Retrograde viral tracing by injection of pseudorabies virus in to iBAT was observed in mice that specifically express GFP in neurons that contain leptin receptors. Viral particles were shown to be co exp ressed with leptin receptor containing neurons in the DMHa, MPOA, and NTS  For this reason at 6 months leptin responsiveness was observed in the DMHa, MPOA and NTS in addition to the arcuate nucleus At six months iBAT weights were similar between all the respective treatment groups while MD and HD lep tin treatment revealed non statistically significant increase in UCP1/g of iBAT tissue These results appear to be somewhat inconsistent with the reduction in STAT3 phosphorylation observed within the DMHa and MPOA with both MD and HD leptin treatment, ho wever it stands to reason that since UCP1 activity is dependent on sympathetic nervous system activity that activation is independent of the leptin STAT3 pathway. Indeed sympathetic nervous system control by leptin has recently been
56 attributed to changes w ithin PI3K and mTOR signaling pathways  Within the NTS no dose dependent changes to STAT3 phosphorylation were observed, however the possibility that the delivery of ac ute leptin into the 3 rd ventricle may not have stimulated t he NTS is a possibility and can not be definitely excluded. In conclusion ou r results demonstrate that in the short term using a higher dose of leptin exerts greater effects in relation t o body we ight loss and increases in energy expenditure T hese positive effects are offset by the long term consequences in the development of leptin resistance which also appears to be dose dependent Thus part of our hypothesis wa s sustained in that high doses o f leptin induce resistance; while lower doses that are able to produce physiological effects, including the MD used in the current study, appear to be equally beneficial in long term effects given that both MD and HD resulted in similar reduced body weight s by 6 months. Although it remains inconclusive whether lower doses are also able to induce leptin resistance as our signaling data did indicate a partial reduction of leptin receptor responsiveness with MD and LD leptin. Future use of leptin in human the rapy should be cognizant of these effects. Especially considering the promising cases of leptin use in patients with hypoleptine m i a, where the therapy has been shown to be effective but in long term use may lead to induc tion of a leptin resistant stat e in these individuals. It may be advisable to alter dosing schedules as presumably if the system is not overly taxed the response may be amenable to res toration Additionally, further experiments into periodic release of leptin should be performed consideri n g that leptin is endogenously regulated under a circadian cy c le. On the other hand, these points could be moot if sensitivity to leptin can be restored which is another continuing area of research.
57 Figure 2 1 Body weight of rats following 3 rd ventricle AAV1 leptin injection. Body weight of rats injected with 1.77x10 10 vg/ rat AAV1 GFP control ( black squares) show a surgical effect following day zero but return to normal growth. AAV1 leptin at 1.76x10 6 vg/ rat ( light grey tr iangles ) exhibits the same surgical effect and growth resumption as control. Middle dose of leptin at 1.76x10 8 vg/rat ( grey triangles) shows no weight gain during the 60 day experimental period, while high dose of 1.76x10 10 vg/rat (bl ack triangles ) shows a decrease in body weight following treatment over the 60 day experimental period.
58 Figure 2 2 Change in b ody weight of rats respective to date of injections. Rats injected with 1.77x10 10 vg/rat of AAV1 GFP control ( black squa res ) decrease weight slightly in response to surgery but resume normal growth. Rats receiving low dose AAV1 leptin at 1.76x10 6 vg/rat ( light grey triangles ) responded similarly to controls. Middle dose leptin at 1.76x10 8 vg/rat ( grey triangles ) did not ind uce a large decrease in body weight but growth was reduced in comparison to controls. Treatment with high dose 1.76x10 10 vg/rat (bl ack triangles ) produced a reduction in body weight over the first 30 days and this reduction was maintained for the 60 day ex perimental period.
59 Figure 2 3 Food intake of rats treated with varying doses of AAV1 leptin. Rats injected with 1.77x10 10 vg/rat AAV1 GFP control ( black squares ) decreased chow intake in response to surgery but exhibited nor mal consumption at 15 days post surgery. Animals receiving LD AAV1 leptin at 1.76x10 6 vg/rat ( light grey triangles ) responded similarly to the control group. Treatment with MD leptin at 1.76x10 8 vg/rat ( grey triangles ) decreased intake in comparison to con trols, while treatment with HD leptin at 1.76x10 10 vg/rat (bl ack triangles ) produced the largest reduction in food intake. The decreased intake of chow in the middle and high dose treated groups is no longer observed by day 30 and all groups consume simila r amounts of chow.
60 Figure 2 4 Cumulative food intake of rats treated with varying doses of AAV1 leptin. A) Total grams of chow diet consumed during the first 30 days post treatment by group. B) Total diet consumed over t he 60 day experimental period. C) Total grams of diet consumed between day 30 and day 60 of the experiment.
61 Figure 2 5 Body composition changes following AAV1 leptin treatment. A) Percentage of body fat was evaluated by TD NMR at 15 day intervals, values were calculated as the change from baseline day 0. Control group (black squares) shows a small increase in body fat over the experimental period and this result is mimicked by low dose leptin group (light grey triangles). Mi ddle dose leptin treatment (grey triangles) results in a small decrease in body fat over the duration of experiment but returns to baseline by the final time point. The high dose leptin treated group (black triangles) exhibits the largest decrease in body fat percentage but begins to accumulate fat again over the experimental period. B) Change in lean mass as percentage of body weight measured by TD NMR with respect to baseline. Results for lean mass are the inverse of that observed for fat bat. C) Total cu mulative wet tissue weight of selected white adipose tissue depots (PWAT, RTWAT and EWAT) measured at sacrifice (day 60). + p<0.001 versus CTRL and LD, p<0.05 versus MD by One Way ANOVA.
62 Figure 2 5. Continued
63 Figure 2 6 Effect of leptin on interscapular brown adipose tissues and levels of UCP1 two months following leptin treatment. A) Weight of iBAT for each treatment group, increasing the dose of leptin reduced the weight of the iBAT depot. p<0.05 for HD compared to CTRL and LD. B) UCP1 expression per microgram of iBAT shown as percent of AAV1 GFP treated control group. C) UCP1 levels related to the size of the overall iBAT depot per group, expressed as percentage of the AAV1 GFP treated control group.
64 Figur e 2 6. Continued
65 Figure 2 7 Thermogenic evaluation of animals treated with increasing doses of leptin observed at day 55. A) Respiratory quotient at day 55 of treatment. B) Heat production in calories per hour of treated groups.
66 Figure 2 8 Phosphorylated STAT3 protein in the mediobasal hypothalamus two months after treatment with leptin vector following acute leptin stimulation. Phosphorylated STAT3 was normalized to levels of total STAT3 and expressed as a percentage of the values obtained in GFP control treatment. All leptin treated groups showed a reduced ability to phosphorylate STAT3 in response to acute leptin stimulation, including low dose (light grey bar), middle dose leptin (grey bar) and high dose leptin (black bar). *p<0.05 for MD and HD in comparison to CTRL.
67 Figure 2 9 Body weight of rats injected with AAV1 leptin vector over the 6 month experimental period. Control rats were injected with 1.77x 10 10 vg/rat AAV1 GFP ( black squares ) and show normal weight gain; initial drop in weight is a surgical effect and the drop in weight at day 60 is due to experimental interventions. Low dose AAV1 leptin at 1.76x10 6 vg/rat ( light grey triangles) resulted in similar growth associated weight gain as in control group within the experimental period. The middle dose of leptin 1.76x10 8 vg/rat ( grey triangles) resulted in a reduction of body weight and a gradual return towards initial starting weight over the experi mental period. The highest dose of leptin used, 1.76x10 10 vg/rat ( black triangles ) produced the most prominent body weight reduction, however this group also showed rapid weight regain and eclipsed the difference observed with the middle dose treatment at 6 months.
68 Figure 2 10 Change in b ody weight over 6 months in rats injected with AAV1 leptin. Control group received 1.77x10 10 viral genomes per rat AAV1 GFP (black squares) and show expected weight gain; the initial drop in weight is due to surgical effect and drop at day 60 is due to experimental interventions. Animals administered low dose AAV1 leptin at 1.76x10 6 vg/rat ( light grey triangles) showed a similar growth pattern to control group. Leptin at 1.76x10 8 vg/rat ( grey triangles) reduced body weight until day 115 where weight regain occurred; body weight even slightly surpassed the starting weight at the conclusion of the experimental period. Treatment with the highest dose of leptin used, 1.76x10 10 vg/rat ( black triang les ) created the largest reduction of body weight; however at day 80 rapid weight regain began, such that this group also exceeded its starting weight by the end of the experiment.
69 Figure 2 11 Chow food consumed following treatment with AAV1 leptin. A) Total grams of chow consumed from surgical day 0 to day 30. B).Total grams consumed from day 30 to day 180. C) Average daily chow consumption in the first 30 days post treatment. D) Average daily chow consumption in the expe rimental period beginning at day 30 until conclusion at day 180.
70 Figure 2 11. Continued
71 Figure 2 12 Body composition of AAV1 leptin treated rats. A) Change in fat mass as percentage of body weight from baseline percent age at intervals of approximately 15 days starting with day 0 (time point 0). B) Change in lean mass percentage from baseline at periodic intervals. C) Wet tissue weight of selected adipose tissue depots including EWAT, PWAT and RTWAT from sacrifice at day 180.
72 Figure 2 12. Continued
73 Figure 2 13 Changes in interscapular brown adipose tissue following 6 months of treatment with AAV Leptin. A) Weight of iBAT for each treatment group. At 6 months all groups have similar i BAT depot weights with the iBAT from middle dose treatment weighing slightly less. B) UCP1 expression per microgram of iBAT at 6 months shown as percent of AAV1 GFP treated control group. C) UCP1 levels related to the size of the overall iBAT depot per gro up after 6 months of treatment with AAV leptin expressed as percentage of the AAV1 GFP treated control group.
74 Figure 2 13. Continued
75 Figure 2 14 Thermogenic evaluation of animals treated with increasing doses of le ptin observed at day 163. A) Respiratory quotient at day 163 of treatment. B) Heat production in calories per hour of treated groups.
76 Figure 2 15 Long term leptin over expression diminishes the ability of acute leptin to induce phosphorylation of STAT3 in select CNS nuclei. A) Phosphorylation of STAT3 in the arcuate nucleus. B) Phosphorylation of STAT3 in the dorsomedial hypothalamic area. C) Phosphorylation of STAT3 in the nucleus of the solitary tract. D) Phosphorylation of STAT3 in the medial preoptic area.
77 Figure 2 15. Continued
78 CHAPTER 3 ALTERNATIVE THERAPEUTIC OPTIONS TO ALTER FOOD CONSUMPTION AND PHYSIOLOGICAL OUTCOMES 3.1 Introduction 3.1.1 Serotonin Agonists and Feeding Use of antidepressants that invoke changes to CNS biogenic amines, including serotonin, have been shown to alter food intake and body weight. The role of serotonin on food intake was established when serotonin was depleted in select brain regions and animals responded by exhibitin g a hyper phagic response eventua lly resulting in obesity  Fur thermore genetic deletion of the 5HT 2C receptor affected food intake and importance of this receptor in relation to development of obesity was established [171, 172] Current and past pharmacological treatments utilize d changes to serotonin to alter feeding. The drug fenfluramin e acts to release 5 HT and its metabolite norfenfluramine is a potent 5 HT 2B/2C receptor agonist  Studies have shown that it is the action on the 5 HT 2C receptor s  specifically in POM C neurons  that mediate the important effect on feeding However, t he lack of specific 5 HT receptor binding results in adverse side effects and has resulted in fenfluramine and other drugs such as the 5 HT and norepinepherine reu ptake inhibitor sibutramine being withdrawn from the market I t was found that agonist activity at the 5 HT 2B receptor results in cardiac dysfunction with fenfluramine while sibutramine was implicated with other cardiovascular problems [129, 175] In order to overcome the issue of adverse effects, development of compounds that are truly selective for the 5 HT 2C receptor has been undertaken. Among the selective 5 HT 2C receptor compounds is the rec ently approved drug lorcaserin which exhibits 18 fold selectivity over the 5 HT 2A receptor and 104 fold
79 selectivity versus 5 HT 2B receptor  Lorcaserin produced long term weight loss in multiple clinical trails  The compound WAY 161503 shows 5 HT 2C receptor agonist activity, however it also has 5 HT 2A & 2B receptor activity  WAY 161503 has also been shown to reduce 2 hour food intake i n overnight fasted mice and rats, while also producing hypophagia and reducing bodyweight when administered over 10 days in SD rats  Both Lorcaserin and WAY 161503 maintain issues with potential action at the 5 HT 2A & 2B receptors ; this is no t the case for ( ) trans PAT which is an agonist at 5 HT 2C receptors but possesses inverse agonist/ antagonist properties at 5 HT 2A & 2B receptors  ( ) Trans PAT has been shown to reduce the in take of palatable dessert treats in a dose dependent fashion in mice  The effect of ( ) trans PAT was related to the time of admi nistration with greater effects when injected immediately prior to testing however the effects were maintained when animals were treated 2 hours prior to testing when a dose of 10 mg/kg was used  Daily injection of ( ) trans PAT over 4 days maintained reduced intake of dessert treat s compared to vehicle treatment, however the reduced intake was attenuated over the progression of t he days suggesting the possibility of desensitization  Based on the successful results of ( ) trans PAT in changing feeding in m ice and its similarity to WAY 161503 we set out to evaluate the effects of ( ) trans PAT in reducing food intake in SD rats. Experiments evaluated both the effect on the re feeding res ponse following fasting and o f continuous delivery directly into the CN S lateral ventricle of rats. Supported by the evidence of WAY 161503 reducing food intake in response to fasting and over a 10 day period with daily injections in SD rats, we predicted that ( ) trans PAT would elicit the same effects. A truly 5 HT 2C recept or
80 selective agonist, with no agonist effects at 5 HT 2A & 2B receptors, able to reduce food intake in multiple rodent systems would warrant intense study ; as such a drug would likely eliminate many of the adverse reactions seen with previous drugs. Studies examining the effects on food intake of selective serotonin agonists are presented in the first section of results within this chapter; while an alternate therapy also implicated in changing feeding, wheel running, is examined in the second part of the re sults section. 3.1.2 Wheel Running and Food Intake Voluntary wheel running elicits important effects on food intake and elucidating the mechanisms behind the changes observed with wheel running may provide new therapeutic target s in the treatment of overweight and obesity. Voluntary wheel running has been shown to reduce caloric consumption and alter food choice between chow and HFD in Fischer 344 x Brown Norway Rats (FBN)  Rats when given free choice of diets will preferentially consume more fat while maintaining constant intake of protein [179 ] In certain strains of rats given voluntary wheel running access caloric intake increases through increased carbohydrate intake  In addition WR was also shown to enhance sensitivity to exogenous leptin stimulation in the ventral tegmental area, but not in other brain areas including the arcuate nucleus, lateral hypothalamus, dorsomedial hypothalamus, ventromedial hypothalamus and substantia nigra  This observation is of interest considering the relation of the VTA to reward related pathways and given that WR may in itself be a rewarding experience  Furthermore differenc es in activity levels were found to be related with differential expression of dopamine 1 receptors and tyrosi ne hydroxylase in the striatum/ nucleus accumb e ns with lower levels of the aforementioned genes increasing voluntary activity levels 
81 E xerc ise induces multiple changes to the brain. Importantly it was shown that a single bout of swimming exercise was able to increase leptin sensitivity, and that the response to leptin followed along a dose responsive profile in exercising rats compared to non exercise controls  Voluntary wheel running, even at minimal levels appeared to restore leptin action in rats made leptin resistant by 5 months of HFD feed ing; when treated with AAV leptin without wheel running, weight gain was exacerbated and WR had minimal effect in rats treated with control vector, but WR com bined with AAV leptin was able to attenuate weight gain  Similar results of WR on maintaining leptin sensitivity were reported in mice on HFD  While WR appears to restore leptin sensitivity leptin reciprocally appears to influence wheel running as leptin treatment in rats increased wheel running, whereas leptin antagonist delivery decreased the amount of voluntary wheel running  Leptin delivered into the ven tricles for 5 days also increased spontaneous activity in SD rats  Wheel running activity has been shown to increase brain derived neurotrophic factor (BDNF) in certain regions of the rat brain  as well as prevent the decrease in BDNF levels observed when rats are fed HFD  Increased BDNF has the potential to change feeding behavior and induce weight loss particularly in t he presence of a HFD  Having observed positive changes in regards to decreased food intake and change in diet preferenc e within a chow versus HFD choice paradigm in Fischer 344 x Brown Norway rats with WR including the resultant changes in leptin sensitivit y we set out to further examine the phenomenon. Considering well known strain differences and changes to wheel running depending on environmental factors [188 190] we first set out to determine if the observed response could be replicated in SD rats. Secondly,
82 considering the changes to WR with leptin treatment and alternatively the changes to leptin sensitivity observed following WR, SD rats were treated with a l epti n antagonist in the CNS and observed in the chow versus HFD choice paradigm. A greater effect in changes to dietary preference was predicted in SD rats considering SD rats typically exhibit higher levels of wheel running; additionally it was hypothesized that leptin antagonist would prevent changes in dietary choice associated with wheel running. 3.2 Results 3.2.1 Central Nervous System Treatment with ( ) Trans PAT 184.108.40.206 Long term infusion of ( ) trans PAT into ventricles of Sprague Dawley rats produces increased weight gain Lateral ventricle cannulae were connected by catheters to mini osmotic pumps delivering either 6.3 g /rat/day of ( ) t rans PAT or 5% DMSO in aCSF (V ehicle ) and the rats were observed for two weeks following initiation of treatment. While rats were on a standard chow diet body weight increased more in the ( ) t rans PAT group. Figure 3 1 shows the change in body weight with regards to the day of pump change. At the conclusion of the chow feeding period the ( ) t rans PAT group had gained 9.57 grams more than the vehicle treated group, but the observed difference was not statistically significant (p=0.12). Once groups were exposed to HFD the groups gained weight at almost equal rates as the difference over the HFD period was only 1.89 grams more in the ( ) t rans PAT group. 220.127.116.11 Prolonged infusion of ( ) trans PAT does not reduce chow nor high fat diet intake Daily intake of chow diet was monitored following treatment with ( ) trans PAT Both the treated and vehicle group displayed a slight reduction in food intake the day following the minor pump change surgery but then consumed normal amounts
83 thereafter ( Figure 3 2 squares). Average daily chow intake was slightly greater in the ( ) trans PAT group, 86.0 2.3 versus 83.2 2.1 kcal in the vehicle group. Since no difference was detected on chow diet, the effect of ( ) trans PAT was evaluated on consumption of HFD. High fat diet was introduced on day 8 and resulted in an immediate hyperphagia in both groups ( Figure 3 2 triangles). While consumption remained slightly elevated in the ( ) trans PAT group compared to the vehicle control 4.9 kcal/day, both groups were able to mount a physiolog ical response to the hyperphagia. The difference in caloric consumption between groups over the experimental period was minimal and can not be attributed to the ( ) trans PAT treatment. 18.104.22.168 ( ) T rans PAT does not change body composition Since a small differenc e in body weight was observed, body composition was evaluated with TD NMR. Measurements of fat, lean and fluid mass were taken prior to pump change surgery on day 0 and again at day 13. Figure 3 3 reveals that ther e was no change between the groups in the percentage of fat mass; both groups increased the percentage of fat as would be expected when consuming a high fat diet. Lean mass as a percentage of body weight decreased in both groups; however, absolute lean mas s as well as fat mass w ere increased, as both groups gained weight over the span of treatment. 3.2.2 Acute T reatment with the Selective Sero tonin 2C Receptor Agonist WAY 161503 in the Central Nervous System 22.214.171.124 Over night fasting induces a re feeding response in Sprag ue Dawley rats To follow up the negative result (i.e., no effect) of continuous ( ) trans PAT delivery, tests using a selective 5 HT 2C receptor agonist, WAY 161503, were developed.
84 Rather than observing long term delivery, an acute dose re feeding respon se was evaluated. Prior to observing the response to WAY 161503, the viability of the re feeding paradigm was evaluated in SD rats preceding the surgical implantation of lateral ventricle cannulae. Multiple time point intervals were evaluated, and data for food intake at 30 minutes, 1 hour, 2 hours, 6 hours and 24 hours after re feeding or matched time of day without food restriction are shown in Figure 3 4 A ; because intervals were not of equal length data was trans formed into 30 minute blocks, while the inset shows the total consumption within the given interval. Rats were used as their own control to account for individual feeding differences and paired t tests were used to evaluate consumption within individual in tervals. Overnight fasting results in significant increase in consumption during the first 30 minutes following presentation of chow; as well as increased consumption for periods of 1 hour to 2 hours and 2 hours to 6 hours. However, consumption is decrease d in the 6 hours to 24 hours re feeding reflecting the adjustment to the increased calories consumed in the prior intervals. The cumulative consumption is increased at all time points when rats were restricted by overnight fasting resulting in a 2.9 gram i ncrease in food intake over a 24 hour period ( Figure 3 4 B). 126.96.36.199 Acute lateral ventricle delivery of vehicle does not change re feeding following overnight fast In order to determine if acute injection into the lateral ventricle of Sprague Dawley rats alters the response in re feeding following an overnight fast, rats were either sham injected or 5 l of aCSF delivered over a 2.5 minute period through an implanted guide cannula. Figure 3 5 A demonstrates that vehicle injection did not significantly alter the re feeding pattern at any given interval after presenting chow diet
85 to rats. No changes were detected in the cumulative consumption of chow at any given time point as would be predicted by the lack of differences within the individual intervals ( Figure 3 5 B ). The increase in cumulative food intake in the 24 hour re feeding period compared to Figure 3 4 B is due to the increase in diet consumption of rats that occurs with normal growth. 188.8.131.52 Lateral ve ntricle delivery of 5 g WAY 161 503 does not change chow intake after overnight fast A 5 g/rat dose of the 5 HT 2C receptor agonist WAY 161503 was given i mmediately prior to re feeding after rats had been subjected to an overnight fast. Food intake measured at given intervals after presentation of food is represented in Figure 3 6A. The 5 g dose of WAY 161503 did not alter consumption at any point over the 24 hour evaluation period and as such there is no change in the cumulative amount of chow consumed between the treatment and vehicle group ( Figure 3 6 B ). 184.108.40.206 Leptin given acutely to lateral ventricle reduces 24 hour food intake in Sprague Dawley rats The lack of effect observed with the WAY 161503 compound was unexpected; therefore to confirm the ability of acute injection of drug into the lateral ventricle to decrease food intake, the known anorexic peptide leptin wa s given at a 5 g/rat dose. While leptin was unable to change the initial re feeding response to overnight fast it did indeed cause a statistically significant decrease in consumption in the 6 hour to 24 hour interval ( Figure 3 7 A p=0.012), resulting in decreased total chow intake over the 24 hour evaluation period ( Figure 3 7 B p=0.006). The observed result is consistent with the known mechanism of leptin action within the central nervous system, as the ability of leptin to decrease food intake involves the induction of intracellular transcription machinery. Furthermore, the positive results from leptin treatment suggested that
86 cannulae were indeed placed in proper location s. Cannulae placement was further confirmed by sectioning brains on a microtome and observing the cannula tract at the conclusion of the experiment. 220.127.116.11 D ecrease in food intake was observed after overnight fasting following acute administration of a 40 g dose of WAY 161503 Rats undergoing an overnight fast were then treated with 40 g/rat of the 5 HT 2C receptor specific agonist WAY 161503 administered to the lateral ventricle. The animals responded with an initial significant decrease in food intake during the 30 minute period after presentation of food. Compared to vehicle delivery, rats consumed 1 gram of chow less in the initial 30 minutes and then maintained slightly decreased consumption for the full 24 hour period of observation ( Figure 3 8 A ). In contrast to the 5 g/rat dose used previously the cumulative consumption remained below that of vehicle treatment. A 1.9 gram reduction in chow intake compared to vehicle treatment was maintained at 24 hours post fasting ( Figure 3 8 B ). Although the reduced intake was not statistically significant at 24 hours after fasting, in contrast to the reduction obser ved with a 5 g dose of leptin, the WAY 161503 compound was rapidly acting a nd was able to reduce chow intake more so than leptin over the initial 6 hour period. It thus appears that agonists in sufficient doses at 5 HT 2C receptors are fast acting and produce reduction of food intake even in a state of high energy demand such as f ollowing fasting; whereas the action of leptin requires a longer time course to produce reductions in chow food intake.
87 3.2.3 Peripheral Injection of WAY 161503 and ( ) Trans PAT 18.104.22.168 WAY 161503 treatment given peripherally is able to decrease chow food intake Spragu e Dawley rats were treated with a 3 mg/kg dose of WAY 161503 given by intraperitoneal (IP) injection and chow food intake was evaluated at periodic intervals following a 12 hour daytime fast. Control rats were similarly fasted, but received 0.9% saline by IP injection immediately prior to the presentation of chow diet. The effect of WAY 161503 was immediate and reduced the initial intake of chow over the first two hours of re feeding; however after the initial reduction intake was similar to vehicle treated rats ( Figure 3 9 A ). Importantly there was no long term hyperphagia, thus the differenc e in cumulative food intake between v ehicle and WAY 161503 treated rats remained statistically significant at 24 hours ( Figure 3 9 B ). Even 48 hours after treatment total intake remained suppressed, with the v ehicle group having consumed 55.0 grams of chow compared to 51.7 grams in the WAY 161503 treated group. 22.214.171.124 No reduction in chow intake d uring re feeding is observed when rats are treated with ( ) t rans PAT Rats were exposed to a 12 hour daytime fast then given an IP injection of 10 mg/kg of ( ) trans PAT prior to presenting chow diet. Consumption of the diet was measured 2, 14, 24 and 48 h ours following the treatment. Consumption within the given intervals as grams per hour is shown in Figure 3 10 A revealing that the ( ) trans PAT treatment did not significantly reduce chow intake at any time point Figure 3 10 B illustrates that there is no significant reduction in cumulative intake of chow diet at any time point. Taken together, these results therefore show that ( ) trans PAT does not have the same activity on food intake as WAY 161503.
88 3.2.4 Voluntary Wheel Running and Diet Preferences in Sprague Dawley Rats 126.96.36.199 Wheel running alters the proportion of diet consumed in the chow versus high fat diet choice paradigm Two variations of a two diet choice paradigm that inclu des voluntary wheel running intervention were tested in young male SD rats. In the first instance rats were introduced to diet choice between a 60% high fat diet (HFD) and standard chow; thereafter running wheels were introduced for a one week period after which they were removed (Choice WR Choice; CWC). In the alternate format rats were first introduced to HFD followed by wheel running for one week prior to chow diet being introduced while HFD remained present; running wheels remained present for an additi onal week then were removed while the diet choice remained (HFD WR Choice; HWC). 188.8.131.52 Diet choice with one week wheel running intervention Figure 3 11 A demonstrates that when HFD is introduced to rats in the presence o f chow, subjects preferentially consume HFD (day 0 6). An initial hyperphagia of HFD is observed but is rapidly attenuated and forms a stable HFD baseline; however the average caloric intake is elevated (91.1 3.2 kcal) compared to that of the same rats w ith access to chow only (68.5 2.3 kcal). Upon the introduction of running wheels HFD consumption decreases and chow intake increases over the days that wheel running is present ( Figure 3 11 A days 7 13). When runn ing wheels are removed the amount of chow consumed is decreased, whereas intake of HFD increases ( Figure 3 11 B days14 21). Averages using the last four days of each phase, choice before wheel running (C1), during w heel running (WR) and choice following removal of running wheels (C2), were calculated and results are represented graphically in Figure 3 11 B The p resence of running wheels significantly increased the amount of c how consumed in the WR phase
89 compared to C1; additionally, although the amount of chow consumed in C2 was less than during WR, intake was still significantly more than during the C1 phase. Similarly, intake of HFD was also significantly changed during the WR phase ( Figure 3 11 B open bars). In the presence of running wheels intake of HFD was reduced by approximately 50%, 8.9 1.0 versus 17.1 0.5 grams; when the running wheels were removed intake returned to 15.0 0.5 grams which was not statistically different from the amount consumed prior to introduction of running wheels. The presence of running wheels clearly alters diet intake and even one week of wheel running appears to produce some latent changes in diet intake after wheels are removed. 184.108.40.206 Rats maintained on high fat diet, introduced to running wheels and then exposed to dietary choice To further evaluate the role of voluntary wheel running on diet choice, the order of diet and wheel running introduction was altered. Rats were first introduced to HFD which results in hyperphagia compared to the animals preceding chow baseline (90.7 2.5 versus 70.4 1.8 kcal). Following the initial extreme hyperphagia on the first day of HFD introduction, a relatively stab le intake was observed ( Figure 3 12 A days 0 8). Upon the introduction of running wheels, consumption is reduced ( Figure 3 12 A days 9 16). Chow diet was introduced to the animals on day 17, with HFD and running wheels remaining present. Introduction of the chow diet following one week of WR on HFD resulted in a dramatic shift in the proportion of the two diets consumed as HFD intake decreased further and there was a strong response in consumption of chow. However, the difference in the proportion of c how diet consumed waned over the week that choice and running wheels were present, in fact HFD consumption on day 22 was equal to the level observed when running wheels and only HFD were present Figure 3 12 A days
90 17 22). Running wheels were removed on day 22 of the experiment and consumption of HFD increased, yet there appeared to be a latent effect of wheel running on chow consumption a s rats continued to consume a part of their daily intake as chow. Figure 3 12 B represents the average daily intake of the two diets during the separate phases of the experiment. When HFD only is present (H) rats co nsume significantly more than in the condition of HFD with running wheels (H/WR). Running wheels with diet choice (C/WR) produced a further significant reduction in HFD intake. W hen runn ing wheels were removed and diet choice remained (C) HFD intake rose to the level observed in the HFD only phase, and the chow diet intake decreased significantly from the C/WR phase. The combined results of the CWR and HWC experiments suggest that the effect of WR on HFD intake is mainly independent of diet choice; however the duration of WR in SD rats appears to have an effect on consumption of chow diet. 220.127.116.11 Wheel running activity in CWC and HWC experiments Wheel running was evaluated in both the CWC and HWC experiments and average daily distance ran by rats can be observed in Figure 3 13 In the CWC experiment ( Figure 3 13 A ) WR distance increases over the duration of the experiment. Total caloric intake also increases slightly over the period of wheel running. The increase in caloric consumption is due to increase d chow intake only, as observed for the corresponding days of WR in Figure 3 12 A In the HWC experiment ( Figure 3 13 B ) wheel running also increases daily over the course of the experiment. Daily increase in wheel running was more prominent over the initial week following introduction of wheel running while rats were on HFD only and appeared to plateau around 1500 m/day during the diet choice phase. The e xperimental design did not allow for the determination of whether the WR increase was attenuated by introduction of diet choice
91 or if the maximum level of WR within the group was simply attained after on e week. Interestingly, total caloric consumption increased when diet choice was offered. In contrast to the increase observed in the CWC experiment the increase in caloric intake is due to an increase in HFD consumption along with decreased chow intake ( Figure 3 12 B days 17 22), rather than an increase in chow. 3.2.5 Central Nervous System Administration of Leptin Antagonist Increases Food Intake but Does Not Prevent Reduced Intake Attributable to Voluntary Wheel Runni ng 18.104.22.168 Increase in chow intake following voluntary wheel running is not prevented by leptin antagonist Sprague Dawley rats that had previously been used in wheel running diet choice experiments underwent lateral ventricle cannulation surgery and mini osmotic p umps were implanted. After recovery from surgical procedure rats were given dietary choice between chow and HFD. After baseline consumption of the diets stabilized, osmotic pumps were changed to leptin antagonist treatment at a dose of 25 g per day while the control group received aCSF as vehicle. Subjects were observed in the diet choice paradigm for one week following the initiation of treatment and then running wheels were introduced for an additional week and change in food intake was observed with wh eel running. Two Way Repeated Measures ANOVA with SNK post test revealed that leptin antagonist treatment did not increase the amount of chow diet consumed within the one week treatment phase, and the introduction of wheel running significantly increased c how consumption in both the control and antagonist treated groups (p=0.026) to a similar degree ( Figure 3 14 A ).
92 22.214.171.124 Leptin antagonist increases high fat diet consumption, but does not prevent wheel running induced red uction of intake In both leptin antagonist and aCSF treated control group the predominant diet consumed was high fat. Two Way Repeated Measures ANOVA for HFD intake revealed that leptin antagonist treatment significantly increased caloric consumption of H FD for the experimental period compared with control (p=0.02). Additionally, a main effect of WR was observed (p<0.001) and SNK post tests revealed statistically significant decreases of HFD intake in both the control group (p=0.002) and leptin antagonist treated group (p=0.008). In fact the reduction in HFD intake was approximately 20 kcal in both groups as demonstrated in Figure 3 14B Total caloric intake for each treatment in both phases of the exper iment is shown in Figure 3 14 C ; because there was an increase in chow consumption in both groups, the total daily caloric reduction attributed to WR is approximately 15kcal. 126.96.36.199 Leptin antagonist treatment did not reduce wheel running activity however leptin antagonist d ramatically increased body weight Wheel running was greater in the leptin antagonist treated group as was the variation between individual animals compared to the control group, and thus the difference in WR was not statistically significant ( Figure 3 15 ). Although there was increased WR in the leptin antagonist treated group with only a total of 10 kcal elevation in food intake per day over 15 days, body weight was significantly increased in the leptin antagonist g roup. Figure 3 16 demonstrates that leptin antagonist causes an increase in body weight, which was exacerbated in response to running wheels. When running wheels were introduced, the body weight gain of leptin anta gonist treated rats was attenuated compared to the rate of growth without running wheels, yet these rats continued to gain body weight in contrast to control treated animals with access to
93 running wheels. Although the change in body weight in Figure 3 16 is with respect to the initial day where treatment was started, even when change in body weight was calculated with respect to starting weight upon the introduction of running wheels the same statistical difference s for days 8 15 were revealed. In addition to leptin antagonist increasing body weight there was a trend for an increase in white adipose tissue depot size in all depots evaluated ( Figure 3 17 ). When the white ad ipose tissue depots that were examined were pooled together, there was a 5.72 gram increase in fat weight in the leptin antagonist treated group versus the aCSF control group The difference in total adipose tissue weight did not reach statistical signific ance (p=0.07), however based on the course of weight gain observed over the duration of the experiment there would have been a clear increase in adiposity either with a longer duration of treatment or had running wheels not been introduced. 3.3 Materials and M ethods 3.3.1 General 188.8.131.52 Animals Guide for the Care and Use of Laboratory Animals and protocols were approved by the University of Florida Institutional Animal Care and Use Committee. Food intake and b ody weight were measured daily. Rats were singly housed in order to make food intake measurements under a 12:12 hour light: dark cycle (07:00 19:00). 184.108.40.206 Statistics Two Way ANOVA (with repeated measures where app ropriate), One Way ANOVA, t tests (paired where appropriate) were p er formed using SigmaStat v3.1 (Systat Software, Inc., San Jose, CA). Student Newman Keuls (SNK) posttests were
94 used for group comparisons where the main effect was found to be significant. The s tatistical signifi cance threshold value was set at p<0.05. 3.3.2 Long T erm I nfusion of ( ) T rans PAT 220.127.116.11 Surgery Two month old male SD rats (Charles River Laboratories Inc., Wilmington, MA) were divided into Vehicle group (n=5) and ( ) trans PAT (n=8). Following acclimation to facilities rats underwent surg ery under isoflurane (VEDCO, St. Joseph, MO) anesthesia at 5% induction and 2% maintenance for the duration of surgery. Cannulae from Alzet Brain Infusion Kit 2 (Durect, Cupertino, CA) were directed at the lateral ventricle with assistance of a stereotaxic frame; coordinates used were 0. 13 mm posterior, 1. 9 mm lateral, 4.0 mm ventral with respect to Bregma  Small screws were affixed to the skull, one rostrally and one caudually of the cannula base S etup was adhered using dental cement. Fourteen day Alzet mini osmotic pumps model 2002 (Durect, Cupertino, CA) filled with artificial cerebrospinal fluid (aCSF: 125mM sodium chloride, 3mM potassium chloride, 1mM disodium phosphate, 1.4mM calcium chloride, 1mM magnesium chloride, 10mM alpha D glucose, pH 7.4 filter sterilized through 0.2 m nylon mem brane) were connected to ventricular cannulae via catheters Osmotic pumps were guided to the dorsal surface of the animals. Rats were allowed to recover for two weeks following cannulation surgery prior to pump change. Under isoflurane anesthesia, a small incision was made in the dorsal surface of the rat and the original implanted mini osmotic pump was replaced with a 14 day Al zet mini osmotic pump model 2002 (Durect, Cupertino, CA) filled with vehicle or treatment
95 18.104.22.168 Treatments (1R,3S) trans 1 phenyl 3 dimethylamino 1,2,3,4 tetrahydronaphthalene (PAT) M.W. 287.83  was provided by Dr. Raymond Booth (Department of Medicinal Chemistry, University of Florida). Diluti on of ( ) trans PAT was made in 37C aCSF to a final concentration of 0.5446 mg/ml. That dilution allowed pumps to deliver 6.3 g per day per rat equivalent to ~18 g/kg/day. The v ehicle group received pumps filled with aCSF. 22.214.171.124 Food c onsumption Rats were ma intained on standard rodent chow diet (Diet 7912 3.1 kcal/g, 17 % kcal Fat, 25% kcal Protein ; Harlan Laboratories Inc., Indianapolis, IN), for the acclimation period, cannulation surgical recovery period and for the first week of experimental observation f ollowing initiation of treatment with ( ) trans PAT. Diet was changed weekly coinciding with cage change, while intake was measured daily accounting for any spillage. After one week of ( ) trans PAT treatment, diets were switched to a 60% high fat diet (Di et TD 12492, 5.24 kcal/g, 60% kcal Fat, 20% kcal Protein ; Research Diets, Inc., New Brunswick, NJ) 126.96.36.199 Body c omposition Time Domain Nuclear Magnetic Resonance (TD NMR) was used to perform measurements of fat, lean and fluid mass. Live unanaesthetized animals were weighed and placed into sampling tubes and measured using a minispec LF90 analyzer ( Bruker Optics, The Woodlands, TX ). Measurements were taken in duplicate for each animal at day 0 and then again at day 13 Fat and lean mass were calculated as a perce ntage of body weight for each individual animal. Data were transformed to reflect the change in percentage over the 13 day experimental period.
96 3.3.3 Acute CNS Treatment Experiments I nvolving WAY 161503, Leptin, and ( ) T rans PA T 188.8.131.52 Surgery Experiments used two mon th old male SD rats (Harlan Laboratories Inc., Indianapolis, IN). Under isoflurane (VEDCO, St. Joseph, MO) anesthesia at 5% induction and 2% maint enance for the duration of surgery, acute guide c annulae ( C312GA/SP Plastics One, Roanoke, V A) were directed at the lateral ventricle. C oordinates used were 0. 08 mm posterior, 1. 6 mm lateral, 4.0 mm ventral with respect to Bregma  Cannulae were secured using dental cement and were fitted with a dummy cap. Cannula placement was verified by sectioning the brains of animals at sacrifice and observing the cannula tract. 184.108.40.206 Treatments Prior to surgical placement of cannulae, rats were used to evaluate difference s in consumption following an overnight fast. Rats were evaluated for foo d consumed at 30 minutes, 1 hour, 2 hours, 6 hours and 24 hours after re fe eding starting at 09:00 following food being removed at lights out (19:00). 16 rats were used as their own controls with a non restricted test occurring 4 days after the overnight fasting test. To ensure that the delivery of 5 l of volume to the lateral ventricle would not interfere with food consumption, a group of 7 rats were sham injected and compared to a 5 l aCSF injection 5 days later. 8,9 Dichloro 2,3,4,4a tetrahydro 1H pyrazino[1,2 a]quinoxalin 5(6H) one hydrochloride ( WAY 161503; W0270, Sigma A ldrich, St. Louis, MO) was dissolved in a solution of 5% dimethyl sulfoxide (DMSO; D8418, Sigma Aldrich, St. Louis, MO) and aCSF to a final concentration of 1 mg/ml. Rats (n=7) were treated with 5 l injection of
97 5% DMSO/aCSF vehicle through the implanted acute guide lateral ventricle cannula over a period of 3 minutes. Immediately following the injection, food was provided and the re feeding response was observed for 24 hours at multiple intervals. 4 days later the same rats underwent overnight fasting and were treated with 5 l of 1 mg/ml WAY 161503 through the guide cannula and the re feeding response was measured. The same procedure was repeated in a group of 7 rats using 5 l 5% DMSO/aCSF as vehicle and 5 l of 1 mg/ml murine leptin (Amgen, Thousand Oak s, CA) given into the lateral ventricle through acute guide cannula over a 3 minute period. The final experiment involved 5 l of 8mg/ml WAY 161503 in 5% DMSO/aCSF providing a 40 g treatment dose. 220.127.116.11 Diets Rats were maintained on standard rodent chow diet (D iet 7912 3.1 kcal/g, 17 % kcal Fat, 25% kcal Protein ; Harlan Laboratories Inc., Indianapolis, IN). For overnight fasting, diet was removed at lights out (19:00) and re feeding occurred immediately subsequent to injections at 09:00. Food intake on test day s was measured at 0 9:30, 10:00, 11:00, 15:00 and 0 9 :00 of the following day. On non test days intake was evaluated daily at 09:00. 3.3.4 Peripheral Injection of WAY 161503 and ( ) T rans PAT 18.104.22.168 Animals Two month old male SD rats (Harlan Laboratories Inc., Indianapol is, IN) were divided into two groups (n=8/group). After one week of acclimation to facilities, rats received daily intraperitoneal (IP) injections of 0.9% saline at 1 ml/kg to prepare animals for experimental treatment.
98 22.214.171.124 Treatments WAY 161503 (W0270, Sigma Aldrich, St. Louis, MO) was diluted to 1.5 mg/ml in 0.9% saline. On the test day rats were fasted beginning at lights on (07:00). IP injection of 0.9% saline or WAY 161503 (2 ml/kg) was given at 19:00, after which food was provided. Intake was measured at 21:00, then at 09:00 and 19:00 the following day. One week after testing the effect of WAY 161503, rats previously used as vehicle controls were treated with 10 mg/kg ( ) t rans PAT, while the WAY 161503 treated rats were injected with 0.9% saline as contro ls using the same intake paradigm described previously. 3.3.5 Wheel Running Diet Choice Experiments 126.96.36.199 Experimental design of choice wheel running choice experiment (CWC) Three month old male SD rats n=7 (Harlan Laboratories Inc., Indianapolis, IN) were maintained on standard chow diet (Diet 7912 3.1 kcal/g, 17 % kcal Fat, 25% kcal Protein ; Harlan Laboratories Inc., Indianapolis, IN). On day 0, rats were introduced to a 60% high fat diet (Diet TD 12492, 5.24 kcal/g, 60% kcal Fat, 20% kcal Protein ; Research Diets, In c., New Brunswick, NJ) while access to standard chow diet remained. Consumption of each respective diet was measured every 24 hours, and the location of diet within the cage was alternated daily. Following one week of diet choice, Nalgene Activity Wheels ( 1.081m circumference, Fisher Scientific, Pittsburgh, PA ) were introduced to the home cage. Wheels were equipped with magnetic switch counters tracking total revolutions and the value was recorded and reset daily when food intake of respective diets and bod y weight was measured. After one week, access to running wheels was stopped and diet intake of the two diets was evaluated for an additional week.
99 188.8.131.52 Experimental design of high fat wheel runnin g choice experiment (HWC) An additional group of 3 month old male SD rats n=7 (Harlan Laboratories Inc., Indianapolis, IN) were maintained on 60% high fat diet (Diet TD 12492, 5.24 kcal/g, 60% kcal Fat, 20% kcal Protein ; Research Diets, Inc., New Brunswick, NJ) On day 9 Nalgene Activity Wheels (1.081m circumference, Fi sher Scientific, Pittsburgh, PA ) were introduced to the home cage and activity was recorded daily. Rats remained on high fat diet only until day 17, whe n in addition to the high fat diet, standard chow diet (Diet 7912 3.1 kcal/g, 17 % kcal Fat, 25% kcal Pr otein ; Harlan Laboratories Inc., Indianapolis, IN) was introduced. Intake of both diets was observed daily and the location of diets within the cage rotated. Running wheels were removed from the cages on day 22 while the choice between chow and high fat di et remained for an additional 12 days. 3.3.6 Wheel Running Diet Choice w ith Leptin Antagonist Treatment 184.108.40.206 Animals A subset of rats from the CWC and HWC experiments were used in the leptin antagonist experiments. Rats were divided into control group (n=6) and lepti n antagonist group (n=5) with an even distribution of rats from the preceding treatment condition. Prior to starting the experiment rats were maintained on chow diet with no intervention for one month. 220.127.116.11 Surgery Animals were anesthetized using isoflurane (V EDCO, St. Joseph, MO) anesthesia at 5% induction and 2% maintenance for the duration of surgery. Cannulae from Alzet Brain Infusion Kit 2 (Durect, Cupertino, CA) were directed at the lateral ventricle using a stereotaxic frame; coordinates used were 0.8 mm posterior, 1.6 mm lateral, 4.0 mm ventral with respect to Bregma  Small screws were affixed to the
100 skull, on e rostrally and one caudually to the cannula base, and the setup was adhered to the skull using dental cement. Fourteen day Alzet mini osmotic pumps model 2002 (Durect, Cupertino, CA) filled with artificial cerebrospinal fluid (aCSF: 125mM sodium chloride, 3mM potassium chloride, 1mM disodium phosphate, 1.4mM calcium chloride, 1mM magnesium chloride, 10mM alpha D glucose, pH7.4 filter sterilized through 0.2m nylon membrane) were connec ted to ventricular cannulae via catheters and guided to the dorsal surface of the rat Rats were allowed to recover for two weeks following cannulation surgery prior to pump change. Under isoflurane anesthesia, a small incision was made in the dorsal surfa ce of the rat and the original implanted mini osmotic pump was replaced with a 14 day Alzet mini osmotic pump model 2004 (Durect, Cupertino, CA) containing either aCSF or leptin antagonist. 18.104.22.168 Treatment A triple mutant (L39A/D40A/F41A) of the full length rat leptin peptide ( Protein Laboratories Rehovot Ltd. Rehovot, Israel) acts as an antagonist of leptin receptors. Leptin antagonist protein was diluted in aCSF to a final concentration of 2.16 mg/ml and mini osmotic pumps were filled and surgically implanted. Antagonist treated animals therefore received approximately 25 g of leptin antagonist per day. 22.214.171.124 Diets Three days prior to initiation of leptin antagonist treatment rats were introduced to choice between standard rodent chow diet (Diet 7912 3.1 kcal/g, 17 % kcal Fat, 25% kcal Protein ; Harlan Laboratories Inc., Indianapolis, IN) and 60% high fat diet (Diet TD 12492, 5.24 kcal/g, 60% kcal Fat, 20% kcal Protein ; Research Diets, Inc., New Brunswick, NJ) Choice between the two diets was maintained for the durat ion of the experiment. Consumption of respective diets was measured daily along with body
101 weight. Additionally, the location of the diets within the home cage was alternated daily. Diets were changed weekly coinciding with cage change. 126.96.36.199 Wheel r unning Eight days following leptin antagonist treatment Nalgene Activity Wheels (1.081m circumference, Fisher Scientific, Pittsburgh, PA ) were introduced to the home cage. Running wheels were connected to a magnetic switch counter that tracked the total number of revo lutions. Data from running wheels was obtained daily and counters were reset at the time of diet intake and body weight measurement. 188.8.131.52 Body c omposition At the conclusion of the experiment rats were sacrificed and select fat depots were excised and weighed to provide a measure of adiposity The epididymal, perirenal and retroper iton e al white adipose tissues were summated to provide values for total white adipose tissue. 3.4 Discussion 3.4.1 Selective Serotonin Agonists Lack of decreased chow intake in response to CNS in fusion of ( ) trans PAT was unexpected considering that previous studies show ed a reduction of dessert treat intake in fed mice where the dose related inhibition of intake for 50% was calculated at 9.2 mg/kg similar to the WAY 161503 compound  While our study investigated the effects of ( ) trans PAT on general feeding we expected a similar result considering that 2 mg/kg WAY 1615 03 daily IP injection in SD rats significantly reduced food intake provided on a 4 hour schedule for up to six days after which food intake remained lower  We were able to replicate the reduced food intake in response to a 3 mg/kg IP injecti on of WAY 161503 in SD rats ( Figure 3 9 B ). One explanation for why CNS
102 infusion of ( ) trans PAT did not produce the expected result is that the method of delivery was directly into the CNS at a dose of 18 g/kg/da y Considering that the rats weighed ~330 grams and the reported ventricular volume for rats of this size is ~135 l  while total cerebrospinal fluid volume is ~250 l with a turnover rate of ~2 l/min  We calculated a concentration of 30.4 nM ( ) trans PAT delivered per minute via infusion by using an estimated cerebrospinal fluid volume of 500 l to account for turnover. The value of 30.4 nM is in excess of EC 50 =19.9 2.22 nM reported for activation of 5 HT 2C receptors by ( ) trans PAT  hence we expected appropriate receptor stimul ation in our system. Since ( ) trans PAT was shown to reduce intake of palatable treats the diet was switched to a palatable HFD as consumption of this diet may activate different neural pathways that may be inhibited by ( ) trans PAT. ( ) Trans PAT howe ver did not reduce HFD intake either. An alternate explanation for the lack of an observed effect is that constant infusion produced tolerance. The effect of ( ) trans PAT on reducing dessert treat intake depended on time, and with repeated daily injection tolerance developed  ; therefore tolerance with continuous delivery is a plausible explanation for the lack of an observed effect. Based on this possibility, we instead gave acute CNS injections In order to drive the rats to consume chow the animals were fasted overnight and then food was presented immediately following the injection of drug into the ventricle. Even using acute inje ction, 5 g WAY 161503 failed to reduce re feeding ( Figure 3 6 ). To confirm that cannulae were properly positioned we delivered 5 g of leptin to which decrease d intake was observed after 24 hours, in agreement wit h the know n pattern of leptin action ( Figure 3 7 ). With a 40 g dose of WAY 161503, a reduction of food intake
103 was observed, but the reduction was only significant in the first 30 minutes of re feeding ( Figure 3 8 ). It is possible that the overnight fast presented rats with an increased drive to feed that could not be overcome; however this contradicts the results observed following a 24 hour fast in which IP injection s of WAY 161503 were able to reduce 2 hour food intake  Based on the difference in route of administration we tested IP injection of both WAY 161503 and ( ) trans PAT. With a 12 hour day time fast, 3 mg/kg IP injection of WAY 16150 3 was able to r educe 2 hour re feeding, and no resultant hyperphagia was observed such that cumulative intake remained reduced after 24 hours ( Figure 3 9 ). This result suggested that there was indeed an issue with direct CNS deli very and potentially dosage, or alternatively peripheral effects of WAY 161503 are necessary to reduce food intake. Building on the successful result of peripheral WAY 161503 administration a 10 mg/kg IP injection of ( ) trans PAT was undertaken. Even wi th th is large 10 mg/kg dose ( ) trans PAT failed to elic it a significant reduction in 2 hour food intake or reduce intake over a 24 hour observation period ( Figure 3 10 ). Several possibilities exist to explain the discrepancies observed in these results. For one, WAY 161503 has some agonist activity at 5 HT 2A/B receptors even though it is more selective for 5 HT 2C receptors. T he actions at 5 HT 2A/B receptors may be necessary to influence decreased consumption as th is pattern is consistent with the therapeutically effective drug lorcaserin while ( ) trans PAT has antagonist/inverse agonist properties for 5 HT 2A/B receptors. The explanation for differences in ( ) trans PAT effects in our studies and those in which it was reported to decrease palatable treat intake may be two fold; presumably it could simply be due to species difference s between mice and rats or ( )
104 trans PAT only responds to palatable food s which were not employed in our study. Presumably it would no t be difficult to test SD rats in the dessert intake paradigm and such an experiment may be worth considering in future investigations Cumulatively our results demonstrating an inability of ( ) trans PAT to reduce chow food intake, along with the toleranc e observed in response to continued treatment with ( ) trans PAT in other experiments, indicate that this compound is unlikely to be an effective monotherapy for overweight and obesity treatment. However potential modification of the compound with varying substituent functional groups may resolve this issue; indeed a modified form of the ( ) trans PAT compound has been shown to also have effects on reducing treat intake in mice  When viewed through a different prism our results are suggestive that ( ) trans PAT and its derivative compounds, known to have antipsychotic effects, are unlikely to be plagued by the adverse effect of weight gain, and therefore may be a therapeutic improvement for such con ditions. 3.4.2 Wheel Running Voluntary wheel running intervention had previously been shown to reduce HFD intake and alter the proportion of diet consumed in a chow versus HFD choice paradigm in FBN rats  The same paradigm was first evaluated in SD rats and similar trends were observed. Introducing HFD to SD rats resulted in a 25% increase in caloric in take compared to 30% increase in FBN rats. When WR was introduced to FBN rats they decreased HFD consumption to an average of approximately 31.5 kcal and increased chow consumption to 18.6 kcal; whereas with SD rats HFD consumption with WR was 46.7 kcal an d chow consumption was 12.5 kcal. Both strains of rats started with similar daily intake prior to whee l running at ~90 kcal, but WR had a greater effect in FBN rats
105 than SD rats and this was reflected in the magnitude of change in diet preference between t he two strains as well. Strain differences were more pr onounced in the HWC paradigm. Initial caloric consumption for both strains w as approximately 90 kcal; however WR in FBN rats reduced daily i ntake to 42.7 kcal whereas in SD rats WR only reduced intak e to 61.4 kcal Both groups increased intake when chow diet choice was added to WR by ~10 kcal, therefore daily kcal consumption remained elevated in SD compared to FBN rats. When running wheels were removed both groups returned intake to approximately 90 kcal per day; interestingly in SD rats following WR the consumption of chow remained slightly elevated but less so than with WR, an effect that disappeared more rapidly in FBN rats following removal of running wheels. There is clear evidence for differenc es between strains in response to WR; however the observed differences are contrary to what was initially predicted. In FBN rats reduction of HFD was correlated with the amount of WR  Whereas FBN rats run in the range of 300 m/day in both the CWC and HWC paradigm the SD rats in the preceding experiments had much higher levels of WR. Within the C WC experiment SD rats ran ~ 1000 m/day, yet HFD consumption was 15 kcal/day greater than intake observed in FBN rats in the same paradigm. Also of interest in the HWC paradigm SD rats on HF D only ran ~1000 m/day, but when choice was introduced WR was incr eased to ~1500 m/day ( Figure 3 13 B ) and an increase in HFD intake was observed in conjunction with the increase in WR. While a decrease in fat intake is seen in both st r ains following WR the amount of running does not correlate with the decrease in male SD rats. Decreased fat preference has been also been observed in female SD rats with treadmill exercise  One potential complication with these studies is that SD rats
106 diverge into two subtypes including the diet induced obesity prone (DIO) and dietary induced obesity resistant phenotype (DR) which have different physiological responses  The SD rats used in the WR diet choice paradigm were not selected for DIO versus DR, a fact that may in part explain why wheel running variability is high and did not directly correlate to reduced HFD consumption. Since the action of leptin has been previously shown to alter levels of WR  and WR led changes in leptin sensitivity [112, 113, 178] we set out to evaluate if WR induced changes would be obse rved in the presence of leptin antagonist Leptin antagonist infusion at 25 g/day was previously shown to increase food intake [165, 196] Leptin antagonist did not prevent the reduction in caloric intake observed with WR or the change in diet preference ( Figure 3 14 ). The antagonist used in the experiment was physiologically effective as caloric consumption with the antagonist was significantly increased compared to vehicle, as was adiposity and body weight. WR reduced intake of HFD equally by 20 kcal in both groups although the initial intake was higher in the antagonist treated group ( Figure 3 14 B ). Of note in control rats with WR chow intake was not increa sed to the extent previously observed without any intervention suggesting a potential surgical effect. It is possible that osmotic pump implant and CNS cannulation led to increased levels of inflammation that interfered with the WR effect on diet preferen ce but this is merely speculation. Also of interest is that the amount of WR, although highly variable was greater in the antagonist treated group than in the control group. However, rats were not evaluated for WR activity prior to grouping or selected f or DR versus DIO subtype; therefore the increased WR in the antagonist group may be due to unbalanced grouping. Even considering these issues
107 the evidence is rather clear that the antagonist did not prevent WR induced changes contrary to our initial pred iction. Furthermore in a separate experiment d elivery of an AAV super mutant leptin antagonist in to the 3 rd ventricle also did not prevent WR from reducing caloric intake and altering diet preference in the diet choice paradigm (unpublished observations). Importantly based on the cumulative evidence it appears that WR is an effective intervention to change diet intake, however further investigation into the mechanisms of action are necessary.
108 Figure 3 1 Central nervous system delivery of ( ) trans PAT results in increased body weight gain in SD rats. Mini osmotic pumps were changed at day 0 to deliver vehicle (open squares) or ( ) trans PAT (closed squares) and weight gain was evaluated while on chow diet until day 8; t hereafter the diet was switched to 60% high fat diet. Figure 3 2 Food Intake of rats treated with ( ) trans PAT on chow and high fat diet. Vehicle treated rats on chow (open squares) consume similar amount as ( ) trans PAT treated (closed squares); when diets were switched to 60% high fat diet, both vehicle (open triangles) and ( ) trans PAT (closed triangles) treated rats normalized the hyperphagic response.
109 Figure 3 3 Change in body compos ition following treatment with ( ) trans PAT for two weeks. No effect of ( ) trans PAT was observed in either percentage of body fat or lean mass, as an increase in adiposity was observed in both groups as would be expected when fed a high fat diet. The ch ange in percentage of body fat is mirrored by a decrease in the percentage of lean mass however absolute mass of both fat and lean increase
110 Figure 3 4 Food Restriction by overnight fasting induces a re feeding response i n SD rats. A) Chow consumption adjusted as 30 minute blocks at multiple intervals for a 24 hour time period after re feeding. Inset shows total consumption for each given interval. B) Cumulative chow intake at given time points following re feeding. p<0. 05, ** p<0.01, *** p<0.001; paired t test.
111 Figure 3 5 Acute injection of 5 l of aCSF vehicle to lateral ventricle does not change re feeding response to overnight fast in SD rats A) Chow consumption adjusted as 30 minut e blocks at multiple intervals for a 24 hour time period after re feeding. Inset shows total consumption for each given interval. B) Cumulative chow intake at given time points following re feeding.
112 Figure 3 6 Acute injec tion of 5 g WAY 161505 into lateral ventricle does not change re feeding response to overnight fast. A) Chow consumption adjusted as 30 minute blocks at multiple intervals for a 24 hour time period after re feeding. Inset shows total consumption for each given interval. B) Cumulative chow intake at given time points following re feeding.
113 Figure 3 7 Acute injection of 5 g of leptin produces a significant decreas e in chow intake in the 24 hour period following an overnight fast. A) Chow consumption adjusted as 30 minute blocks at multiple intervals for a 24 hour time period after re feeding. Inset shows total consumption for each given interval. B) Cumulative chow intake at given time points following re feeding. *p<0.05, *p<0.01, paired t test.
114 Figure 3 8 Acute lateral ventricle injection using a 40 g dose of WAY 161503 produces a decrease in re feeding response following overnight fast. A) Chow consumption adjusted as 30 minute blocks a t multiple intervals for a 24 hour time period after re feeding. Inset shows total consumption for each given interval. B) Cumulative chow intake at given time points following re feeding. ***p<0.001, paired t test.
115 Figure 3 9 Intraperitoneal 3 mg/kg WAY 161503 inhibits chow food intake following 12 hour daytime fast. A) Food intake adjusted to grams consumed per hour for 0 2, 2 14, 14 24 and 24 48 hour intervals following a 12 hour daytime fast. IP WAY 161503 shows rapid action as it decreases food intake during the initial interval and no resultant hyperphagia is observed at later time points. Inset shows total grams consumed during given interval. B) Cumulative grams of chow consumed at 2, 14, and 24 hour time point foll owing re feeding. The initial reduction of food intake is maintained for 24 hours. *** p<0.001, p<0.05, t test.
116 Figure 3 10 Intraperitoneal injection of 10mg/kg ( ) trans PAT has no effect on food intake following a 12 hou r daytime fast. A) Food intake adjusted to grams consumed per hour for 0 2, 2 14, 14 24 and 24 48 hour intervals following a 12 hour daytime fast. Inset represents total grams consumed within individual intervals. B) Cumulative grams of chow consumed at 2, 14, and 24 hour time point following re feeding.
117 Figure 3 11 Voluntary wheel running changes the amount of diet consumed in two diet choice paradigm in SD rats. A) Daily intake in grams of chow (closed squares) and HFD (open squares) during two diet choice; pro portion of diet consumed changes when running wheels were introduced between days 7 13. B) Average intake of chow (black bars) and HFD (open bars) during initial choice phase (C1), with wheel running (WR) and choi ce phase after wheel running (C2). *p<0.05, ***p<0.001, Repeated Measures One Way ANOVA.
118 Figure 3 12 Voluntary wheel running while on HFD reduces intake and produced a shift in consumption toward chow in response to the t wo diet choice paradigm. A) Daily food intake of HFD (open squares) and chow diet (closed squares) before, during and after introduction of running wheels (days 9 22). Running wheels reduce HFD intake and shift intake towards chow when two diet choice wa s introduced following one week of WR on HFD (day 17). B) Average daily food intake during distinct phases of wheel running diet choice experiment. Intake of HFD (open bars) is reduced when running wheels are present (H/WR) compared to HFD only (H ); when cho w diet (black bars) wa s introduced in the choice paradigm with running wheels (C/WR), equal amounts of the two diets w ere consumed. When running wheels are removed but the choice rema ins (C), intake of HFD increased yet rats continue d to consume some chow diet. *p<0.05, **p<0.01, ***p<0.001, Repeated Measures One Way ANOVA.
119 Figure 3 13 Average wheel running of Sprague Dawley rats in CWC and HWC experiments. A) Wheel running (squares) increases over the duration of the whe el running phase yet there is no significant change in total kilocalories consumed (diamonds). B) Daily wheel running increases when rats are on HFD only (black squares) but when rats are given choice between HFD and chow (grey squares) the daily increase in wheel running is attenuated. Total caloric consumption (diamonds) decreases slightly with WR on HFD but when choice is introduced intake increases.
12 0 Figure 3 14 Leptin antagonist increases caloric intake but does not prev ent decreased intake in response to wheel run ning. A) Average daily caloric consumption of chow diet during the one week leptin antagonist treatment phase while sedentary ( SED ) and with treatment and wheel running (WR).Wheel running increased intake of cho w diet in both control (open bars) and in leptin antagonist treated group s (black bars) B) Average daily caloric intake of high fat diet with leptin antagonist treatment without wheels ( SED ) and treatment with running wheels (WR). Intake of HFD is greater in antagonist treated (black bars) than in control group (open bars) but wheel running reduced intake in both groups. C) Total daily caloric intake of both chow and high fat diet during the one week diet choice phase with leptin antagonist treatment witho ut wheels ( SED) and in presence of running wheels (WR).
121 Figure 3 14. Continued
122 Figure 3 15 Wheel running activity does not change with leptin antagonist treatment in SD rats. Wheel running levels are higher in rats tre ated with leptin antagonist (closed squares) compared to control aCSF treated rats (open squares) but variability between individual animals is also much greater. Figure 3 16 Change in body weight following leptin antagonist treatment. Control rats (open squares) show a minor surgical effect and resume growth while leptin antagonist treated rats (closed squares) show increased weight gain. Running wheels were introduced to both groups on day 7 and resulted in a slight decreas e in body weight that was maintained while running wheels were present in control group, w hereas antagonist treated rats continued to gain body weight albeit at an attenuated rate compared to weight gain without running wheels. p<0.05, **p<0.01, ***p<0. 001; t test.
123 Figure 3 17 Wet tissue weight of multiple adipose tissue depots collected after two weeks of leptin antagonist treatment. Leptin antagonist treated group (black bars) exhibits increased adiposity in all depots examined including perirenal (PWAT), retroperitoneal (RTWAT) and epididy mal (EWAT) white adipose tissue The cumulative weight of the white adipose tissue depots is given in the SUM WAT column.
124 CHAPTER 4 BLOCKADE OF CENTRAL NERVOUS SYSTEM MELANOCORTIN 3/4 RE CEPTORS IN FISHER 344 X BROWN NORWAY RATS 4.1 Introduction Melanocortin receptors play an important role in feeding regulation. Of the five sub types of melanocortin receptors (MCRs) the melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) are vit al for regulating food intake and energy expenditure and are expressed in the CNS [197, 198] Products of the POMC gene MSH are endogenous agonists of MCRs while Ag RP is the endogenous antagonis t /inverse agonist of MCRs [199 201] The expression of MCRs occurs in multiple brain areas [202, 20 3] Depending on localization differential effects of the receptors are observed as evidenced by the physiological reactions when AgRP was over expressed in various brain areas  and upon selective re expression of MC4R in MC4R null mice  In addition to influencing food intake, MCRs also play a significant role in re gulating energy expenditure [206 208] can alter peripheral lipid metabolism  a nd change locomot o r activity  Another important role of MC4Rs is to regulate intake of dietary fat; MC4R null mice significantly increase intake on a higher fat diet while wild type mice are able to regulate intake to levels similar to low fat diet  Additionally the MC4R null mice did not increase activity on running wheels when exposed to a higher fat diet although the null and wild type mice had similar levels of wheel running activity when on a low fat diet  Voluntary wheel running in MC4R null mice shows different changes depending on age of mice. In young mice the initial weeks of WR allow MC4R null mice on running wheels to keep the same body weight as wild type mice with WR although after initially resp onding to WR with hypophagia, after one month they become hyperphagic [212 ]
125 The increased food intake becomes more pronounced when running wheels are removed, and a rapid gain in body weight occurs that increases body weight to the levels observed in MC4R null mice that never had running wheel access  Interestingly WR activity is higher during lights on in MC4R null mice than wild type but this pattern is reversed during lights off; yet total WR activity is slightly reduced in MC4R null mice [212, 213] The effect of voluntary wheel running can still be produced in MC4R null mice at 47 weeks of age where WR reduced body weight and decreased food inta ke, although this effect remained transient as with young mice  Furthermore WR in MC4R null mice prevents the decrease in the ratio of lean to fat mass that develops in these mice over time  In experiments with SD rats when AAV POMC was delivered to the hypothalamus or the NTS, the effect of POMC on red ucing food intake was transient with the hypothalam ic injection but sustained in the NTS  Injection of AAV POMC into the NTS also increased WR distance, which was not observed in the hypothalamic i njected group  When POMC was over expressed in both the arcuate nucleus and NTS in FBN rats, WR distance was also increased  In addition to the effects of POMC on wheel running the consumption of dietary fat is influenced by MC4Rs Using MC4R selective ag onists and evaluating macronutrient intake in mice, the a gonists only decreased dietary fat intake in wild type mice but not in MC4R knockout mice  Furthermore, the endogenous ant agonist/inverse agonist of MC4R AgRP, was shown to influence respo nse for a fat reinforcer in a conditioned stimulus experiment in rats but not for carbohydrate reinforcer 
126 Based on the influence of th e melanocortin system on both wheel running activity and on the selection of dietary fat we wanted to exp lore the role of the MCRs in the two diet choice paradig m. SHU9119 is an antagonist of the MC3R and MC4R  and administration of SHU9119 increases feeding  therefore it was used in our experiment to observe if it would block the effects of WR. Since HFD was present in our experiment and past studies showed that MC4Rs were critical for the intake of HFD we predicted that WR would not be able to reduce HFD intake in the SHU9119 treated grou p. Furthermore a group pair fed to control animal intake was included to observe the effects of SHU9119 on body composition and to determine if the caloric consumptio n was a primary factor in WR distance. 4.2 Results 4.2.1 SHU9119 Treatment Increases Body Weight Gain in Presence of High Fat Diet that is Attenuated by Voluntary Wheel Running Previous studies have shown that the presence of running wheels can alter the amount of c how and high fat diet (HFD) that is consumed in a two diet choice paradigm; to observe if this effect involved the melanocortin responsive feeding pathway, rats were treated with an intraventricular melanocortin 3/4 receptor antagonist and exposed to the t wo diet choice paradigm with and without functioning running wheels. Daily measurements of body weight were taken and change in body weight was calculated with regard to the initial day of drug treatment and is shown in Figure 4 1 All rats were initially maintained on a chow diet and had a similar response to pump change surgery, resulting in a slight body weight decrease. On day 4, HFD was introduced in addition to the continued presence of chow, while access to functioning running wheels was introduced on day 11 in the wheel running (WR) group; in the sedentary (S ED ) group,
127 the running wheels were locked. Control rats respond with increased body weight gain after access to HFD while the body weight response wa s m uch greater in rats treated with SHU9119. Pair feeding of SHU9119 rats to control diet consumption resulted in an initial small reduction in weight gain, but this group recovered and eventually exceeded weight gain compared with the control sedentary rats by the conclusion of the experiment. Two Way ANOVA for treatment and exercise for the entire span of the experiment showed significant effects for each factor (p<0.001) with no interaction. SNK post tests revealed significant differences for treatment bet ween SHU9119 and aCSF a s well as SHU9119 pair fed groups (p<0.001) but not between SHU9119 pair fed to controls. Exercise effect was significant in both SHU9119 and SHU9119 pair fed (p=0.013 and 0.007, respectively) but not statistically significant in aC SF (p=0.095). SHU9119 treated animals had significantly increased bodyweight compared to both control and SHU9119 pair fed rats during the period of HFD feeding prior to starting WR (p<0.001) but no difference was observed between control and SHU9119 pair fed. Two Way ANOVA for ch ange in body weight was also per formed for the one week period following introduction of running wheels, as well as for the period of running wheel access until conclusion of the experiment. For the week following introduction of running wheels both treatment and exercise had significant effects (p<0.001). SNK post tests revealed that SHU9119 treated animals differed from both aCSF and SHU9119 pair fed groups (p<0.001) but the SHU9119 pair fed group was not different from control s; this is in contrast to the result for the entire duration in which all treatment outcomes are significantly different from each other (p <0.001). A One Way ANOVA was
128 per formed on day 28 at the conclusion of the experiment by combining treatment and WR st atus into a single factor. SNK post test for group comparisons revealed significant differences between both SHU9119 S ED and WR to all ot her groups (p<0.001 and p=0.013 between the respective groups ) SHU9119 pair fed S ED was not different from aCSF S ED bu t when compared to aCSF WR and SHU9119 pair fed WR differences did exist (p=0.012 and p=0.019, respectively). SHU9119 pair fed WR was not different from either aCSF group and additionally no statistical difference existed between aCSF groups. Cumulatively these resu lts suggest that differences under WR conditions manifest rapidly. Furthermore, WR access may indeed compensate for caloric drive as the SHU911 9 pair fed S ED group showed increased weight gain compared to aCSF S ED by One Way ANOVA when consider ing body weight change over the diet choice phase (p=0.048), addressed in depth within the discussion section. 4.2.2 SHU9119 Increases Food Consumption and Delays Homeostatic Regulation of Caloric Consumption, While Voluntary Wheel Running Accelerates Regulation of Caloric Intake Food intake of rats treated with SHU9119 was observed daily over the 28 day experimental period. Dietary choice between a standard chow diet and HFD was introduced on day 4, and voluntary wheel running was introduced on day 11.While we h ypothesized that wheel running would act to alter dietary consumption patterns in the dietary choice paradigm this effect was not observed; however voluntary wheel running did have an effect on the temporal normalization of the heightened caloric intake as sociated with HFD hyperphagia as is demonstrated in Figure 4 2 HFD introduction resulted in hyperphagia in all groups compared to chow consumption (days 0 3). In control animals the initial hyperphagia is reduced within 3 days to a stable baseline value, yet caloric consumption remains elevated over intake on chow only. SHU9119
129 treated animals responded to the presence of the HFD similarly to control animals three days post introduction, however thereafter, these a nimals increased intake of the HFD and then established a relatively stable intake one week following the introduction of the diet. The resulting elevated intake of HFD is present for 9 days (days 9 17), after which intake of HFD is steadily reduced ( Figure 4 2 grey closed triangles). Two Way Repeated Measures ANOVA statistical testing followed by SNK post test when main effect was significant was performed for average daily intake of both chow and HFD before and a fter introduction of running wheels, using stable baseline average pre wheel running (Phase A) and the time period one week post wheel running introduction (Phase B). For chow consumption, both treatment and presence of running wheels were statistically si gnificant (p<0.001 and p<0.05, respectively). SHU9119 increased baseline consumption of chow in Phase A (p<0.05). In Phase B, SHU9119 WR chow consumption was increased versus both aCSF Sed and aCSF WR groups (p<0.001, and 0.015 respectively); however SHU91 19 SED chow consumption was greater than aCSF SED (p=0.008) but not significantly different from aCSF WR (p=0.082). WR was able to increase chow consumption in the SHU9119 WR group (p=0.031) but WR did not reach the statistical threshold in the aCSF WR gro up (p=0.063); there was no difference in either treatment group under sedentary conditions, as would be expected since no intervention was performed in those groups. The small increases in chow intake with the addition of running wheels are unlikely to hav e any significant physiological effect. Of greater importance was that large decreases in HFD consumption were observed in both control and SHU9119 treated animals in the presence of running
130 wheels as is demonstrated in Figure 4 3 Prior to WR the baseline average HFD caloric consumption for controls was 88.1kcal/day while SHU treated animals consumed 164.9 kcal/day. Two Way Repeated Measures ANOVA revealed significant differences for both treatment and time (p<0.00 1) as well as interaction (p=0.028). SNK post tests revealed no effect on HFD consumption in either of the sedentary treatment groups as would be expected, but WR significantly reduced HFD consumption in the control group with a 26.1 kcal/day reduction (p< 0.001) and a 19.8 kcal/day reduction for the SHU9119 treated group (p=0.003). Both SHU9119 treatment groups were different from controls (p<0.001), and SHU9119 WR showed a significant difference from SHU9119 SED following WR (p=0.008). The 18 kcal differen ce between aCSF WR and aCSF SED group approached statistical significance but this did not exceed the preset threshold (p=0.058). On day 28, One Way ANOVA for HFD consumption maintains a significant treatment effect, SHU9119 SED and SHU9119 WR were signifi cantly different from aCSF WR (p<0.05), but no other comparisons were statistically significant. 4.2.3 SHU9119 Reduces Wheel Running but Pair Feeding Attenuates Wheel Running Reduction Running wheels were added to the home cages of rats on day 11 and remained un til the conclusion of the experiment on day 28. Rats in the sedentary conditions were similarly exposed to running wheels; however the wheels were locked in place. There was minimal change in the amount of wheel running per day within each individual group so the data is represented as the daily average wheel running in meters over the wheel running period in Figure 4 4 One Way ANOVA revealed a significant effect of treatment and SNK post test showed that SHU9119 treatment significantly reduced wheel running compared to control group (65 8 v. 206 27 m/day, mean SEM,
131 p<0.001). Pair feeding SHU9119 treated animals resulted in a non statistically significant increase in WR (109 17 m/day) compared to SHU9119 (p =0.171), yet WR remained significantly decreased compared to control group (p=0.003). As previously mentioned there was no significant variation in amount of daily wheel running, but of note, the direction of change was different between control and both g roups that received SHU9119. Control rats showed an increase in WR as the overall daily average was approximately 38 meters greater than that observed in the initial days of exposure; whereas in both SHU9119 and SHU9119 pair fed groups the overall average was decreased by 38 meters compared to the initial days of WR exposure. 4.2.4 SHU9119 Treatment Increases Adiposity and Reduces Lean Mass Body composition was assessed by Time Domain Nuclear Magnetic Resonance (TD NMR) on day 0 for baseline and then again prior to sacrifice. The change in both adiposity and lean mass as a percentage of body weight over the experimental period of 28 days was calculated and is represented in Figure 4 5 A & B Since all groups consumed primar ily HFD throughout the experiment, every group shows an increase in adiposity as would be expected. The SHU9119 treatment group shows the largest increase in adiposity and is significantly different from both aCSF and SHU9119 pair fed groups (p<0.001). Int erestingly, even though SHU9119 pair fed animals only consumed the caloric value of their respective control group, the presence of SHU9119 in these animals contributed to a significant increase in adiposity compared to aCSF groups (p<0.001). Running wheel s attenuated the increase in adiposity as the main effect (Two Way ANOVA p=0.006), but SNK post test for group comparisons revealed that the only significant difference occurred between aCSF groups (p=0.008). Increase in adiposity
132 led to a decrease in the proportion of lean mass as a percentage of body weight. The change in lean mass is virtually the inverse of adiposity, as fluid percentage was not altered in any group. Even though as a percentage of body weight lean mass decreased in all groups, the absol ute lean mass in grams increased in every group. To further confirm the adiposity changes, selected white adipose tissue (WAT) depots were removed when animals were sacrificed. The average cumulative wet tissue weight of perirenal, retroperitoneal and epid idymal WAT depots for each treatment group is displayed in Figure 4 6 Results from the excised WAT depots corroborate the TD NMR body composition data, as there was a significant difference in adipose tissue with SHU9119 treatment compared to aCSF control (p<0.001); while pair feeding SHU9119 treated animals to aCSF control caloric intake attenuated the increase in adiposity in comparison to SHU9119 treated animals (p<0.001) a significant increase in adipose tissu e was observed compared to control group (p<0.001). The increase in adiposity of the SHU9119 pair fed group suggests that SHU9119 contributes to adipose tissue deposition by other means than increased food intake. Two Way ANOVA also indicated a main effect of WR (p=0.01), but SNK posttests did not reveal this effect to be significant within individual groups. The percentage of reduction in adipose tissue compared to sedentary within each treatment group, correlated with the amount of wheel running observed. 4.2.5 SHU9119 Treatment Increases Interscapular Brown Adipose Tissue Size and Changes the Expression of Uncoupling Protein 1 Brown adipose tissue plays an important role in body weight maintenance and thus the interscapular brown adipose tissue (iBAT) was studi ed. Treatment with SHU9119 increased the size of iBAT in rats and the increa se in size appears to be
133 depende nt on caloric intake as rats pair fed to control consumption did not show an increase in iBAT size as demonstrated in Figure 4 7 Furthermore, voluntary wheel running decreased the size of iBAT as Two Way ANOVA revealed a significant main effect (p<0.05) of wheel running use, but no statistical differences between groups using SNK posttests. In addition to ob serving the size of the iBAT depot, uncoupling protein 1 (UCP1) levels were evaluated in the iBAT by Western blotting. SHU9119, irrespective of pair feeding, decreased the level of UCP1 expression in comparison to aCSF control in both sedentary and wheel r unning groups ( Figure 4 8 A, B ). UCP1 expression in the sedentary group s was reduced to 27% and 31% of aCSF control with SHU9119 and SHU9119 pair fed treatment, respectively (p<0.001). In the WR groups, UCP1 express ion was reduced to 33% and 32% of aCSF control in the SHU9119 and SHU9119 pair fed treated groups respectively (p<0.001). In separate Western blots the effect of WR was compared within individual treatment groups ( Figure 4 9 ) Wheel running reduced the expression of UCP1 in aCSF control to 48% of sedentary control (p=0.002 ; Figure 4 9 ). In the SHU9119 treated group, which had significantly less WR activity, UCP1 was reduced to 77% of SHU9119 sedentary control ( Figure 4 9 B ) ; however this result was not statistically significant in part due to high unequal variances between samples demanding a Mann Whitney Rank Sum test. Similarly in the SHU9119 pair fed treatment group WR reduced UCP1 expression to 43% of the SHU9119 pair fed sedentary control, but again the difference was not statistically significant due to high variability in the groups ( Figure 4 9 C ) The power of the test in both the SHU9119 and SHU9119 pair fed groups was much lower than desired and
134 contributed to the lack of detecting a statistical ly significant difference in t he reduction of UCP1 expression. I ntuitively the results indi cate that WR reduced UCP1 expression in iBAT. 4.3 Materials and Methods 4.3.1 Animals Guide for the Care and Use of Laboratory Animals and protocols were approved by the University of Florida Instituti onal Animal Care and Use Committee. Six month old male Fischer 344 x Brown Norway rats were obtained from the National Institute of Aging Colony at Harlan Laboratories (Prattville, AL). Rats were received and maintained on a standard chow diet for one week prior to cannulation surgery. Food intake and body weight were measured daily. Rats were singly housed in order to make food intake measurements. 4.3.2 Surgery Animals were anesthetized using isoflurane (VEDCO, St. Joseph, MO) anesthesia at 5% induction and 2% maintenance for the duration of surgery. Cannulae from Alzet Brain Infusion Kit 2 (Durect, Cupertino, CA) were directed at the lateral ventricle using a stereotaxic frame; coordinates used were 0.8 mm posterior, 1.6 mm lateral, 4.0 mm ventral with respect to Bregma  Small screws were affixed to the skull, one rostrally and one caudually of the cannula base, and the setup was adhered using dental cement. 14 day Alzet mini osmotic pumps model 2002 (Durect, Cupertino, CA) filled with artificial cerebrospinal fluid (aCSF: 125mM sodium chloride, 3 mM potassium chloride, 1mM disodium phosphate, 1.4mM calcium chloride, 1mM magnesium chloride, 10mM alpha D glucose, pH 7.4 filter sterilized through 0.2 m nylon membrane ) were connected to ventricular cannulae via catheters and guided to
135 the dorsal surfa ce of the animals. Rats were allowed to recover for two weeks following cannulation surgery prior to pump change. Under isoflurane anesthesia, a small incision was made in the dorsal surface of the rat and the original implanted mini osmotic pump was repla ced with 28 day Alzet mini osmotic pump model 2004 (Durect, Cupertino, CA). 4.3.3 Treatments Ac Nle Asp His D Nal(2) Arg Trp Lys NH 2 Amide Bridge: Asp 3 Lys 8 known as SHU9119 (Phoenix Pharmaceuticals Inc., Burlingame, CA) was diluted in aCSF so that 0.2 nmol/day/rat would be delivered during the experimental period via mini osmotic pump. Control rats received infusion of aCSF. 4.3.4 Diet Choice Paradigm and Pair Feeding On day 4 following pump change surgery high fat diet ( Research Diets, Inc., New Brunswick, NJ TD 12492, 5.24 kcal/g, 60% kcal Fat, 20% kcal Protein) was introduced, while rats continued to have access to chow diet (Harlan Standard Rodent Chow Diet 7912, 3.1 kcal/g, 17 % kcal Fat, 25% kcal Protein ). Diets (40 g each) were given every day and data for food intake was obtained by calculating the difference in weight of both HFD and chow for the 24 hour period accounting for any spillage into the cage Location of the two diets was alternated each day. For rats in the pair fed groups, th e average amount of both chow and HFD consumed in the respective aCSF control group the previous day was provided. Pair fed rats always consumed all diet provided. 4.3.5 Wheel Running Nalgene Activity Wheels (1.081 m circumference, Fisher Scientific, Pittsburgh, PA) equipped with a magnetic switch counter for determination of revolutions were introduced on experimental day 11. Revolution counters were connected to a computer running VitalView acquisition software (Mini Mitter, Inc., Bend, OR). In animals that wer e
136 designated to the sedentary groups, access to the running wheels was present but the wheels were locked preventing functional use. 4.3.6 Body Composition Time Domain Nuclear Magnetic Resonance (TD NMR) was used to perform measurements of fat, lean and fluid ma ss. Live una naesthetized animals were weigh ed and placed into sampling tubes and measured using a minispec LF90 analyzer (Bruker Optics, The Woodlands, T X ). Measurements were taken in duplicate for each animal at day 0 and again at day 28. Fat and lean mas s were calculated as a percentage of body weight for each individual animal. At sacrifice, select fat depots were collected and weighed to provide a secondary measurement for alterati ons in body composition. The epididy ma l, perirenal and retrop e r iton ea l wh ite adipose tissues were summated to provide values for total white adipose tissue. 4.3.7 UCP1 Interscapular brown adipose tissue was dissected and weighed. An approximately 20 mg sample was removed from the iBAT and homogenized in 0.300 ml buffer (10 mM Tris HC l, 2% SDS pH 6.8) by sonication. Remaining cellular debris was removed by centrifugation and the supernatant was transferred to fresh tubes. In order to remove lipid component, the supernatant was passed through a 0.45 m syringe filter (Whatman Inc., Flor ham Park, NJ). Protein concentration of the samples was determined by detergent compatible protein assay (Bio Rad Laboratories, Hercules, CA). 5 g of protein samples was electrophoresed on 10% Tris HCl polyacrylamide gels and subsequently transferred to n itrocellulose membranes. UCP1 expression was detected using primary antibody to UCP1 (ab10983, A bcam, Cambridge, MA) and horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology Inc.,
137 Danvers, MA). The blots were developed using Amer sham ECL Plus reagent (GE Healthcare Biosciences, Piscataway, NJ) and scanned using a Storm 860 Phosphorimager (GE Healthcare Biosciences, Piscataway, NJ). ImageQuant5.0 software (Molecular Dynamics Inc., Sunnyvale, CA) was used for densitometry analysis o f bands. UCP1 expression was adjusted for the size of the iBAT depot from the individual rat sample. Results were expressed as the percentage of expression in the appropriate control treated rats. 4.3.8 Statistics Two Way ANOVA (with repeated measures where appr opriate) or One Way ANOVA for endpoint assessments, in which treatment and exercise condition were combined as a single factor, were p er formed using SigmaStat v3.1 (Systat Software, Inc.,. San Jose, CA). Student Newman Keuls (SNK) posttests were used for g roup comparisons where main effect was found to be significant. Statistical signifi cance threshold value was set at p<0.05. For simple two group compar isons, t test or Mann Whitney Rank S um te st were per formed where appropriate. 4.4 Discussion The use of the M C3/4R antagonist SHU9119 produced physiological effects in increased body weight and food intake in accordance with previous results. However SHU9119 did not prevent WR induced reduction in food intake, contrary to the initial proposed hypothesis. The effe ct of SHU9119 did not present until rats were given access to a HFD, but since HFD was introduced shortly after delivery of SHU9119 began, it is possible that SHU9119 would have increase d food intake and body weight on chow diet as well. Once HFD was intro duced, body weight rapidly increased in the SHU9119 treated group, whereas a body weight increase was also observed in control
138 rats as expected, but at a reduced rate of gain ( Figure 4 1 ). The rats treated with SHU 9119 but pair fed initially gained less weight than the control rats, but this was likely due to the timing of pair feeding, as all diet was presented at the time of weighing, Since SHU9119 most likely increased the drive to eat, rats in the pair fed group consumed their allotted calories earlier in the 24 hour cycle than the ad libitum fed controls; therefore more food likely had passed fully through these animals, and as such they present at lower body weights when measured. However in the sedentary pair fed group, body weight gain actually surpassed the control rats in the long term, suggesting that SHU9119 lowers metabolic rate independent of caloric intake in the long term. The introduction of WR was able to attenuate the rate of weight gain in all grou ps. In the control rats, when HFD was introduced the initial hyperphagic response was rapidly reduced, however in the SHU9119 treated group HFD intake continued to increase for one week until it stabilized; eventually HFD intake began to plateau and then d ecrease approximately two weeks after introduction ( Figure 4 2 grey triangles). Similar to previous experiments, this result suggests that MCRs are important for regulating HFD intake, however we show that even in SHU9119 treated ra ts other physiological processes are eventually able to reduce the caloric intake. In both control and SHU9119 treated groups, WR was able to cause a significant reduction in HFD intake in the week following introduction of running wheel s, an effect not observed in either sedentary group ( Figure 4 3 ). Unlike in MC4R null mice where the WR effect was transient  the reduction in HFD was maintained, but HFD intake was reduced in both the control and SHU9119 treated sedentary groups over time and therefore the WR dif ference was no longer significant. While WR did increase the level of chow diet
139 intake even with SHU9119, the increased intake was a minor component of total food intake and therefore likely had no physiological effect. While it was expected that chow inta ke would increase with WR in the control group, no such effect was observed; similar to the effect observed in the leptin antagonist experiment in Chapter 3, we speculate that the lack of increase is due to a surgical effect. Blockade of MC3/4Rs resulted i n decreased WR activity in FBN rats. The observed decrease in WR activity with SHU9119 suggests that endogenous activation of MC3/4Rs play s a role in activity, and further supports previous results that increases in POMC, and therefore presumably increased activation of MCRs, increases WR activity [214, 215] In the group pair fed to control animal intake but treated with SHU9119, there was an increase in WR activity compared to SHU9119 treatment, although overall activity was lower than in non treated controls The increase is potentially due to the reinforcing nature of wheel running observed in states of food deprivation  in animals receiving SHU9119 and being pair fed a virtual state of deprivation may have been induced. Food restriction results in increased wheel running activity  Leptin effectively reduces the increase in wheel running under deprivation  considering that the SHU9119 pair fed rats had higher adiposity, it is likely that they also had increased levels of leptin, thus explaining why WR activity remained reduce d compared to control rats. Adiposity was increased in rats treated with SHU9119, even in the rats that were pair fed to control treated diet intake. This result is in agreement with the effects of SHU9119 on increasing the expression of genes involved in lipogenesis and fatty acid storage within white adipose tissue  The increase in adiposity occurs without
140 increased food intake or weight gain in MC3R knockout mice  however with the MC3/4R blockade using SHU9119 there was increased food intake (SHU9119) and weight gain (SHU9119 and SHU9119 pair fed), so the increase in adiposity cannot be a ttributed to a specific receptor pathway. The expression of UCP1 protein in iBAT was reduced in response to SHU9119 treatment in both sedentary animals and those with access to WR, and was independent of caloric intake. Similarly, UCP1 expression was reduc ed in Wistar rats in response to SHU9119 infusions  Without functional MC4Rs the activation of UCP1 is reduced, even in response to leptin stimulation  When UCP1 expression was evaluated with WR within each individual treatment condition, WR reduced UCP1 expression, although t he effect was only statistically significant in control animals ( Figure 4 9 ). Since WR also reduces caloric intake, the reduced UCP1 appears to be logical as there would be less demand for a thermogenic response. T he amount of reduction also appears to be related to caloric intake, as UCP1 expression was reduced to a lesser degree in the SHU9119 treated group than in the SHU9119 pair fed group. Based upon the results from our current experiments, WR maintains the ab ility to redu ce HFD intake even in the presence of CNS MC3/4R blockade. Although WR was reduced by treatment with MCR blockade, even low levels of WR were able to produce the changes in physiological responses, furthering the argument that even minimal lev els of WR are able to induce physiological effects  Surprisingly, neither melanocortin r eceptor blockade or leptin antagonist treatment (Chapter 3) prevented the WR induced reductions in caloric consumption, suggesting that the effects of WR may act through alternate mechanisms. One p otential explanation is that BDN F levels are
141 changed by WR. Exercise has been shown to increase levels of BDNF that are reduced with HFD intake  and the effect of BDNF lies downstream of MCRs  BDNF levels were not measured in these experiments, but it remains a possibility that part of the WR effect was to increase BDNF levels independently of MCRs. Other potential mechanisms involved with WR induced changes include reduced endoplasmic reticulum stress and decreased inflammation. We attempted to evaluate various inflammatory mar kers with in the hypothalamus of WR expose d rats but were unable to detect significant differences, and many of the markers we intended to evaluate were below the limit of detection of the assay. Future studies directed at characterizing the changes to both CNS inflammation, as well as endoplasmic reticulum stress, in response to WR should be conducted. In conclusion, voluntary wheel running is an effective method of reducing caloric intake in rats. The mechanism of action underlying WR induced alterations t o diet preference and overall caloric intake remains to elucidated, however if accomplished, may indeed provide a viable therapeutic target for the treatment of overweight and obesity.
142 Figure 4 1 Daily change in body weigh t with SHU9119 treatment during dietary choice with introduction of running wheels. Control rats (black closed squares SED, open WR), SHU9119 (grey closed squares SED, open WR), and SHU9119 pair fed (grey closed diamond SED, open WR) had pumps changed at d ay 0, high fat diet was introduced on day 4 and running wheels were introduced on day 11 for the remainder of the experiment. All groups showed increased weight gain upon introduction of high fat diet and introduction of running wheels reduced weight gain.
143 Figure 4 2 Daily intake of chow and high fat diet in aCSF and SHU9119 treated rats with or without access to running wheels. Intake of chow diet is represented in squares while caloric consumption of HFD is displayed with triangles. C losed symbols are representative of animals maintained in the sedentary state during the wheel running phase while open symbols represent animals with access to running wheels following the introduction on day 11. Control animals are shown usi ng Black and SHU9119 treated animals are represented by grey symbols, SHU9119 pair fed animals are excluded for clarity but match control values for caloric intake of each diet with respect to the corresponding pair fed group.
144 Figure 4 3 Average daily caloric intake of high fat diet in aCSF and SHU9119 treated groups at baseline (Phase A) and the one week period following the introduction of running wheels (Phase B). Control aCSF animals with locked running wheels (black bar s) show no difference compared to baseline, while control aCSF animals with functional running wheels (open bars) have reduced intake. SHU9119 treated animals with locked wheels (grey bars) show no change of caloric intake in the presence of running wheel s while SHU9119 treated animals with functional wheels (grey dotted bars) show reduced intake. ** p<0.01, *** p<0.001 by SNK post test on Two Way Repeated Measures ANOVA.
145 Figure 4 4 Average daily wheel running activity in S HU9119 treated diet choice paradigm. SHU9119 treatment reduces the distance rats run daily in running wheels (middle bar, dark grey). Pair feeding rats treated with SHU9119 (right bar, light grey) decreases the magnitude of reduction in wheel running when compared to SHU9119 treatment, however wheel running is still significantly reduced in comparison to control animals. ** p<0.01, *** p<0.001 to aCSF control.
146 Figure 4 5 Change in body composition following one month of S HU9119 treatment assessed by TD NMR. A) Adiposity as a percentage of body weight increases in all groups and SHU9119 treatment independent of caloric intake promotes deposition of adipose tissue; however the increase in adiposity is attenuated in groups wi th access t o running wheels (open bars). B) The change in lean mass as a percentage of body weight responds in an inverse fashion to changes in adiposity. Bars denoted by different letters demonstrate statistically significant differences (p<0.001, except a/b p<0.01).
147 Figure 4 6 Cumulative wet tissue weight of selected white adipose tissue depots from rats treated for 28 days with SHU9119. SHU9119 treatment (dark grey bars) resulted in an increased deposition of white adipo se tissue compared to aCSF control (black bars). Moreover, SHU9119 promoted the increase in fat independently of caloric consumption as values are elevated in SHU9119 pair fed groups (light grey bars). Wheel running (open bars) is able to attenuate increas e in fat deposition when compared to sedentary groups (closed bars). Bars denoted by different letters demonstrate statistically significant differences (p<0.05).
148 Figure 4 7 Interscapular brown adipose tissue (iBAT) depot s ize following 28 day treatment with SHU9119. Rats treated with SHU9119 (dark grey bars) showed an increase in iBAT size however pair feeding SHU9119 treated rats prevented the increase in iBAT size (light grey bars) compared to control aCSF group (black b ars). Voluntary wheel running (open bars) reduces the size of the iBAT depot. Bars denoted by different letters demonstrate statistically significant differences (p<0.001).
149 Figure 4 8 Uncoupling Protein 1 expression in iBA T depots. A) SHU9119 treatment independent of caloric intake decreases the expression of UCP1 in sedentary animals as the decrease compared to aCSF control (black bar) was similar in both SHU9119 (dark grey bar) and SHU9119 pair fed animals (light grey bar ). B) Similar to sedentary conditions, SHU9119 treatment decreases UCP1 expression in rats when animals had access to functioning running wheels; SHU9119 (dark grey open bar) and SHU9119 pair fed animals (light grey open bar) UCP1 expression was reduced by approximately 70% in comparison to aCSF control (black open bar). *** p<0.001 compared to respective control.
150 Figure 4 9 Effect of voluntary wheel running on UCP1 expression in iBAT depots. Expression of UCP1 with wheel ru nning was compared within each in dividual treatment condition. A) WR reduced UCP1 expression by approximately 50% in control aCSF animals. B) In SHU9119 treated animals WR reduced UCP1 exp ression by approximately 25%. C) WR produced a reduction of approxim ately 65% in SHU9119 pair fed rats. ** p<0.01 by t test.
151 CHAPTER 5 CONCLUDING REMARKS Overweight and obesity are major global healthcare issues in current times. Even though the rate of increased prevalence may indeed be slowing in the United States, the increase d prevalence observed in the developing world is alarming and more than enough to necessitate vigorous investigation into the mechanisms associated with the onset of, as well as developing effective therapeutics against overweight and obesity. Furthermore, even without increased prevalence, maintenance of the status quo at the current level of overweight and obesity is both medically and economically troublesome. The preceding chapters highlight some of the complications associated with current therapies, a nd also present evidence for some potentially new directions for treatment. Clearly there is a substantial amount of work yet to be done in order to understand the regulation of energy balance at the molecular, cellular, and organ systems level Considerin g the numerous inputs involved with overall energy homeostasis, the future work proposed below is limited within the framework of the preceding text; though plentiful alternatives exist that are equally significant in addressing the overall obes ity problem The evidence suggests continual stimulation with leptin results in a resistance which is the major obstacle for the use of exogenous leptin therapy, especially considering the already increased endogenous levels of leptin present in the overweight state. The evidence presented in Chapter 2, that high dose leptin treatments will produce a resistant state even in lean animals, further highlights this issue. While we suggest that a lower dose may allow for a more prolonged beneficial effect, intermittent lep tin delivery may be considerab ly more effective. This may be i n part due to the
152 potential for recovery of the leptin system, as intermittent delivery would allow for the negative feedback mechanisms involved with continual stimulation to be reset and there fore re sensitize the receptors. Furthermore, intermittent fasting has been shown to improve physiological parameters and increase lifespan just as is observed with calorie restriction  Considering the effects of leptin on food intake, intermittent leptin treatment would presumably mimic an intermittent fasting state Such experiments should be readily feasible using viral mediated leptin delivery in a tetracycline on/off system; a system that has previously been demonstrated to be effective in rats  Additionally, normal physiological leptin concentrations are subject to circadian regulation. Leptin is only a singular component of energy regulation; therefore when leptin is infused or over expressed by viral methods, constant leptin expression is produced, which is not reflective of the natural state. Development of leptin viral vectors under the control of circadian promoters is currently underway D elivery of leptin by this meth od may potentially produce a m ore significant physiological effect, as well as preventing the onset of leptin resistance due to the projected cyclic variation of leptin levels. It would obviously be a gross oversight not to mention that either of these methods would depend on the subje cts being sensitive to leptin in the first place. However based on the progress made in sensitizing leptin action, as evidenced by amylin pairing in pharmacological treatment [114, 150, 151] with wheel running [112, 226] and hopefully additional agents in the future, altering the pattern of leptin delivery may yet be shown to be effective in all populations. The experiments on the development of leptin resistance from different viral loads were terminated after 6 months ; h owever this t ime frame did not provide a
153 complete picture. While it is financially and experimentally difficult to conduct long term animal experiments a more complete view of lower dose leptin treatments over time would be valuable A two year longitudina l study could potentially provide further insight into the rate of onset with regards to leptin resistance, and discern if such a phenomenon occurs even at lower doses of leptin treatment. In retrospect, the rats in these studies could have been subjected to alternative methods for evaluating the state of leptin resistance withi n the 6 month experimental time frame. While feeding rats a HFD would potentially produce a unique physiological profile, this can be an effective short term leptin challenge. In norm al leptin sensitive rats feeding normalizes rapidly within a one week period ; however leptin insensitive rats have a delayed normalization as is evidenced when administering leptin antagonist and evaluating the feeding response to HFD  Since the effect manifests rather rapidly, this could allow for repeated evaluation of resistance due to different doses within the compressed timeframe and due to the short nature of the test period animals could rapidly be returned to chow diet between testing. Such a strategy would also be advisable for future experiments involving the alternative forms of leptin delivery proposed in the preceding paragraph. As the mapping of leptin responsive neural pathways progresses, it may become possible to selectively target specific actions of leptin. The evidence that the energy expenditure effe ct of leptin is spared from resistance much longer than the effect on food intake (Chapter 2 and  ), suggests that if leptin treatment could be directed at a distinct neural population, the effects of leptin resistance may be minimized. To explore these actions the use of neural subtype specific promoters driving leptin expression
154 could be considered While such experiments depend on the results o f ongoing neural mapping projects, they remain on the horizon and will hopefully provide additional insight into leptin action. In the interim, more descriptive localization of leptin action could be evaluated by using immunohistochemistry techniques from isolated brain slices following acute leptin stimulation. Such methods still allow for quantification of pSTAT3 signals, but also provide increased neuroana tomical context opposed to the micropunch and Western blotting methods that were performed in the cu rrent experiments Further more, the addition of a GFP sequence, or alternative tag sequence recognizable via an antibody, to leptin could be evaluated; thereby allowing both the determination of neuronal activation due to leptin through evaluation of pSTAT 3 and the localization of virally produced leptin peptide. Although there are drawbacks to the use of the afore described immunohistochemical technique it certainly is an option that should be strongly considered in the design of further experiments. Eve n though the specific mechanism as to the changes induced by wheel running to food intake were not determined in the course of these studies the observed phenomenon remains an area of research that can be enthusiastically pursued. The findings that neithe r leptin antagonist nor melanocortin receptor blockade prevents wheel running induced reduction of high fat diet intake are exiting in and of themselves. While the past strategies involved a deductive form of evaluation, it is advisable to conduct future e xperiments in an inductive fashion. Based upon past evidence that wheel running revealed enhanced leptin signaling in the VTA  a sensible direction would be to evaluate the effect of whe el running on reward processes.
155 While n umerous potential studies evaluating the effect of wheel running on reward can be conceived, the current evidence would sugg est beginning by observing the impact of high fat diet reward. Past studies have shown that feeding and recreational drug use stimulate similar neuronal pathways allowing for the use of comparable behavioral paradigms to be used in observing food reward [227, 228] Protocols involving both fixed ratio and progressive ratio responding to rewards, including natural reinforcers, work in rat models ; considering the use of pharmacological agents as modifiers for responding ha ve been extensively used in the past  it is not a stretch to think that behavioral modifications such as wheel running could also change the response pattern. The effect of wheel running also appears to maintain some latency so animals exposed to wheel running could be subjected to behavioral testing following a bout of running. Variation in both the type of reinforcer i. e. HFD, chow diet, sucrose diet etc. and duration of wheel running (hours to days pretreatment) should be evaluated in addition to observing the CNS dopamine profile of experimental animals at the end of testing. If evidence for positive changes associated with wheel running are found then alternative methods of exercise activity should also be evaluated as wheel running itself is known to constitute a rewarding activity and can alter the mesolimbic dopamine reward pathway  While determining the mechanism of action of wheel running induced changes to diet intake i s interesting and of significance lack of understanding the mechanism of ac tion does not necessitate postponing experiments evaluating the action of wheel running on leptin sensitivity. Experiments using wheel running in combination with pharmacological low dose leptin, in comparison to high dose leptin and wheel running, are cur rently being designed.
156 Effective solutions are due to the sum of individual incremental advances. Although the results of experiments provided within this text demonstrate that many obstacles remain with current obesity therapies utilizing leptin and serot onin mechanisms, there is hope for such therapies in the future. The evidence that wheel running is able to initiate changes to dietary intake independently of both leptin and melanocortin pathways, provides the basis for discovery of novel therapeutic tar gets. In the interim, this work adds further evidence that even minimal levels of exercise should be employed in the treatment of obesity, as activity produces physiological changes independent of the energy expended by the activity. While effective pharma cological treatments alone are still unlikely to completely solve the current crisis, the pairing of effective treatments with behavioral modifications could potentially increase patient compliance and adherence to comprehensive weight loss programs. Given the increased attention to the issue of overweight and obesity within recent years, there is immense hope that the cumulative efforts of the scientific community will soon relegate the issue of overweight and obesity to the past.
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175 BIOGRAPHICAL SKETCH Kevin Yannick Emanuel Strehler was born in Zurich, Switzerland. He received his high school diploma from Mayo Senior High School Rochester, Minnesota in 2005 and graduated summa cum laude with a Bachelor of Science emphasis in human b iology from Minnesota State University, Mankato in 2009. His undergraduate research involved determining p rotein interactions of the beta1 and beta2 isoforms of actin capping protein. He also completed a summer undergraduate research fellowship at the University of Texas Southwestern Medical Center in Dallas studying lipolytic pathways in Drosophila m elanogast er For his undergraduate research he was awarded three university level research grants along with a department sponsored grant. In fall of 2009 h e enrolled in the Interdisciplinary Program in Biomedical Sciences at the University of Florida a s an Alumni and Linton E. Grint er Fellow. His Ph.D. was obtained from the College of Medicine under the tutelage of Dr. Philip Scarpace in the Department of Pharmacology and Therapeutics.