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The Role of Endocannabinoid Receptor Activity in Young and Aged Rats with High-Fat Feeding

Permanent Link: http://ufdc.ufl.edu/UFE0041049/00001

Material Information

Title: The Role of Endocannabinoid Receptor Activity in Young and Aged Rats with High-Fat Feeding
Physical Description: 1 online resource (145 p.)
Language: english
Creator: Judge, Melanie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aged, am251, antagonist, cannabinoid, cb1, chow, endocannabinoid, fat, high, hypothalamus, leptin, palatability, preference, receptor, resistance, young
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Two-thirds of adult Americans are overweight/obese, which increases the risk of developing other serious diseases. Leptin, a hormone produced in adipose tissue that acts within the hypothalamus, increases energy expenditure and decreases food intake. Endocannabinoids are produced on-demand and act on central cannabinoid-1 (CB1) receptors to stimulate food intake and fat storage. This dissertation examined leptin and CB1 antagonist responses in young adult and aged rats with or without high-fat (HF) feeding to better understand the dysregulation of these two signaling systems in the aged and/or obese state. First, we demonstrated that all aged rats, as opposed to only some young adult rats, are susceptible to the detrimental effects of a HF diet. When given ad libitum access to a HF diet, aged rats display exacerbated hyperphagia and body weight gain, characterized by a disproportionate gain in fat versus lean mass. Additionally, we demonstrated that young adult rats display dose-dependent reductions in food intake and body weight in response to peripheral leptin infusions while aged rats remain unresponsive to exogenous leptin. Next, we showed that daily i.p. administration of AM251, a CB1 antagonist, reduced the intake of the HF diet to a greater extent than normal chow during short-term exposure do the diets. AM251 stimulated greater anorectic responses, characterized by decreases in caloric intake and body weight gain, in aged rats, which was further enhanced with short-term HF feeding. In accordance with the decrease in body weight, AM251 treatment induced a reduction in adiposity and serum leptin levels in young adult and aged rats. However, AM251 was unable to change the preference for the diets tested. We next investigated responsiveness to AM251 after long-term dietary exposure. Again, AM251 induced greater anorectic effects with age and HF-feeding, measured by increased sensitivity and maximal efficacy. These results appeared to be related to the diet and established obesity as well as the development of leptin resistance. Further studies are needed to confirm the connections between leptin resistance and the dysregulation of the endocannabinoid signaling system that is believed to occur in aged and/or obese states.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melanie Judge.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Scarpace, Philip J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041049:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041049/00001

Material Information

Title: The Role of Endocannabinoid Receptor Activity in Young and Aged Rats with High-Fat Feeding
Physical Description: 1 online resource (145 p.)
Language: english
Creator: Judge, Melanie
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aged, am251, antagonist, cannabinoid, cb1, chow, endocannabinoid, fat, high, hypothalamus, leptin, palatability, preference, receptor, resistance, young
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Two-thirds of adult Americans are overweight/obese, which increases the risk of developing other serious diseases. Leptin, a hormone produced in adipose tissue that acts within the hypothalamus, increases energy expenditure and decreases food intake. Endocannabinoids are produced on-demand and act on central cannabinoid-1 (CB1) receptors to stimulate food intake and fat storage. This dissertation examined leptin and CB1 antagonist responses in young adult and aged rats with or without high-fat (HF) feeding to better understand the dysregulation of these two signaling systems in the aged and/or obese state. First, we demonstrated that all aged rats, as opposed to only some young adult rats, are susceptible to the detrimental effects of a HF diet. When given ad libitum access to a HF diet, aged rats display exacerbated hyperphagia and body weight gain, characterized by a disproportionate gain in fat versus lean mass. Additionally, we demonstrated that young adult rats display dose-dependent reductions in food intake and body weight in response to peripheral leptin infusions while aged rats remain unresponsive to exogenous leptin. Next, we showed that daily i.p. administration of AM251, a CB1 antagonist, reduced the intake of the HF diet to a greater extent than normal chow during short-term exposure do the diets. AM251 stimulated greater anorectic responses, characterized by decreases in caloric intake and body weight gain, in aged rats, which was further enhanced with short-term HF feeding. In accordance with the decrease in body weight, AM251 treatment induced a reduction in adiposity and serum leptin levels in young adult and aged rats. However, AM251 was unable to change the preference for the diets tested. We next investigated responsiveness to AM251 after long-term dietary exposure. Again, AM251 induced greater anorectic effects with age and HF-feeding, measured by increased sensitivity and maximal efficacy. These results appeared to be related to the diet and established obesity as well as the development of leptin resistance. Further studies are needed to confirm the connections between leptin resistance and the dysregulation of the endocannabinoid signaling system that is believed to occur in aged and/or obese states.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melanie Judge.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Scarpace, Philip J.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041049:00001


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THE ROLE OF ENDOCANNABINOID RECEPT OR ACTIVITY IN YOUNG AND AGED RATS WITH HIGH-FAT FEEDING By MELANIE KAE JUDGE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Melanie Kae Judge 2

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To my patient, loving husband Michael Adam Judge Adam, you are the icing on my cake. 3

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ACKNOWLEDGMENTS The research described in this dissert ation was made possible through the help and support of University of Florida facu lty, fellow graduate students, post-docs, scientists, and other collaborators of the Philip J. Scarpace laborat ory. First, I would like to thank Dr. Scarpace for all his guidance an d support. He has been integral in my scientific maturation and has provided me the oppor tunity to explore areas that interest me as well as attend important annual meetings in the field. It has been a pleasure to work with him for the last few years. Each and every member of the Scarpace l ab has contributed to these experiments as well as my development as a scientist. Without Michael Matheny, the lab would be a dull, dismal, quiet place. While giving endless scientific aid, Mike kept everyones spirits up with singing, yodeling, excellent conversa tion, the occasional debate, and laughter. On a more personal note, Mi ke offered me much-needed em otional support when I felt like giving up on an assay or experiment. Jiejin Zhang continuously offered her scientific expertise during trouble-shooting or planning an experiment. I also want to thank her for helping to expand my small world by entertaining my many questions about the Chinese culture. Dr. Yi Edi Zhang has provided priceless experimental advice. Her wealth of knowledge in obesit y research and science in general always amazes me. Kit-Yan Thomas Cheng has been a life-saver for me, weighing rats and helping in as many ways as possible. T homas, a fellow soccerand chocolate-lover, never raised the courage to yodel in lab, but I m sure he practiced at home all the time. In Dr. Lourdes Andino, I found a kindred spir it. We immediately bonded through our love of baking and our experiences as newlyweds. Her advice during experiments and when preparing this dissertation has been great ly appreciated. Dr. Alexandra Shapiro 4

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has provided much support on present ations, posters, and experiments. Simultaneously, she has served as a good example for me on how to successfully balance raising a family and pursuing a career. I owe a debt of gratitude to many facu lty members who gave advice and guidance through my years as a graduate student. As the coordinator of Women in Science meetings, Dr. Nihal Tumer has given me the opportunity to network with other women in the science field as well as hear their c oncerns and beliefs. Dr. Tumer has also participated in many lab meetings and prov ided valuable guidance on my experiments. Dr. Kathleen Shiverick, also a member of the Women in Science group, has helped open my eyes to the issues facing females in the scientific field. Dr. Neil Rowland has continually provided an intellectual chal lenge through his questions, comments, and courses taught. Dr. Christy Carter has an amazing knowledge of animal physiology, especially in aging models. Although I was unabl e to use it for my dissertation, Dr. Michael King kindly took the time to teach me immunohist ochemistry. Dr. Timothy Garrett has provided invaluable resources for attempting to measuring hypothalamic endocannabinoid levels. Above all else, though, I would like to thank my family and friends for providing a firm foundation of support throughout my life. I thank my husband Adam for being a quiet listener when I had to get something o ff my chest and helping me put things back into perspective. He has listened to count less PowerPoint presentations, though very little of which he fully understood. Through everything, he has been a shoulder to cry on, a hand to hold, and a best friend to laugh wit h. I want to thank my parents, Bob and Sue, for their unconditional support and love throughout my life. They have always been my biggest (and loudest) fans at sporti ng events, formal ceremonies, and in every5

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day life. My sister, Robin, a member of People for the Ethical Treatment of Animals (PETA), also deserves a special note of grat itude as it was particularly hard for her to know I worked with rodents. We had an unspok en agreement that I would not tell her about my experiments and she would not bombard me with PET A propaganda. However, it was hard to resist not throwi ng a couple of teasing comments her way every once in a while. I also thank my grandparent s both paternal and maternal. They were always interested in how my research was pr ogressing and how many rats I had to feed every day. Throughout my life, my grandpar ents have supported every decision I made and believed I could accomplish anything I set my mind to. Lastly, I thank the members of my Girls Night group for helping me escape from the world of science every once in a while. Even though the conversation often turned back to science during our outings, it was nice to vent out frustr ations and celebrate accomplishments with a good group of friends. 6

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ..................................................................................................4 LIST OF TABLES ..........................................................................................................10 LIST OF FIGURES ........................................................................................................11 LIST OF ABBREVIATIONS ...........................................................................................13 ABSTRACT ...................................................................................................................15 1 BACKGROUND AND S IGNIFICA NCE................................................................... 17 The Obesity Epidemic .............................................................................................17 Health Consequences Associated with Obesity ......................................................17 Whole Body Energy Homeostasis ..........................................................................18 HF Diets ...........................................................................................................18 Complex Mechanisms for Energy Homeostasis ...............................................18 Leptin ......................................................................................................................20 Role of Leptin in Energy Regulation .................................................................20 Congenital Leptin Deficiency ............................................................................20 Other Physiological Functions of Leptin ...........................................................21 Leptin Receptor ......................................................................................................22 Isoforms ............................................................................................................22 Leptin Expression in the Brain ..........................................................................22 Leptin-Leptin Receptor Binding ........................................................................23 Intracellular Domain of the Leptin Receptor .....................................................24 Leptin Receptor Deficiency ...............................................................................24 Leptin Receptor Signal Transduction ......................................................................25 JAK2-STAT3 Pathway ......................................................................................26 ERK Pathway ...................................................................................................26 PI3K-cAMP Pathway ........................................................................................27 Tyr1077-STAT5 Pathway .................................................................................27 Negative Regulators .........................................................................................27 Downstream Leptin Signaling & Neuropept ide Regulation in the Hypothalamus ....28 Brown Adipose Tissue (BAT) and Uncoupling Protein 1 (UCP1) ............................30 Leptin Resistance ...................................................................................................31 Leptin Treatments in Human Obesity ...............................................................31 Mechanisms .....................................................................................................31 Role of Elevated Leptin in Leptin Resistance ...................................................32 Age-Related Obesity and Leptin Resistance ....................................................34 Caloric Restriction Reverses Leptin Resistance ...............................................34 Summary of the Leptin Signaling System ...............................................................35 The Endocannabinoid System (ECS) .....................................................................35 Cannabinoid-1 (CB1) Receptors ......................................................................36 7

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Cannabinoid-2 (CB2) Receptor ........................................................................37 ECs ..................................................................................................................38 Central ECs ......................................................................................................39 Peripheral ECs .................................................................................................40 Interactions of Leptin and the ECS .........................................................................41 CB1 Receptor Antagonists & the Treatment of Obesity ..........................................43 Summary of the ECS ..............................................................................................45 Central Hypothesis .................................................................................................45 2 GENERAL METHODS AND MATERI ALS..............................................................47 Experimental Animals .............................................................................................47 General Experimental Design .................................................................................47 Experimental Diets ..................................................................................................48 Subcutaneous Leptin Infusion .................................................................................49 Intraperitoneal (i.p.) CB1 An tagonist Administration ...............................................49 Body Composition Measurement ............................................................................50 Serum Leptin ..........................................................................................................50 Physical Performance Tests ...................................................................................50 Wheel Running .......................................................................................................50 Tissue Harvesting and Preparation .........................................................................51 Western Analysis ....................................................................................................51 RNA Isolation and Reverse Transcription ...............................................................51 Relative-Quantitative PCR ......................................................................................52 Statistical Analysis ..................................................................................................52 3 UNEXPECTED PROLONGED HYPERPHA GIA WITH HIGH-FAT FEEDING CONTRIBUTES TO EXACERBATED WEIG HT GAIN IN RATS WITH ADULTONSET OBEISTY...................................................................................................54 Introduction .............................................................................................................54 Experimental Design ...............................................................................................55 Results ....................................................................................................................56 Body Weight Change with HF-Feeding ............................................................56 Caloric Intake with HF-Feeding ........................................................................57 Body Composition ............................................................................................58 Serum Leptin and Adiposity ..............................................................................59 Wheel Running & Physical Performance Tests ................................................59 Hypothalamic Measures of Leptin Action .........................................................60 BAT UCP1 Levels ............................................................................................60 Dose Response to Peripheral Leptin Infusion ..................................................61 Discussion ..............................................................................................................62 A NOVEL STUDY OF HIGH-FAT DIET-INDUCED HYPERPHAGIA AND RESPONSES TO CB1 ANTAGONIST, AM 251, IN YOUNG AND AGED RATS....77 Introduction .............................................................................................................77 Experimental Design ...............................................................................................79 8

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Experiment 1 ....................................................................................................79 Experiment 2 ....................................................................................................80 Results ....................................................................................................................80 Experiment 1 ....................................................................................................80 Experiment 2 ....................................................................................................83 Discussion ..............................................................................................................85 5 RESPONSES TO THE CANNABINOID RECEPTOR-1 ANTAGONIST, AM251, ARE MORE ROBUST WITH AGE, WITH ESTABLISHED HIGH-FAT FEEDINGINDUCED OBESITY, AND WITH LEPTIN R ESISTANCE......................................95 Introduction .............................................................................................................95 Experimental Design ...............................................................................................96 Results ....................................................................................................................97 Biochemical Indicators .....................................................................................99 Comparisons of Young Adult and Aged Rats with Chow and HF Feeding .....100 Discussion ............................................................................................................101 6 GENERAL DISCUSSION AND CONCLUS ION....................................................111 Major Finding and Conclusions .............................................................................112 Future Directions and Potential Improvements .....................................................120 Conclusions ..........................................................................................................127 LIST OF REFERENCES .............................................................................................129 BIOGRAPHICAL SKETCH ..........................................................................................145 9

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LIST OF TABLES Table page 3-1 Hypothalamic PTP1B protein levels hypothalamic SOCS-3 mRNA levels, and BAT UCP-1 protein levels at sacrifice from 3and 30-month old rats following chow or HF feeding. .............................................................................73 3-2 Serum leptin levels in young and aged ra ts on a chow diet after 7-day saline or leptin infusion. ................................................................................................74 4-1 Adiposity, lean mass, and serum leptin levels afte r respective AM251 doses in young rats during hyperphagia. ......................................................................90 4-2 Adiposity, lean mass, and serum leptin levels afte r respective AM251 doses in aged rats during hyperphagia .........................................................................92 5-1 Change in body composition and biochem ical markers of young rats during daily i.p. vehicle or AM251 injections. ...............................................................106 5-2 Change in body composition and biochem ical markers of aged rats during daily i.p. vehicle or AM251 injections. ...............................................................108 10

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LIST OF FIGURES Figure page 3-1 Body weight gain in young and aged rats during chow or HF feeding ................67 3-2 Body weight change in rats of differing ages on chow or HF diet .......................68 3-3 Caloric intake of 3-, 6-, and 30-month old rats on a HF diet ...............................69 3-4 Change in fat mass, lean mass, and the ratio of fat-to-lean mass over time in 3and 30-month-old rats on either a HF or a standard chow diet .......................70 3-5 Serum leptin at day 60 and white adi pose tissue at sacrifice in 3and 30month-old rats on chow or HF diets ....................................................................71 3-6 Voluntary wheel runni ng over a 4-day period. ....................................................72 3-7 Change in body weight and food intake during a 7-day peripheral leptin infusion in 3-month-old chow-fed rats .................................................................75 3-8 Change in body weight and food intake during a 7-day peripheral leptin infusion in 30-month-old chow-fed rats ...............................................................76 4-1 Change in caloric intake and body wei ght in young rats dur ing daily i.p. 0.83 mg/kg or 2.78 mg/kg AM251 administration .......................................................89 4-2 Change in caloric intake and body wei ght in aged rats during daily i.p. 0.83 mg/kg or 2.78 mg/kg AM251 administration .......................................................91 4-3 Caloric intake and change in body wei ght in young rats during food choice and i.p. vehicle or 0.83 mg/kg AM251 treatment ................................................93 4-4 Caloric intake and change in body weight in aged rats during food choice and vehicle or 0.83 mg /kg AM251 treatment .............................................................94 5-1 Decrease in body weight at day 10 fo llowing peripheral vehicle or leptin infusion in chow or HF fed rats .........................................................................104 5-2 Change in cumulative caloric intake and body weight in young rats during 7day daily i.p. administration of AM 251 (0.45 mg/rat/day; 1.2mg/kg/day) ..........105 5-3 Change in cumulative caloric intake and body weight in aged rats during 7day daily i.p. administration of AM 251 (0.45 mg/rat/day; 0.8 mg/kg/day) .........107 5-4. Percent change in body weight in res ponse to peripheral leptin infusion versus peripheral AM251 injections in y oung and aged rats with and without HF feeding ..............................................................................................................109 11

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5-5 Change in body weight in AM251-tr eated rats compared to vehicle-treated rats on Day 5 of the respective experiments ....................................................110 12

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LIST OF ABBREVIATIONS 2-AG 2-arachidonoylglycerol AEA andandamide; N-arac hidonylethanolamine AgRP agouti-related protein Arc Arcuate nucleus BAT brown adipose tissue BBB blood brain barrier BMI body mass index BRET bioluminescence resonance energy transfer cAMP cyclic adenosine monophosphate CART cocaineand amphetam ine-regulated transcript CB1 cannabinoid receptor-1 CB2 cannabinoid receptor-2 CRH corticotropin-related hormone CSF cerebrospinal fluid DIO diet-induced obese DMH dorsomedial hypothalamus DR diet resistant EC endocannabinoid ECS Endocannabinoid System ERK extracellular signal-related kinase EWAT epididymal white adipose tissue FAAH fatty acid amide hydrolase F344xBN Fischer344 xBrown Norway 13

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GABA -aminobutyric acid GI gastrointestinal HF high-fat i.p. intraperitoneal JAK janus kinase LCD liquid crystal display LHA lateral hypothalamic area MAPK mitogen-activated protein kinase MGL monoglycerol lipase MSH melanocyte stimulating hormone NPY neuropeptide Y PDE3B phosphodiesterase 3B PFA perifornical area PI3K phosphatidylinositol 3-kinase POMC pro-opiomelanocortin PVN paraventricular nucleus PWAT perirenal white adipose tissue RTWAT retroperitoneal white adipose tissue SOCS3 suppressor of cytokine signaling 3 STAT3 signal transducer and activator of transcription 3 TD-NMR time domain nuclear magnetic resonance THC (-)9-tetrahydrocannabinol UCP-1 uncoupling protein VMH ventromedial hypohtalamus WAT white adipose tissue 14

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy THE ROLE OF ENDOCANNABINOID RECEPT OR ACTIVITY IN YOUNG AND AGED RATS WITH HIGH-FAT FEEDING By Melanie Kae Judge December 2009 Chair: Philip J. Scarpace Major: Medical Sciences Physiology and Pharmacology Two-thirds of adult Americans are overwei ght or obese, which increases the risk of developing other serious diseases. Leptin is a hormone produced in adipose tissue that acts within the hypothalamus to increase energy expenditure and decrease food intake. In contrast, endocannabinoids are produced on-demand and act on central cannabinoid-1 (CB1) receptors to stimulate food intake and fat storage. This dissertation examined leptin and CB1 antagonist responses in both young adult and aged rats with or without highfat (HF) feeding in order to better understand the dysregulation of these two signaling syst ems in the aged and/or obese state. First, we demonstrated that all aged rats, as opposed to only some young adult rats, are susceptible to the detrimental e ffects of a HF diet. When given ad libitum access to a HF diet, aged rats display ex acerbated hyperphagia and body weight gain, characterized by a disproportionate gain in fat versus lean mass. Additionally, we demonstrated that young adult rats display dose-dependent reductions in food intake and body weight in response to peripheral leptin infusions while aged rats remain completely unresponsive to the exogenous leptin. 15

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Next, we showed that daily i.p. admin istration of AM251, a CB1 antagonist, reduced the intake of the highly palatable HF diet to a great er extent than normal chow during short-term exposure do the diets. AM251 stimulated greater anorectic responses, characterized by decreases in caloric intake and body weight gain, in aged rats, and this effect was fu rther enhanced with short-term HF feeding. In accordance with the decrease in body weight, AM251 trea tment induced a reduction in adiposity and serum leptin levels in young adult and aged rats. However, AM251 was unable to change the palatability or prefer ence of the diets tested. After characterizing AM251 responsiveness during short-term di et exposure, we investigated responsiveness after long-term exposure. Again, AM251 induced greater anorectic effects with age and HF-feeding, m easured by increased sensitivity and maximal efficacy. These results appeared to be related to the diet and established obesity as well as the development of leptin resistance. Further studies are needed to confirm the connections between leptin re sistance and the dysregulation of the endocannabinoid signalin g system that is believed to occur in aged and/or obese states. 16

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CHAPTER 1 BACKGROUND AND SIGNIFICANCE The Obesity Epidemic Overweight and obesity are defined as the accumulation of fat that may cause impaired health (4). Physi cians often use body mass inde x (BMI), measured by a persons weight in kilograms divided by hi s/her square of the height in meters, to measure an individuals adiposity level, where a BM1 of 25-29. 9 is considered overweight and a BMI of 30 or higher is obese (4). According to the World Health Organization (WHO), approximately 1.6 billion adults were overweight and at least 400 million adults were obese in 2005. These figures are expected to further increase to 2.3 billion overweight and 700 million obese by 2015. At least 20 million children less than 5 years old were overweight in 2005, and these numbers are expected to increase as well (164). In addition, some believe that the current economic recession may exacerbate these epidemic trends becaus e when finances are tight, people often choose less nutritional, calorically dense fast foods over healthy supermarket purchases and are forced to give up memberships at gyms and sports clubs (95). Health Consequences Associated with Obesity Obese individuals suffer both emotional, caused by social prejudices, and physical consequences (4). In fact, obesity is likel y caused by multiple factors like diet, genes, and psychological factors (122). Many diseas es are believed to be secondary to the establishment of obesity, including type 2 di abetes, uterine cancer, gallbladder disease, osteoarthritis, stroke, hypertension, cor onary heart disease, breast cancer, and colon cancer (4). 17

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Whole Body Energy Homeostasis Obesity results from an imbalance between energy intake and energy expenditure. A positive energy balance is characterized by a change in energy intake without a compensatory and parallel chang e in energy expenditure. For example, it can be caused by an increase in energy intake without a corresponding increase in energy expenditure. Similarly, a positive energy balance can also be caused by a decrease in energy expenditure, as with aging, without a corresponding decrease in energy intake. Moreover, a prolonged positive energy balance may result in increased body weight gain and adiposity. HF Diets The ever-spreading obesity epidemic is often attributed to lifestyle changes, especially in Western societies (129). The typical Western diet is often characterized by expanding portion sizes and rela tively inexpensive foods hi gh in fat and sugar (129). When provided a highly palatable HF diet, rats display diet-induced hyperphagia, which increases caloric intake and promotes body weight gain (41). Mo reover, chronic HF feeding can alter the hormonal signaling invo lved in regulating energy homeostais, whether the rats become obese or not (50, 172). Complex Mechanisms for Energy Homeostasis Under homeostatic conditions, energy intake is metabolized to fulfill the bodys fuel needs, and excess energy is stored as fat for later use (88). Thus, the body requires a signaling system to assess the nutritional state of the animal and adjust energy intake/output as necessary, including t he inhibition of food intake when fuel requirements are met or the increase energy expenditure with excess food consumption. This system is often called the homeostatic regulation of body weight and 18

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involves interacting signals from both t he central nervous system (CNS) and peripheral organ systems (88). Nutrient s ensing begins with oral taste receptors and continues in the gastrointestinal (GI) tr act, pancreas, liver, muscle, and adipose tissue, which all involve crucial communication sy stems with the brain. For example, ghrelin is secreted in the mucosa of an empty stomach in preparation of food intake, but its secretion is abruptly inhibited after ingestion (88). In contrast, pancreatic in sulin secretion is increased after a meal, and the adipocyte-produc ed leptin, which is released as a signal of the bodys adiposity level, acts in the brain to reduce energy intake (156). White adipose tissue (WAT) has also become wi dely accepted as an important endocrine organ, secreting adipokines like adiponectin, resistin, adipsin, visfatin, and leptin. Studies in rodents have shown that peripheral administration of adiponectin doesnt alter food intake, but decreases body weight by increasing energy expenditure (123). Although conflicting evidence exists as to whet her resistin levels increase, decrease, or remain the same in obesity and type II di abetes, stimulation of macrophages with an endotoxin stimulates resistin production and re lease, suggesting that resistin is a key mediator in the insulin resi stance associated with certain in flammatory conditions (45). Adipsin, which is primarily expressed by adipocytes in mice and monocytesmacrophages in humans, is decreased in mu rine models of obesity and increased or unchanged in human obesity (45). Visfatin, the most recent ly discovered adipokine, is primarily produced in visceral white adipose ti ssue and binds to the insulin receptor to produce insulin-like effects (45) However, since its discov ery in 1994, leptin has been the most actively in vestigated adipokine. 19

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Leptin Role of Leptin in Energy Regulation Leptin, a 16 kD hormone, is produced by the obese (ob) gene and is named for the Greek word leptos meaning thin (53). It is prim arily produced in adipose tissue and circulates in proportion to whole body adiposity, but it is also synthesized in placenta, gastric fundic mucosa, skeletal muscle, and mammary epithelium (2). Human and rat leptin are 83% homologous (2). Leptin appe ars to be involved in both shortand longterm regulation of energy homeostasis. In ro dents, leptin levels increase within hours after ingestion, and in humans, leptin leve ls are increased days after overeating. However, leptin levels normalize hours after fasting begins in both species (2). In addition, leptin is rapidly depleted from the stomach of rodents after a meal, suggesting some activity in t he short-term regul ation of food intake (53). Congenital Leptin Deficiency Mice with homozygous mutations in the ob gene are characterized by early onset obesity. These ob/ob mice lack leptin, c ausing them to be hyper phagic, hypothermic, and obese in addition to other metabolic and neuroendocrine abnormalities (2). While congenital leptin deficiencies in humans ar e rare, a few cases have been reported. Two Pakistani cousins, who were severely obese at an early age although the family history included no obesity, were found to be homo zygous for a frame-shift mutation that resulted in the deletion of a guanine nucleot ide in codon 133 and produced a premature stop codon in the leptin message. As a re sult, the truncated lept in protein was not properly secreted, and the child ren had undetectable serum leptin levels (105). Another mutation, involving a single nucleotide substitution in codon 105 of the leptin gene, was found in a Turkish family. The affected i ndividuals were characterized by obesity, 20

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hyperphagia, hypogonadism and low serum leptin levels (148). Daily subcutaneous administration of leptin in t hese individuals caused a dramatic reduction in body weight, 98% of which was adipose tissue, attenuatio n of hyperphagia, and a steady reduction in the levels of plasma insulin, trig lycerides, and serum cholesterol (47). Other Physiological Functions of Leptin In addition to its energy homeostasis roles, leptin is an active participant in many other physiological functions, including reproduction, bone growth, metabolism, immunity, angiogenesis, and blood pressure r egulation. Mutations in the ob and db genes in rodents and humans result in hypo gonadism, but administr ation of leptin restores puberty and fertility ( 23, 47). Leptin deficient ob/ ob mice have various skeletal bone abnormalities, including dec reased bone length and increased spongy bone mass, when compared to wild-type controls, but this condition is rectified when leptin is administered to these ob/ob mice (78). Leptin decreases both glucose and insulin levels in ob/ob mice before weight loss o ccurs and stimulates lipolysis and fatty acid synthesis in the liver (3, 26). It has rec ently been shown to have many actions related to immune responses, including direct acti ons on T-cells, a regulatory action on natural killer cells, and induction of cytokines in macrophages (114). Leptin stimulates angiogenesis and has been found in the placenta, a highly angiogenic tissue (124). Regional sympathetic outflow is stimulat ed by endogenous leptin, even in the obese state, but short-term subcutaneo us infusions of low doses of leptin have been shown to significantly reduce blood pr essure in rats (82). 21

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Leptin Receptor Isoforms The leptin receptor, originally cloned in 1995, is a single membrane-spanning receptor, a member of the cl ass I cytokine receptor family, and has multiple isoforms (Ob-Ra, Ob-Rb, Ob-Rc, Ob-R d, Ob-Re and Ob-Rf ) (53, 149, 150). These isoforms result from alternative RNA splicing at the most C-terminal coding exon, meaning that all isoforms are identical throughout the ent ire extracellular portion and the differences in isoforms arise from diffe ring lengths and sequencing composit ions in the intracellular portions (149). The long-form of the receptor Ob-Rb, has an intracellular domain of 303 amino acids and is capable of intracellula r signal-transduction (149). This isoform is expressed predominantly in the hypothalamus, but lower le vels can be found in other brain regions, testes, and adipose tissue (86, 149). The shor t-form of the receptor, ObRa, which contains only a sm all portion of the intracellu lar domain, has been implicated in participating in transporting leptin from the blood into the cerebrospinal fluid (CSF) (149). Ob-Re, the soluble le ptin receptor, contains no intracellular portion and thus circulates in the bloodstr eam (86). Expression of Ob -Re has been located in the hypothalamus, testes, heart, and adipose tissue (86). Leptin Expression in the Brain Leptin receptors are expre ssed throughout the brain, bu t patterns of expression appear to be isoforms-specific. The short form of the receptor ha s been localized to the choroid plexus and moreover, the microvesse ls, which make up the blood brain barrier (BBB) (11). This evidence supports the possi bility that these Ob-Ra receptors function, at least partly, in transporting leptin ac ross the BBB through a high-affinity, saturable transport system (178). The long form of the receptor is primarily located in the 22

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hypothalamus and cerebellum. In fact, cerebellar expression which is believed to be involved in somatic motor ac tivity, muscle tone regulation, and equilibrium maintenance, surpasses expression in all other brain r egions (11). Within the hypothalamus, the highest concentrations of the leptin receptor are found in the arcuate nucleus (Arc), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH), and ventral premamillary nucleus (43, 90). These nuclei are all associated with feeding centers in the brain, providing further evidence for leptin action in the hypothalamus. The leptin receptor is only moderately expr essed in the paraventricular nucleus (PVN) but highly expressed in brain regions that pr oject to the PVN (i.e. DMH, VMH, and Arc), suggesting that leptin activation of the PVN is both direct and through innervation (43). Leptin-Leptin Receptor Binding All of the alternative splicing products of the leptin receptor have the same extracellular and transmembrane domains, but the mechanism of leptin binding to its receptor is still largely unknown (13). The extracellular porti on of the receptor is made up of 820 amino acids and contains two im munoglobulin-cytokine receptor homologous regions (CRH) separated by an Ig-like domai n and followed by two fibronectin III-like domains (34, 118). The CRH domain closes t to the membrane has been identified as the high affinity binding site on the receptor (52, 118). The Ig-like and two fibronectin IIIlike domains are not integral for leptin bind ing, but are required for leptin receptor activation (52, 118). Experiments based on quantitative bioluminescence resonance energy transfer (BRET) suggest that two leptin molecules bind to a pr e-existing receptor dimer in a 2:2 ratio to induce a rec eptor conformational change and stimulate downstream signaling. In contra st, the short form of the re ceptor primarily exist as monomers (31). This finding was supported by Devos, et al., who further described the 23

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leptin receptors as homodime rs, rather than heterodimer co mplexes where the different isoforms interacted (34). Intracellular Domain of the Leptin Receptor The isoforms of the leptin receptor have a long (Ob-Rb), short (Ob-Ra, Ob-Rc, ObRd, and Ob-RF) or no (Ob-Re) intrace llular domain (86). The membrane-bound isoforms share a common 29 amino acid sequenc e called Box 1, which is also highly conserved among other members of the cytokine receptor family (13). The second motif, called Box 2, is usually located within the first 50-60 amino acids in the cytoplasmic domain of cytokine family recept or members, and is only found in the ObRb leptin receptor isoforms (13). These motifs are nec essary for the intracellular interaction of the leptin rec eptor with tyrosine kinases. Be cause all other forms of the leptin receptor, aside from Ob-Rb, do not contain both of these motifs, they are signaling inactive and are considered incapable of mediating downstream signaling (13, 31, 86). Leptin Receptor Deficiency Leptin receptor mutations result in a phenotype characterized by obesity, hyperphagia, hypercholesterolemia, hyperlipidemia, and hyperglycemia (48, 119). In C57BL/K db/db mice, the mutation is caused by a single nucleotide substitution, which introduced a premature stop codon in the cytoplasmic region. This causes a replacement of Ob-Rb with Ob-Ra in these rodent s (86). In Zucker fatty (fa/fa) rats, a missense mutation causes a Glutamine to be replaced with a Proline on codon 269 of the extracellular domain (69). Surprisingly, these rats are still capable of binding leptin, suggesting that the mutation does not allow for proper dimerization of the leptin receptors, thus inhibiting in tracellular downstream signaling (119). However, mutations 24

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in the leptin receptor are extremely rare in humans. Clement, et al. studied a family with a strong prevalence of morbid obesity and found a single nucleotide substitution on exon 16. Both parents and 4 children were het erozygous for this allele, two additional unaffected children were homozygous for the wild type allele, and the remaining 3 children were homozygous for the mutation. The heterozygous children displayed normal BMIs, eating behavior, growth patterns, and sexual maturation, suggesting that one functional copy of the leptin re ceptor gene seems to be enough for normal physiology (25). Leptin Receptor Signal Transduction The leptin receptor is similar to all other members of the cla ss 1 cytokine receptor family in that it has no intrinsic kinase activity. Instead, it is dependent on cytoplasmicassociated Janus kinases (JAKs) for intrac ellular signaling to occur (173). Leptin binding to the long form of the leptin recept or stimulates activation of JAK2 by crossphosphorylation, which then phosphor ylates specific tyrosine residues (Tyr 985, 1077, and 1138) on the intracellular domain of the receptor (173). Leptin receptor activation stimulates many different in tracellular signaling pathways. Leptin Intracellular Signaling Cascade: 25

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JAK2-STAT3 Pathway Tyrosine 1138 phosphorylation recruits signal transducer and activator of transcription 3 (STAT3) and acts as a docki ng site, where the STAT3 molecules are phosphorylated by JAK2. Then the activated STAT3 proteins dimerize and translocate to the nucleus where they bind to DNA to r egulate gene transcription (175). This JAK2STAT3 pathway is crucial for leptin regulat ion of energy homeostasis. Bates, et al. created mice with a homozygous substitution of a serine residue at the Tyr1138 site. These mice (s/s) were hyperphagic and obese. Physiologically, the s/s mice were very similar to db/db mice except the db/db mice were infertile, s hort, and diabetic while the s/s mice were fertile, long, and less hyperglycaem ic (7). This suggests that while the regulation of feeding behavior is dependent on this pathway, reproduction, fertility, and glucose homeostasis are r egulated via another pathway. ERK Pathway Phosphorylation on Tyr985 recruits the tyro sine phosphatase SHP-2, which is then phospohorylated and activates the extracellu lar signal-related kinase (ERK) signaling pathway (163). The ERK pathway entails a set of serine/threonine kinases that are involved in cellular physiology and gene transcr iption regulation (6). Phosphorylation at Tyr985 also increases the levels of c-fos, which may be responsible for activation of neurons in the arcuate nucleus, and suppressor of cytokine signaling 3 (SOCS3), which acts as a negative regulator of leptin re ceptor-mediated signaling (6). Mice with a homologous leucine substitution at Tyr985, which blocks SHP2/SOCS3 recruitment, have lower energy intake levels, lower adiposity, and increased sensitivity to leptin. These results, in addition to those obtained from cultured cells, confirm a role of Tyr985 phosphorylation in the inhibition of leptin receptor signaling (14). 26

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PI3K-cAMP Pathway Another leptin signaling pathway is mediated by JAK2 phosphorylation, independent of leptin receptor intracellular tyrosi ne phosphorylation. JAK2 phosphorylation leads to the phos phorylation of the insulin receptor substrate (IRS) protein. This recruits phosphatidylinositol 3-kinase (PI3K), which activates phosphodiesterase 3B (PDE3B) and dec reases cyclic adenosine monophosphate (cAMP) levels (68). Central administration of a PDE3B inhibitor or cAMP blocks leptins anorectic effects and stimulates feeding, re spectively (176). These experiments suggest that the PI3K-PDE3B-cAMP pathway is integral for leptins hypothalamic control of energy balance. Tyr1077-STAT5 Pathway Of the three intracellular tyrosines phosphor ylated on the leptin receptor, the least is known about Tyr1077. However, recent evidence shows that phosphorylation at this site is necessary for phosphorylation and transcr iptional activation of STAT5 (55, 61). While STAT5 signaling is used by many growth factors and cytokines, it may also play an important role in leptin-mediated energy homeostasis. Mice with whole-body knockout of the STAT5 protein display many severe phenotypes due to the versatility of this signaling protein, but mice with only a central nervous system knockout of STAT5 are relatively normal besides a case of m oderate obesity and elevated leptin levels (2, 12). Negative Regulators SOCS3. Of the many negative regul ators of the leptin re ceptor-signaling cascade, SOCS3 activity is the most studied and charac terized. SOCS3, which contains an SH2 domain, inhibits specific signal transduction pathways, either by targeting the complex 27

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for degradation or by direct inhibition of JAK2 activity (12). SOCS3 is both induced by STAT3 signaling and inhibits it by binding to Tyr985 via its SH2 domain (107). SOCS3 knockout mice displayed greater anorectic responses to exogenously administered leptin. In addition, when these knockout mice were challenged with a HF diet, they consumed less food and gained less wei ght than wild-type controls (177). PTP1B. Another negative regulator of leptin signaling is protein tyrosine phosphatase 1B (PTP1B), which binds directly to and dephosphorylates JAK2. Mice without PTP1B display increased leptin sensitivity, increased energy expenditure, and resistance to both HF diet-induced weight gain and in creasing triglyceride levels (88). Downstream Leptin Signaling & Neurope ptide Regulation in the Hypothalamus Within the hypothalamus, the arcuate nucle us (ARC) is believed to be the primary integration site of various peripheral and c entral nutritional signals (2, 88, 156). The long form of the leptin recept or is coexpressed with two subpopulations of neurons in the arcuate nucleus (88). The first subpopul ation of neurons releases the orexigenic (appetite-stimulating) pepti des neuropeptide Y (NPY) and agout i-related protein (AgRP) (88). Leptin acts to inhibit the synthesis and re lease of these peptides (50). Ablation of the AgRP neurons in adult mice causes severe self-starvation and death (53). Ob/ob mice have increased NPY RNA, but the obesity and other detrimental physiological characteristics of ob/ob mice are improved with NPY knockout (88). The second subpopulation of neurons in the arcuate nucleus coexpresses the anorexigenic (appetite-suppr essing) peptides pro-opi omelanocortin (POMC) and cocaineand amphetamine-regu lated transcript (CART) (121). Leptin acts on these neurons to increase the synt hesis of these peptides. The POMC peptide is further processed to create adrenocorticotrophin, -endorphin, and -, -, and -melanocyte28

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stimulating hormones (MSH). The POMC-derived peptides activate the melanocortin receptors to produce a wide range of biological responses, including roles in skin pigmentation, adrenal steroidogenesis, thermoregulation, and appetite regulation (167). The most important of these pepti des with respect to obesity is -MSH. Specifically, MSH binds to downstream melanocortin-3 and -4 receptors to decrease food intake (50). Melanocortin-4 receptor knockout mi ce are obese and resemble leptin deficient mice (88). Leptin-Mediated Neuropeptide Regulation in the Hypothalamus: The NPY/AgRP and POMC/CART respond to circulating leptin, glucose, fatty acids, and amino acids, and their output signals on food intake interact both directly and on downstream neurons (88). NPY neurons can directly inhibit POMC neurons through local axon collaterals. However, the POMC neurons lack a reciprocal inhibitory axon to NPY, which suggests that appetite stimulation may be the fa il-safe default action (16, 35, 88). 29

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Secondly, the neurons have overlapping projections to second-order neurons located in the paraventricular nucleus (PVN), zona incert a, perifornical area (PFA), dorsomedial nuclei (DMH), and the lateral hypothalamic area (LHA). Each of these areas has been implicated in regulating feeding (133). Brown Adipose Tissue (BAT) a nd Uncoupling Protein 1 (UCP1) Whole body energy expenditure is compris ed of obligatory expenditure, which is required for normal cellular function, physica l activity, and adaptive thermogenesis. It entails the conversion of substrates (i.e. food, stored fat, stored protein, or stored glycogen) and oxygen to carbon dioxide, water, and energy (i.e. heat or work). Leptincontaining hypothalamic nuclei have sympat hetic neural connections to BAT, white adipose tissue (WAT), and muscle. Lipolysis in t hese tissues frees fatty acids to act, in turn, as substrates in BAT and muscle for adaptive thermogenesis, which is one mechanism for regulating body temperatur e and weight after cold exposure and hyperphagia, respectively (171). BAT contai ns uncoupling protein-1 (UCP1) that is expressed in the intermitochondrial membr ane and act as proton transporters, allowing protons to leak across the membrane and le ading to a loss of the electrochemical gradient the mitochondria usually use to create adenosine triphosphate (ATP) (171). This UCP1-mediated uncoupling of cellular respiration fr om ATP formation produces heat (171). The BAT receives sympathetic si mulation from brain regions that act on adrenergic receptors on surface of the BAT cells increasing cAMP levels in BAT. The increased cAMP levels, through a complex me chanism apparently involving free fatty acids, activate UCP1 to produce heat (109). 30

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Leptin Resistance Leptin Treatments in Human Obesity With the discovery of leptin in 1994, sci entists and physicians had high hopes that exogenously administered leptin would induce satiety and weight loss in obese humans. Indeed, leptin treatment improv ed adiposity levels and satiety in leptin deficient rodents and humans (109). In addition leptin treatment was efficacious in humans with lipodystrophy and eating disorders, conditions characterized by hyperphagia, low body fat content, and low leptin levels (109). However, these conditions represent only a small percentage of the populatio n. In contrast, the majo rity of obese humans have elevated leptin levels and a c ondition known as leptin resi stance, where they are not responsive to endogenous or exogenously administered leptin (42, 136, 137). Mechanisms The underlying mechanisms of leptin resi stance are still under debate, but it is believed to be multifactorial. Two of the dom inant hypotheses are t he inability of leptin to reach leptin receptors in the brain bec ause of its limited tr ansport across the blood brain barrier (BBB) and impaired central l eptin signal transduction. Central and, moreover, peripheral leptin administration produces blunted anorectic responses in rodent models of diet-induced and aged-related obesity (136, 137). Thus, it has been proposed that leptin resistance has a c entral and a peripheral component, where peripheral leptin resistance develops first. Studies have shown that both diet-induced and age-related obese rodents display reduced STAT3 phosphorylation, possibly due to diminished leptin receptor number or reduced affinity of leptin for its receptor (109). In addition, elevated leptin levels in obese rode nt models stimulate SOCS3, which further attenuates leptin signaling (2). 31

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Role of Elevated Leptin in Leptin Resistance Leptin levels in the blood and cerebrospinal fluid (CSF) rise with increasing adiposity in both humans and rodents (99). Evidence suggests that chronically administered exogenous leptin may lead to t he development of leptin resistance. For example, male Long-Evans rats given a continuous subcutaneous leptin infusion for 21 days lost the anorectic response to leptin 2 weeks into the infusion period and remained resistant to exogenous leptin during a subsequent peripheral high-dose challenge (127). In another experiment, chow-fed, male Sprague-Dawley rats were centrally infused with leptin. The leptin treatment initially r educed food intake by 50-60%, but food intake levels began gradually increasing on Day 4 until the leptin-treat ed and vehicle-treated rats were isocaloric on Day 13 (58, 132, 159, 165). Interestingly, both dietand agerelated obesity are associated with elevated leptin levels and leptin resistance (139, 140). Collectively, this suggests that the elevated leptin levels may significantly contribute to the development of leptin resistance in these animals. This is further supported by studies in young adult and aged rats inducing central leptin resistance by leptin overexpressi on through gene delivery in the brain in the absence of obesity or elevated se rum leptin levels. In thes e experiments, the leptininduced anorectic effects, decrease in adi posity, and increase in oxygen consumption completely attenuated over time, and the rats were completely unresponsive to a central challenge of a supra-physiological dose of leptin (143) Thus, central leptin overexpression can induce lept in resistance without influences from obesity or elevated serum leptin levels. This suggests that elev ated central leptin is a causative factor in the development of leptin resistance. 32

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In addition, studies have recently disco vered a link between fructose intake and leptin resistance (66, 87, 106, 125). While acute fructose ingestion failed to stimulate leptin release, chronic fructose intake in both rats (85) and humans (131) is accompanied by an increase in plasma leptin le vels, which is often a predictor of leptin resistance (87, 106, 125). In fact, this increase in plasma leptin levels may precede the development of obesity (42, 169). Al though the mechanism is still unknown, researchers have implicated both defective cent ral leptin signaling an d the inability of leptin to cross the BBB as possible contributors. Leptin resistance is a trademark of di et-induced obesity in rats and occurs naturally in obese humans. Diet-induced leptin resistance is associated with diminished hypothalamic leptin signaling an d reduced hypothalamic leptin receptor levels (42, 83, 155). This reduced signaling capacity may be a result of the inability of peripheral leptin to cross the BBB and/or diminished STAT 3-leptin receptor binding within the hypothalamus (174). The condition of leptin resistance predi sposes rats to display exacerbated hyperphagia and body weight gain on a HF die t. When young adult rats are provided a HF diet ad libitum, they immediately exper ience an increase in caloric intake that normalizes in approximately 6 da ys (130, 143). The restoration of caloric intake to preHF diet levels is dependent on leptin recept or activity. For ex ample, when young adult rats are centrally infused with a leptin re ceptor antagonist, they are unable to normalize the HF diet-induced hyperphagia. This suggests t hat leptin resistance in rats results in an exacerbated hyperphagia during t he introduction of a HF diet that may, in turn, cause an exaggerated body weight gain. In fact, similar studies in rats with central leptin 33

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overexpression, aged-related obesity, and fructo se-induced leptin resistance show that these leptin resistant animal s display an elevated caloric intake and body weight gain when exposed to a HF diet (93). Age-Related Obesity and Leptin Resistance In both rodents and humans, body weight a nd adiposity steadily increase with age until early senescence, which is then followe d by a decline later in life (93, 131). Twenty-four month old male F344xBN rats hav e 3-4 times greater serum leptin levels than 3 month-old rats (131). This increase in body fat wit h age cannot be accounted for by an increase in food intake, nor is it due to deficient leptin syn thesis or peripheral serum leptin levels. In fact, there is an in crease in leptin with age that should normally serve to lower body weight. But despite thei r elevated leptin levels, obesity continues and worsens in these rats, suggesting the relationship between leptin, adiposity, and food intake is altered with age (20). In addition, aged rats typically demonstrate impaired physical performance and activity (131) and have impaired leptin modulation of NPY and AgRP expression. Afte r central or peripheral admin istration, the aged rats display blunted responses, as measured by food intake and energy expenditure (140). Similarly, leptin gene therapy produces modes t and transient effects on food and body weight in aged rats (169). Caloric Restriction Reverses Leptin Resistance In rats with diet-induced obesity, unstimulated leptin signaling, measured by hypothalamic STAT3 phosphorylation levels, is approximately 3 times higher than chowfed controls. However, maximal signaling is greatly diminished in these obese rats which is accompanied by a parallel reduction in leptin receptor expression level (49, 169). Interestingly, caloric re striction increases hypothalamic leptin receptor expression 34

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and improves leptin signaling capaci ty in young adult diet-induced obese and agedobese rats (49). In fact, calo ric restriction dramatically dec reases serum leptin levels but only modestly reduces adiposity levels in rats (28). Further research may help clarify whether it is the reduction in serum lept in or adiposity, or bo th, that helps restore leptin responsiveness in these rats. Summary of the Leptin Signaling System Leptin, produced in peripheral adipose tissue, crosses the BBB to act on hypothalamic satiety centers, inducing an increase in energy expenditure and a decrease in food intake. Age-related, diet-induced, and genetic models of obesity are associated with leptin resist ance, where neither endogenous nor exogenous leptin is able to produce its effects on energy homeostasis. Because proper leptin receptor function is required for the normalization of ca loric intake upon HF diet initiation, this predicts that aged leptin resistant animals will display an exaggerated and prolonged caloric intake when introduced to a HF diet. As a result of this prolonged hyperphagia, these rats will likely experi ence a dramatic increase in adiposity and body weight. Testing these predictions in aged rats is one objective of this thesis. The Endocannabinoid System (ECS) For centuries, Cannabis sativa and (-)9-tetrahydrocannabinol (THC) have been used to increase appetite, particularly fo r sweet and palatable foods. However, pharmacological exploitation of the ECS has been largely i gnored until the 19 th century (65). To date, two cannabinoid receptor s have been cloned and identified where the differences between the two receptors include their signaling mechanisms and tissue distributions (32, 40, 168). Current ther apeutic uses for cannabinoid agonists include reducing nausea in cancer pat ients, preventing weight loss in AIDS patients, and 35

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treating pain, asthma, glaucoma. In c ontrast, cannabinoid antagonists have been investigated for the treatment of cardiac disease and obesity (65). Cannabinoid-1 (CB1) Receptors Both of the cannabinoid receptors have seven transmembrane domains and are coupled to G i/o proteins, positively to mitogen-activated protein kinase (MAPK) and negatively to adenylate cyclase (65). In addition, CB1 receptors are coupled through G i/o proteins to certain potassium and calcium channels and through G s proteins to activate adenylate cyclase (142). Within the brain, CB1 receptors are located in the presynaptic membrane to inhibit release of neurotransmitters, including dopamine, noradrenaline, serotonin, glutamate, and -aminobutyric acid (GABA) (116). Cloned in 1990, CB1 receptors were orig inally believed to be found only in the central nervous system, but they have recently been located in many peripheral tissues. CB1 receptors have been located in human s and rodents in adipose tissue, liver, skeletal muscle, the GI tract, and the pancre as (116). CB1 rec eptors are the most abundantly expressed G-protein-coupled rec eptors in the mammalian brain, expressed in areas like the olfactory bulb, cortical regions (neocortex, pyriform cortex, hippocampus, and amygdala), parts of the basa l ganglia, cerebral cortex, brainstem, and many thalamic and hy pothalamic nuclei (40). In fact, the ECS-mediated influence on energy homeostasis may occur by regulation of the expression of hypothalamic anorexic and or exigenic mediators. CB1 receptors co-localize with corticotr opin-releasing hormone (CRH), melaninconcentrating hormone, and pre-pro-orexin in the PVN, LHA, and VMH, respectively (29). CB1 receptor knockout mice express elevated leve ls of CRH, implicating ECS inhibition of this anorectic mediator (63). CB1 receptor activation sensitizes orexin-1 36

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receptors when the two are lo cated within the same cell and may enhance the action of orexins (38). In c ontrast, CB1 receptors were not f ound to co-localize with NPY, but CB1 activation downstream of NPY mediates some of its orexigenic effects, which were blunted with both genetic and pharmacological in hibition of CB1 signaling. However, CB1 antagonists are equally effective as anorectic agents in both wild-type and NPYdeficient mice (108). Collectively, these dat a suggest that the stimulation of food intake by endocannabinoid (EC) action is not medi ated by NPY, and the normal food intake observed in NPY-deficient mice is not due to an EC compensatory mechanism. Cannabinoid-2 (CB2) Receptor The second cannabinoid receptor was cloned in 1993, and it has 68% homology to the CB1 receptor within the transmembr ane domains and 44% hom ology throughout the total receptor (65, 110). It is primarily expressed in immune tissues but has recently been discovered in brain microglial cells, especially in stress and immune response conditions (17). In fact, CB2 rec eptors have been observed both preand postsynaptically in rodent st riatum, midbrain, hippocampus, brain stem, cerebellum, and substantia nigra (80, 145). Some of these regions also express high levels of CB1 37

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receptors. Thus, it is plausible th at these CB1 and CB2 receptors work both independently and/or cooper atively in the various neuronal populations. Mice with genetic deletion of the CB 2 receptor have reduced bone mass, and humans with CB2 gene polymorphisms have been associated with osteoporosis and autoimmune disorders (111). P harmacological activation of the CB2 receptor reduces locomotor activity, and pharmacological block ade inhibits food intake in some, but not all rodent strains (72). In addition, CB2 re ceptor gene expression is reduced in rodents with developed alcohol prefer ence (72). Acute administration of alcohol also downregulates CB2 receptor gene expression in the ventral midbrain of mice (28). These data, combined with data about CB1 receptors, suggest that CB1 receptors directly regulate food intake by affecting t he desire for the food while the CB2 receptors indirectly regulate food intake, possibly by altering the activity of the digestive system during stress, addition, or immune response. ECs The best characterized endogenous cannabino ids are N-arachid onyl ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG). AEA, which consists of an amide tail on arachydonic acid, was disco vered in 1992 and named for the Sanskrit word ananda that means bliss (28). 2AG, characterized by an est\er tail on arachydonic acid, was discovered in 2001 and is the most abundant EC in the brain (28). ECs are produced ondemand and are released from neurons after membrane depolarization and Ca 2+ influx to act by retrograde signaling on presynaptic receptors (39). After acting on the cannabinoid recept ors, AEA and 2-AG are rapidly internalized and degraded by fatty acid amide hydrolas e (FAAH) and monoglycerol lipase (MGL), 38

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respectively (32). AEA and 2-AG have different affinities and concentrations in different tissues, but they generally cause similar reacti ons in response to receptor binding (32). Central ECs ECs and their receptors are found throughout the brain, including areas like the hypothalamus that regulates energy homeostasis and the limbic forebrain which evaluates the hedonic value of food. Generally, the central ECS is believed to have an anabolic tone, in that ECS acti vation in the brain stimulates energy intake and storage (81). Hypothalamic and limbic forebrain EC levels fluctuate with nutritional status. For example, 2-AG levels are increased during fasting and normalize shortly after refeeding (38). CB1-deficient mice consume signific antly less food than wild-type controls after a period of fasting (27, 59, 73, 81, 126, 144, 166). Both central and peripheral administration of CB1 agonists in duces feeding in rodents. In fact, administration of a CB1 receptor antagonist reduc es food intake of both palatable and normal food in ad libitum-fed animals but only normal diet in food -restricted animals (3 8, 67). In addition, the ECS interacts with neurotransmitters hormones, and neuropeptides involved in energy regulation. For example, while EC production in the hypothalamus is inhibited by leptin, the ECs themselves inhibi t orexin neurons and stimulate melaninconcentrating hormone (154). These data suggest that central CB1 receptors act by 1) reinforcing the motivation to find and consume palatable foods, possibly through interactions with the mesolimbic reward pathw ay and 2) transiently regulating the levels and actions of other energy homeostasis regulator s to induce appetite. Furthermore, it suggests that hyperactivity within the ECS may contribute to obesity and other symptoms associated with t he metabolic syndrome. 39

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Peripheral ECs Chronic peripheral administra tion of a CB1 receptor antagonist causes a transient reduction in food intake, but the metabolic effects (body weight loss, adiposity reduction, decreased triglyceride levels, improved gluc ose and insulin sensitivity, and increased adiponectin) continue for several weeks (15, 37). This suggests that the peripheral ECS, which is likely hyperactive during obe sity, plays an important role in energy homeostasis. EC levels are elevated in plasma and adipose tissue of obese animals and humans, but these levels normalize after weight loss (29, 79, 101, 115). In fact, CB1 receptors have been found in many peripheral tissues, including adipose tissue, liver, skeletal muscle, the GI tract, and the pancreas. The receptor ex pression level changes with nutritional status and obesity in each ti ssue. Adipose tissue contains all the essential ECS elements, including the EC s, CB1 receptors, and the enzymes that degrade the ECs. Stimulation of the adipose tissue CB1 receptors induces formation and storage of triglycerides, downregulates adiponectin expression, and increases glucose uptake (112, 113). In t he liver, CB1 receptor activation stimulates the activity of lipogenic factors, causing an increased fatty acid synthesis and the development of fatty liver (94). Pharmacologica l blockade of CB1 receptor activation in skeletal muscle increases the rate of glucose uptake in mice (19, 36). CB1 receptors in the GI tract and vagal nerves are believed to be involved in re laying satiety signals from the gut to the brain. For example, CB1 receptor activation reduces satiation caused by cholecystokinin and enhances the ghrelin-induced stimulation of food intake (101). Both cannabinoid receptors are expr essed in the pancreas, and EC s are negatively regulated by insulin (76). Studies have shown that the ECS becomes hyperactive during 40

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hyperglycemic periods, which may aid in the dev elopment of hyperinsulinemia that is a common characteristic of obesity (38). Interactions of Leptin and the ECS Rodents with genetic leptin or leptin re ceptor deficiencies (obese Zucker rats, db/db mice, and ob/ob mice) display elevated hypothalamic EC levels, which are drastically reduced in ob/ob mice with acute intravenous leptin administration (75, 97). Studies suggest that this negative regulation occurs through post-synaptic leptin receptor action downregulating EC biosynthesis (18) In fact, Buettner et al recently discovered that central adminis tration of leptin is able to downregulate EC levels in peripheral WAT (39, 81). Interestingly, the authors found that th is downregulation is independent of STAT3 signaling. Instead, they found that FAAH, the enzyme primarily involved in metabolizing anandamide, is induced by central leptin administration and is, therefore, likely responsible fo r the peripheral reduction of ECs. Changes in hypothalamic EC levels and blood leptin levels during nutritional fluctuations are inversely correlated. Fo r example, hypothalamic 2-AG is elevated during fasting and reduced after refeeding while blood leptin levels are decreased during food deprivation and increased after food intake (39). Di Marzo, et al propose that these fluctuations in ECs occur as a consequence of the presence of leptin. They suggest that as leptin levels decrease durin g fasting, ECs are allowed to be elevated because of the lack of leptin -mediated downregulation of EC synthesis (84). This is confirmed by studies in genetically obese r odents, in which admin istration of a CB1 receptor antagonist reduced food intake suggesting that enhanced EC activity contributed to the hyperphagia normally seen in these animals (147). 41

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HF feeding, which is known to increase ci rculating leptin levels, also generally increases EC levels in many different tissues For example, Starowicz et al., reported elevated pancreatic AEA and 2-AG levels in mi ce fed with a HF diet compared with lean controls, and suggested that this was due to t he dysregulation of the biosynthetic and degradative enzymes of ECs (112). Simila rly, reduced FAAH expression with HF feeding has been implicated in elevated liver EC levels in mice and in circulating EC levels in obese humans (102). Matias et al., showed that onset, duration and extent of EC dysregulation is dependent on the type of HF diet, s pecifically its fatty acid composition (10). These data are supported by data showing increased brain AEA levels in piglets fed a HF diet rich in l ong-chain polyunsaturated fatty acids (101). HFfed mice have 2.5-fold higher epididymal white adipose tissue (EWAT) EC levels compared with lean mice (101). Interesti ngly, the ECS is upregulated immediately before adipocyte differentiation in fat cells. It is proposed that the ECS is then turned off by leptin, which is produced fr om the matured adipocytes (128). Model of ECS and Leptin Interactions in a Normal, Leptin Responsive Animal: HF feeding stimulates leptin syn thesis and leptin receptor activity to inhibit food intake. In a parallel, and possibly interacting pathway the HF diet stimulates brain reward 42

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centers, which increases EC synthesis and CB 1 receptor activity to stimulate food intake. In a normal, leptin responsive animal, the increas ed leptin receptor activity should downregulate EC synthesis to prev ent the ECS-mediated increase in food intake. Model of ECS and Leptin Interacti on in a Leptin Resistant Animal: However, in a leptin resistant animal, this increased leptin receptor activity is likely unable to downregulate EC synthesis. Theref ore, the HF diet may stimulate brain reward centers, which causes a further in crease in EC synthesis and CB1 receptor activity to further stimulate food intake. T he dysregulation of the ECS may contribute to diet-induced hyperphagia and obesity. CB1 Receptor Antagonists & the Treatment of Obesity Many CB1 receptor antagonists have been shown to have a high binding affinity for the CB1 receptor, a small degree of selectiv ity for CB1 over CB2, and act as inverse agonists upon binding to CB1 rec eptors (21, 64, 154). Specif ically, peripheral or oral administration of both SR141 716 (rimonabant) and AM251 in r odents stimulates gastric 43

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motility, suppresses food intake, and induces long-term body weight loss that continued even after food intake resumed normal levels (33, 120, 141, 157, 158). Several doubleblind clinical trials, Rimonabant in O besity, have been conduc ted with chronic administration of rimonabant in obese humans with or without type 2 diabetes or hyperlipidemia (32). Daily treatment with 20 mg rimon abant, combined with a 600calorie-deficient diet and increased physical activity for one year, caused increases in high-density lipoprotein cholesterol and adi ponectin; reduced body weight, waist circumference, plasma trigl ycerides, fasting insulin; and improved glucose tolerance when compared with placebo-treat ed subjects. At the end of the first year, the rimonabant-treated subjects were re-randomized to either continue to receive 20 mg rimonabant or begin to receive placebo for one year. While the placebo-treated group experienced weight regain, continued rim onabant treatment at this dose produced sustained body weight reductions and improv ements in many metabolic parameters. However, the rimonabant-treat ed subjects displayed a 2-fold increase in the risk of psychiatric adverse events, including depres sion, sleep disturbances, and anxiety. For this reason, the Food and Drug Administration recently declined to approve rimonabant for the treatment of obesit y, and the European Medicines Agency also acknowledged that rimonabant treatment is contraindi cated in patients with depression (93). 44

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AM251 SR141716 Summary of the ECS In contrast to leptin, ECs bind to CB1 receptors to increase food intake and fat storage, and obesity is often associated with an overactive ECS. Leptin downregulates both central and peripheral EC levels. This sugges ts that in a state of leptin resistance, the leptin negative modulation of EC levels will be ineffe ctive, allowing the ECS to become overactivated and induce an increase in food intake as well as body weight and adiposity. This may cause the CB1 re ceptors to become hyper-responsive to antagonism. Because aged rats are leptin resi stant, this predicts that they will display enhanced responsiveness to a CB1 antagonist. Central Hypothesis The major objective in this doctoral disse rtation was to further understand the role of leptin and the ECS in energy regulation in the context of both aging and HF feeding. The development of leptin resistance wit h both aging and HF feeding continues to puzzle researchers. Specifically, we inve stigated the possible link between HF induced and age-related leptin resistance and the ECS. Therefore, we put forth three major hypotheses. First, the leptin resistance associated with age -related obesity results in a prolonged hyperphagia during HF feeding, contributing to exacerbated weight gain. Second, the overactive ECS associated wit h obesity and likely with age-related obesity contributes to the exacerbated hyperphagia and weight gain observed in aged-obese rats during HF feeding. Third, long-term HF-feeding or aging leads to leptin resistance, therefore it is likely t hat the leptin negative m odulation of CB1 receptor activity is blunted or absent in obese rodent models. As a result, we predict that CB1 receptor antagonism will be enhanced in young adult rats with di et-induced obesity due to long-term HF 45

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feeding and this exacerbated CB1 receptor antagonism will be further enhanced in longterm HF fed aged rats. The goals of this di ssertation are to, first, characterize physiological responses to a HF diet in rats of varying ages; second, to examine the effects of a CB1 receptor antagonist on body weight and caloric intake in young adult and aged rats with or without HF feedi ng both shortand long-term. 46

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CHAPTER 2 GENERAL METHODS AND MATERIALS Experimental Animals The F344xBrown Norway (F344xBN) rat, whose weight gain with age parallels that observed in humans, is a rodent model for la te-onset obesity, whose weight gain with age parallels that observed in humans. This rat strain demonstrates a steady gain in adiposity into early scenescence, (approxim ately 24 months of age), followed by a decline beginning at 30 months that continues into late life (93). This increase in body fat with age cannot be accounted for by an increase in food intake, nor is it due to deficient leptin synthesis or peripheral serum leptin levels (136). In fact, there is an increase in leptin with age that should normally serve to lower body weight. But despite their elevated leptin levels, these rats become obese, suggesting the relationship between leptin, adiposity, and food intake is altered with age. These rats experience a reduced responsiveness to leptin, including an impaired anorexic response and little increase in energy expenditure, indicative of l eptin resistance (151). Rats of ages 3 to 6 months are considered young adult, 12 to 18 months are adult, and 24 to 30 months are aged. The median age of this rat strain is 34 months of age (US National Institutes of Health National Insitute on Aging www.nia.ni h.gov Aged Rodent Colony Handbook). For the purpose of this dissertation, all male F344xBN rats were purchased from the National Institute on Aging. General Experimental Design Rats were housed in individual unventila ted cages with corn cob bedding under a light-dark cycle of 12 hours of light and 12 hours of darkness with an ambient temperature of 22 o C. Food intake (Teklad Rat Chow or Research Diets D12492 60% 47

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HF Diet) and animal weight was measured daily At the conclusion of each experiment, rats were sacrificed under a Xylazine/Ke tamine (75mg/kg ketamine plus 7mg/kg xylazine) anesthesia cocktail. The hypot halamus, cerebellum, interscapular BAT, and epididymal, retroperitoneal and peenal white adipose tissue were removed and weighed at animal sacrifice. Adiposity was assess ed by 3 methods: 1) adiposity index (the sum of the weights of the three WAT depots divided by the rat body weight multiplied by 100) at sacrifice, 2) Time-Domain Nuclear Magnet ic Resonance (TD-NMR, MiniSpec, Bruker Optical) on a weekly basis, and 3) serum leptin levels in blood collected from the tail during the experiment and by heart puncture at sacrifice. Experimental Diets All animals were fed either a chow or HF diet as specified for each experimental design. The chow and HF diets were pur chased from Harlan Teklad and Research Diets, respectively. Both diets were prov ided to the rats at room temperature and replaced every 7 days. Harlan Teklad 7012 Chow Diet: 48

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Kcal Comparison of Chow and HF Diet: Subcutaneous Leptin Infusion Rats were anesthetized with 5% isoflu rane inhalation and maintained on 2.5% isoflurane when surgical plane of anesthes ia was reached. An osmotic minipump (model 2001, Durect, Cupertino, CA) containing either muri ne recombinant leptin or saline was implanted in a subcutaneous pocket on the dorsal surface of the rat, and the incision was closed with sutures. Intraperitoneal (i.p.) CB1 Antagonist Administration Rats were held with gentle restraint wit hout the use of anesthesia. The syringe needle was inserted in the abdominal area appr oximately one inch fr om the base of the right hind leg. Care was taken to ensure that no organs or vein s were struck with the needle. Either vehicle (6% DMSO, 5% Tween 80, and 89% Saline) or varying doses of AM251 in the vehicle were administered by i.p. injection. 49

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Body Composition Measurement Body composition was determined by time domain-nuclear magnetic resonance measurements on restrained but awake and alert animals (TD-NMR, Minispec, Bruker Optics, The Woodlands, TX, USA). The Mi niSpec provides three components of body composition (fat, free body fl uid, and lean tissue) by acqui ring and analyzing TD-NMR signals from all protons in the sample area. Validation of TD-NMR methodology has been provided (20). Serum Leptin Blood was collected by tail nick and a gentle milking motion in restrained rats without anesthesia or by cardiac puncture in rats under anesthesia at sacrifice. Total blood samples were centrifuged at 12,000g for 10 minutes and serum was frozen at 80 o C until ready to be analyzed. Seru m leptin was measured using rat radioimmunoassay (RIA) kits (Linco Research). Physical Performance Tests Forelimb grip strength was measured using an automated grip strength meter (Columbus Instruments, Columbus, OH) as described previously (20). Data were expressed as kilograms of fo rce/kilograms of body weight. Muscle tone and endurance were determined by use of an inclined plane as described previously (24). Data are presented as latency time/kg body weight. Wheel Running Voluntary wheel runni ng was measured automatically on Nalgene Activity Wheels (1.081 meters circumference; Fisher Scient ific, Pittsburgh, PA) by a magnetic switch and counter with liquid crystal display (LCD). Data are presented as meters ran per day for each age group. 50

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Tissue Harvesting and Preparation Rats were killed between 9:00AM and 4:00PM by thorocotomy under anesthesia (ketamine (75 mg/kg)/xylazine (7 mg/kg cocktail) or 5% is ofluroane inhalation). Blood samples were collected by cardiac puncture, and serum was separated by centrifugation in serum separator tubes. Th e circulatory system was then perfused with 30 ml of cold saline. The epididymal, perirenal, and retroperit oneal white adipose tissues (EWAT, PWAT, and RTWAT, respec tively), hypothalamus, and BAT were excised. For removal of hypothalamus, an incision was made medi al to the piriform lobes, caudal to the optic chiasm and anterior to the cerebral crus to a depth of 2-3 mm. The hypothalamus was sonicated in 10 mM Tris-HCl (pH 6.8), 2% SDS, and 0.08 g/mL okadaic acid plus protease inhibitors. Prot ein concentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA ). BAT samples were prepared using a similar protocol but were filtered through a 0.45m syringe filter (What man, Clifton, NJ) to remove lipid particles before m easuring the protein concentration. Western Analysis Homogenate samples were boiled and separated on Tris-HCl pol yacrylamide gel (Bio-Rad) and transferred to a nitrocellu lose membrane. Immunoreactivity was assessed on separate membranes with antibodies specific to hypothalamic STAT3, both unphosphorylated and phos phorylated (Cell Signaling), and BAT UCP-1 (Linco Research). Immunoreactivity was visualiz ed by a chemiluminescent detection system (GE Healthcare, Piscataway, NJ) and quantif ied by ImageQuant TL (GE Healthcare) RNA Isolation and Reverse Transcription Cellular RNA was extracted using TRI reagent (Sigma-Aldrich, St. Louis, MO), using a modified method originally published by Chomczynski (137). T he integrity of the 51

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RNA was verified by running it in a 1% agarose gel stained with ethidium bromide and quantified using multiple dilu tions of each sample by s pectrophotometric absorption at 260 nm. The RNA was then treated with a DNA -free kit (Ambion, Austin, TX), and a first-strand cDNA was synthesized from 1 ug RNA using random primers (Invitrogen) containing 200 units of M-MLV reverse transcriptase (Invitrogen). Relative-Quantitative PCR Using a Quantum RNA 18S Internal Standard kit (Ambion), relative quantitative PCR was completed by multiplexing target gene primers and 18s primers and coamplifing for a specific number of cycles in the linear range of the target. For example, the primer sequences for SOCS-3 are sense 5 ACCAGCGCCACTTCTTCACA-3 and antisense 5-GTGGAGCATCATACTGGTCC-3. The optimum ratio of 18S primer to com petimer was 1:9, and t he primers used were sense PCR was performed at 94 o C denaturation for 90 s, 59 o C annealing temperature for 60 s, and 72 o C elongation temperature for 120 s for 28 cycles. The PCR product was then electrophoresed on a 5% Tris-Bor ate EDTA acrylami de gel (BioRad) and stained with SYBR green (Molecular Probes). Gels were scanned using a STORM fluorescent scanner (GE Healthcare ) and quantified using ImageQuant (GE Healthcare). Statistical Analysis Data were analyzed by one-way and two-way ANOVA. When the mean effect was significant, a post-hoc test (Newman-Keuls, Bonferroni, or Dunnett) was applied to determine individual differences between the means. Body composition values were analyzed by paired and non-paire d T-tests. A value of p < 0.05 was considered significant. 52

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For all objectives, the decision to assign 6 animals per group was based on experience using a statistical power analysis from preliminary studies in our laboratory. In the preliminary data, the ratio of diffe rence to standard deviation was always greater than 2.5. Therefore, statis tical power analysis indicates the number of animals needed for significance at = 0.05 (two-tailed) and = 0.01 is 6 rats per group. In experiments with aged rats, extra animals may be include d in each group to account for natural death or disease-onset that is typical with senescence. 53

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CHAPTER 3 UNEXPECTED PROLONGED HYPERPHA GIA WITH HIGH-FAT FEEDING CONTRIBUTES TO EXACERBATED WEIGHT GAIN IN RATS WI TH ADULT-ONSET OBESITY Introduction The typical western diet contains an exce ss of fat, and this is believed to be one contributor to the prevalenc e of obesity (130). Dietary obesity in adult humans and adult rats has been the subject of intense res earch, but the role of a HF diet with aging has largely been ignored. In rode nts, upon initiation of a high fat diet, there is a transient increase in caloric intake that returns to pre-treatment levels usually within a week despite continuation of the HF diet (174). One factor necessa ry for this normalization of caloric intake after high fat feeding is leptin receptor ac tivity (174). We previously established that blockade of the leptin recept or with a specific leptin receptor antagonist prevents the normalization of caloric inta ke after HF feeding (130, 139, 140). Furthermore, rats made leptin resistant by ce ntral overexpression of leptin also display a prolonged hyperphagia with HF feeding and an exaggerated weight gain compared with leptin responsive rats fed a HF diet ( 93). Collectively, these studies suggest that the normalization of caloric intake is mediated by leptin action. The F344xBrown Norway (F344xBN) rat, whose weight gain with age parallels that observed in humans, is a rodent model for late-onset obesity. This rat strain demonstrates a steady gain in adiposity in to early senescence, (approximately 24 months of age), followed by a decline beginning at 30 months that continues into late life (93). This increase in body fat with age can not be accounted for by an increase in food intake, nor is it due to deficient leptin synthesis or peripheral serum leptin levels (136). In fact, there is an in crease in leptin with age that should normally serve to lower 54

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body weight. But despite their elevated l eptin levels, these aged rats become obese, suggesting the relationship bet ween leptin, adiposity, and food intake is altered with age. These rats experience a reduced responsiveness to leptin, including an impaired anorexic response and little increase in ener gy expenditure, indicative of leptin resistance (136). Because aged-obese rats are leptin resistant (89), these data predict that they will have a delayed normalization of caloric in take and exaggerated weight gain when provided a high fat diet. In order to evaluat e this hypothesis, we HF-fed rats of various ages and examined caloric intake and body weight over a 5-month period. Experimental Design Six(n=5), twelve(n=5), eighteen(n=5 ), twenty-four(n=7), and thirty-month old rats (n=5) were provided a HF diet (60% fa t; 5.2 kcal/g D12492; Research Diets, New Brunswick, NJ, USA) ad libitum for up to 5 months, and an additional twenty-four-month old rats (n=7) were fed a standard rat chow (15% fat; 3.3 kcal/g diet 2018; Harlan Teklad, Madison, WI, USA) ad libitum. Body weight and food intake were recorded daily. A separate group of three(n=32) and thirty-month-old (n =23) rats were placed on either a standard chow or HF diet for 60 da ys and killed for assessment of serum leptin levels, hypothalamic PTP1B levels, hypothal amic SOCS-3 mRNA expression levels, BAT UCP-1 levels, and fat depot sizes. Body composition (fat/lean mass) was det ermined before noon prior to initiation of the experimental diets and perio dically thereafter. Towa rds the end of the 150-day experimental period, physical performance tests and voluntary wheel running were assessed. 55

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Additional groups of three(n = 36) and thirty-month-ol d rats (n =24) on a chow diet were used to examine a 7-day dose response to peripheral leptin infusion. Results Body Weight Change with HF-Feeding Normally, when young adult rats are provi ded with a HF diet, they spontaneously divide into two groups, diet-induced obese (D IO) prone and diet resistant (DR), based on the amount of weight gained (46, 89, 91). This phenomenon has been well described in the Sprague-Dawley rat strain (170), and recently described in the F344xBN rat strain (2). In the present study, the 3-mont h-old rats fed the HF diet spontaneously divided in two distinct groups, consisting of the top 75% of weight gainers (DIO prone), and the lowest 25% (DR, Fi gure 3-1, A), which were more similar to the chow-fed as measured by the maximized value of the log likelihood. Surprisingly, this phenomenon was not seen in the aged rats. All of the aged rats provided the HF diet gained similar and considerable amounts of weight, and thus in comparison to young adult rats, aged rats are all susceptible to the weight-gaining effects of HF feeding (Figure 3-1, B). 56

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This age-related susceptibility to HF feeding was also evident in the body weight gain across different rat ages. In particularl, age exacerbat ed the initial body weight gain seen with HF feeding (Figure 3-2). Both the six-month-old young adult rats and the twenty-four-month-old mature rats steadily gained weight on the HF diet and continued to gain weight throughout the experimental per iod, but the weight gain in the 24-monthold rats was significantly greater throughout the study period except during the last 2 weeks. This dietand age-related weight gain was most pronounced in the oldest age group, especially during the initial phase of HF feeding. In the first 30 days of HF feeding, the 30-month-old rats gained the most weight, but this increase in body weight reached a plateau by day 30 and at approximately 31 months of age. It should be noted that initial body weights, prior to initiation of HF-feeding were greater with increasing age (3-month-olds: 324.35 3.68 g, 6-month-olds: 385.26 11.28 g, 12-month-olds: 435.58 14.24 g, 18-month-olds: 500.86 13.47 g, 24-montholds: 551.23 9.47 g, 30-m onth-olds: 568.33 9.17 g). Caloric Intake with HF-Feeding The disproportionate age-related increase in initial weight gain suggests that this may be related to the hyperphagia normally observed after introduction of a HF diet. When three-, six-, and thirty-month old rats were provided the HF diet, all ages experienced an immediate increase in daily caloric intake that gradually normalized to basal levels, or near basal level in the case of the oldest age group, over time (Figure 33, A). For ease in analyzing the data, the daily caloric intake was divided into two phases, with phase 1 representing the peak calori c intake upon initiation of the HF diet, and phase 2 being the days necessary to normaliz e the elevated caloric intake back to levels prior to HF feeding. It is important to note that the animals of all ages consumed 57

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a similar amount of calories on the regular chow diet prior to the introduction of the HF diet (Figure 3-3, A, data points prior to Day 0). The peak daily caloric intake with HF feeding increased with age (R 2 = 0.85, P = 0.009), as is evident in a comparison of P hase 1 across ages (Figure 3-3, B). The three-month-old rats reached a peak caloric intake of 100 kcal. The six-, twelve-, and eighteen-month old rats all reached a peak of approximately 115 kcal, and the twentyfour-month-old rats reached a peak caloric inta ke of 125 kcal. The thirty-month-old rats, the oldest age tested in this experiment, r eached a maximal caloric intake peak of 142 kcal. A similar trend was seen in Phase 2 (Figur e 3-3, C), where the days required to normalize caloric intake to basal levels increased with age (R 2 = 0.93, P = 0.002). In this F344xBN rodent strain, the 3-month-old rats experi enced a complete normalization of caloric intake by day 6. The 6-monthold rats and the 12-month-old rats required 9 days and 17 days, respectively, whereas, the 18and 24-month-old rats did not experience normalization until day 25. In contra st to the younger ag es, the 30-month-old rats were unable to normalize to pre-HF calo ric intake, but stabilized at a new slightly elevated plateau by day 29. Body Composition Absolute fat and lean body mass were greater in the 30-month-old rats compared with the 3-month-old rats at the beginning of the study (f at mass: 175.67.38 g vs. 73.56.86 g, P < 0.0001; lean mass: 339.78 5.33 g vs. 199.05.17 g, P < 0.0001). By examining the change in body composition, the increase in fat and lean mass was significantly greater in both young adult and aged HF-fed groups compared to their agematched chow-fed controls (Figure 3-4, A & B). In addition, the HF-fed aged rats 58

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gained significantly more fat mass than the HF-fed young adult rats. Conversely, the HF-fed young adult rats gained significantly more lean mass than the HF-fed aged rats. This resulted in a significantly larger fa t-to-lean mass ratio in the aged, HF-fed rats compared to all other age/diet gr oups (Figure 3-4, C). In cont rast, the ratio of fat-to-lean mass was relatively constant over time in the chow-fed aged rats (Figure 3-4, C). Serum Leptin and Adiposity With HF feeding in young adult rats, by day 60, serum leptin levels rose by more than two-fold compared with the chow-fed count erparts. Interestingly, after 60 days of HF feeding in the young adult, serum leptin reached the same levels as the aged chowfed rats (Figure 3-5, A). Similar to the young adult, the HF-fed aged rats had serum leptin levels more than two-fold greater th an the aged chow-fed rats by day 60. Serum leptin levels at Day 60 paralleled that of the amount white adipose tissue (sum of PWAT, RT WAT, and EWAT) present in each gr oup at sacrifice (Fi gure 3-5, B). The young adult HF-fed and aged chow-fed rats had the same amount of white adipose tissue, while the young adult chow-fed rats had significantly less and the aged HF-fed rats had significantly more. This is consistent with previous reports that leptin circulates in proportion to whole body fat depots (170, 174). Wheel Running & Physical Performance Tests Two to 5 months after HF or chow feedi ng, 3 performance tests were administered wheel running and two measures of forelimb grip strength. Each age group was provided access to running wheels for 4 co nsecutive days, but the rats were not provided any training or encouragement to run. The youngest chow-fed rats ran greater than 1000 meters/day (Figure 3-6). In contras t, the oldest chow-fed rats ran 8-fold less, only 136 meters/day during the same period. HF feeding also im pacted wheel running, 59

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but not to the same degree as age. The younges t HF-fed rats ran two-fold less than the corresponding chow-fed. With increasing age and HF feeding, wheel running declined with the oldest HF-fed rats running onl y 81.6 meters per day (Figure 3-6). Because wheel running may be dependent on muscle strength, we subjected the twenty-four-month-old rats to a grip str ength and inclined plane te st at approximately day 130 on the standard chow or HF diet. T here was no significant difference in either grip test or ability to perform in the inc lined plane test between the diet groups in the 24month old rats (data not shown). Hypothalamic Measures of Leptin Action We examined two factors that participate in leptin signaling, SOCS-3, a negative regulator of leptin signa ling, and PTPIB, a phosphatase that dephosphorylates activated components in the leptin signaling cascade. Consistent with models of leptin resistance, both SOCS-3 mRNA levels and PTP1B protein le vels were significantly increased in aged rats compared to young adul t rats (Table 3-1). In addition, both hypothalamic SOCS-3 expression and PTP1B le vels are significantly elevated with HF feeding (Table 3-1). BAT UCP1 Levels The induction of UCP1 in BAT is a marker for enhanced thermogenes is in rodents, and is often used as an indicator of energy ex penditure. Consist ent with our previous findings (42, 92, 130), HF feeding increased UCP1 protein levels in BAT in young adult rats (Table 3-1). Surprisingly the HF diet also induced in creased UCP1 protein levels in BAT in the aged-leptin resistant rats. 60

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Dose Response to Peripheral Leptin Infusion Because previous data suggest that leptin resistance prolongs or prevents the renormalization of caloric intake following HF feeding, we evaluated the responsiveness to leptin prior to initiation of the HF feeding in both young adult and aged rats. The doses used were 0.03, 0.05, 0.07, 0.1, and 0.5 mg/day (0.1, 0.167, 0.233, 0.33, and 1.67 mg/kg/day, respectively) in the young adult rats and 0.05, 0.1, and 0.5 mg/day (0.091, 0.182, and 0.909 mg/kg/da y) in the aged rats. In t he young adult rats (initially 3 months old and weighing 308.79 3.19 g), a ll doses greater than 0.03 mg/day of peripheral leptin significantly increased serum leptin levels compared with saline-infused rats (Table 3-2). The leptin-infused rats displayed a dose-dependent body weight and food intake reduction in response to a peripheral leptin up to a dose of 0.07 mg/day, above which there was no additional effect (F igure 3-7, A and B, respectively). The decrease in body weight for each of the lept in-infused groups was significantly greater than that of the saline-infused group. In addition, the reduction in cumulative food intake due to leptin infusion was greater wit h each leptin dose except the lowest dose compared to the saline-infused group. The anorectic and weight reduction responses to doses of 0.07, 0.1, and 0.5 mg/day were not statistically different from each other and appear to represent the maximum response to leptin. In contrast, the aged rats (initially 30 m onths old and weighing 553.39 5.48 g) did not respond to even the largest dose of l eptin. In particular, cumulative food consumption was unchanged across leptin doses (Figure 3-8, B). All of the aged rats lost weight, likely in response to the detrim ental effects of surgery (Figure 3-8, A). However, the leptin-infused rats did not differ from the saline-infused rats with respect to either body weight or food intake reducti on, even though both the doses of 0.1 and 0.5 61

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mg/day of leptin significant increased seru m leptin compared to saline-infused rats (Table 3-2). Discussion Rodents provided with a highly palatable calorically dense di et initially consume an elevated level of calories, but within several days adjust their total food consumption, such that their diet becomes isocaloric to that of chow-fed groups (174). Leptin resistant animals, however, display impaired normalization of caloric intake with HF feeding (130). Because aged-obese rats are also leptin resistant (70, 71), we predicted that aged rats would also fail to properly normalize caloric intake after exposure to HF feeding. The present invest igation confirms this hy pothesis by examining the physiological effects of a HF diet on rats of various ages between 3 and 33 months of age, and provides several salient findings. First, our data are consistent with several previous studies indicating that aging increases the susceptibility to obesity and fat storage (70). Iossa, et al. (1999) showed that young male Wistar rats that are natura lly growing to maturity have the ability to store both proteins and lipids. However, as the rats ag e, from 1 month to 6 months old, the protein deposition eventual ly becomes almost nonexis tent and all excess energy consumed is stored as fat (70, 71). T hey propose that this is one mechanism underlying age-associated obesity. Moreover, these adult rats were more prone to obesity when fed a HF diet than younger counterparts (170). It is reasonable that these trends toward obesity continue as rats age ev en further. Supporting this hypothesis is our demonstration that aged rats do not divi de into DIO and DR, as previously and currently seen in young F344xBN rats (57, 134, 175). Whereas young rats are either susceptible or resistant to we ight gain, all aged rats are susceptible to this negative 62

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effect of a HF diet. Moreover, body composit ion analysis indicates that the older rats gain a disproportionate amount of body fat compared with younger counterparts when provided a HF diet. Hence, these data suppor t the concept that energy storage shifts towards fat deposition with aging, implicat ing one mechanism underlying age-related obesity. Moreover, aged F344xBN rats, in our case both 24and 30-month old rats, provided a HF diet gain more weight t han correspondingly fed young rats. Together, these data indicate that all aged rats, compared to only some young rats, are prone to develop obesity on a HF diet, and furthermore the degree of weight gain is greater in the older rats. Second, our data indicate that the nature of the transient increase in caloric intake upon initiation of HF feeding is dependent on age. Both t he peak increase in caloric intake upon initiation of HF feeding and the time to normalization increase with age. Moreover, this hyperphagia was a specific result of the HF diet: prior to initiation of the HF diet, all rats, regardless of age, consum ed the same amount of chow diet, confirming earlier studies (174). Thus, the greater initial body weight gain in the 24and 30-month-old rats appears to be a consequence of this failure to normali ze caloric intake after initiation of HF feeding, and we suggest the latt er is a direct result of l eptin resistance in these aged animals. Previous experiments in younger animals demonstrated that the simultaneous administration of a leptin receptor antagonist along with HF feeding prevents the normalization of caloric intake after HF feeding, indicating that leptin receptor activity is necessary for this normalization (137). Ol der rats have reduced numbers of leptin receptors and diminished leptin signaling (138 ). In addition, in the present study, we 63

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found increases with age in both SOCS-3, a negativ e regulator of leptin signaling, and PTPIB, a phosphatase that dephosphorylates activated components in the leptin signaling cascade. These chan ges with age likely impair the native responses to the endogenous elevation in leptin triggered by HF feeding. We examined the status of leptin resistance at the point prior to initiation of the HF feeding by assessing the leptin dose response decrease in food consumption and body weight over the course of seven days in young adult and aged rats. As expec ted, the young adult rats responded in a dose-response fashion, whereas there we re no responses in the aged rats, thus confirming that prior to initiating HF feeding the aged rats were unresponsive to leptin. As such, these leptin resistant animals display a delayed normalization of caloric intake on a HF diet, strongly suggesting that pre-existing leptin re sistance is causal to the exacerbated weight gain with age. It should be noted, however, that these measures of leptin responsiveness were examined only in the 3and 30-month-old rats. While they demonstrate impaired leptin re sponsiveness by 30 months of age, we cannot dismiss the possibility that the leptin resistance may be fully manifested prior to this age. If this is the case, the leptin resistance may be only one factor in the progressive exacerbated weight gain with age to HF feeding. Our data indicating that leptin receptor acti vity is necessary for the normalization of caloric intake predict that any impaired normalization should be proportional to the degree of leptin resistance and thus may never occur in aged rats that are fully leptin resistant. Our data support this prediction. The delay in normalization is proportional to advancing age with the oldest group achieving onl y a partial normalization. Similarly, leptin resistance is greater in 30-month-ol d rats compared with 18 months (152). We 64

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suspect the partial normalization in the oldes t age group represents some residual leptin receptor activity or compensation by another anorexic pathway. Subsequent to the caloric normalization, ra ts of all ages continued to gain weight suggesting that energy expenditure must be di minished. However, any such decrease in energy expenditure does not appear to be re lated to the thermic effect of food, because both the young adult and aged HF-fed rats responded equally with an increase in UCP1 protein level in BAT. In addition to thermogenesis, an important component of energy expenditure is physical activity levels There is an inverse relationship between body weight and physical activity (152), and a decrease in locomotor activity, including volitional activity, may be an important cont ributor to age-related obesity. Voluntary wheel running is one form of volitional activi ty involving motivati onal, exploratory, muscular, age, and body size components (152). Data indicate that locomotor activity declines both with age and obesity (22). We hypothesized that both age and HF feeding would impact voluntary wheel running, and this hypothesis proved correct, wheel running activity declined with both age and HF feeding, the latter especially in young adult rats. It has previously been reported th at Sprague Dawley and S5B/P1Ras rats on a high carbohydrate diet volunt arily run more than those fed a HF diet, but there was no comparison to rats on a standard chow diet (41). Another group showed that the introduction of sweet milk plus standard ch ow decreased voluntary wheel running in female rats, but not male rats (30). Research with hamsters indicated that aged hamsters run significantly less than young ones (77). These data are consistent with our findings that aged F344xBN rats run si gnificantly less than young adult F344xBN rats on a standard chow diet. In addition, HF feeding can further reduce voluntary 65

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66 wheel running activity in young adult rats Interestingly, the aging and HF feeding suppressive effect on voluntary wheel r unning do not appear to be independent; for instance, in the oldest age gr oup, that ran the least, HF feeding had little additional suppressive effect. Collectively, these data s uggest that the propensity for inactivity with age may be one contributory factor in age-related obesity, and the inactivity with HF feeding may accelerate the rate of diet-induced obesity.

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67 3-Month-Olds 10 20 30 40 50 -25 0 25 50 75 100HF-fed DIO-prone HF-fed DR Chow-fed A DaysChange in Body Weight (g) Chow HF-DR HF-DIO 40 60 80 100 120Change in Body Weight (g) 24-Month-Olds 0 10 20 30 40 50 -25 0 25 50 75 100Chow-fed HF-fed B DayChange in Body Weight (g) Figure 3-1. A) Body weight gai n in young adult rats on chow (open symbols) or HF (closed sym bols) diet. Data represent mean s.e.m. of HF-fed DIO-prone (n =24, triangles), HF-fed DR (n=8, closed squares), Chow-fed (n=10, open squares). INSET: Young adult rats were either prone or resistant to the e ffects of the HF diet. We used the maximized value of the log likelihood as a measure of model fit (larger va lues indicate be tter fit). When we assumed one normal distribution for the chow-fed together with the HF-fed DR rats, and a separate normal distribution for the HF-fed DIO-prone, the model-fit yielded a log likelih ood value of -530 compared with -542 when one normal distribution was assum ed for the chow-fed rats and one norma l distribution for the all the HFfed rats (DIO-prone plus DR). This s uggests that HF-fed DR rats are more similar to the chow-fed rats than the HF-fed DIO-prone rats, and thus, the latter should be cons idered a separate group. By day 3 on the diet, the body weight gain in HF-fed DIO-prone animals was significantly greater than that of the Chow-fed group (P < 0.0001 by t-test). The change in body weight was significant between t he two HF-fed groups by day 15 (P < 0.05 by ANOVA). B) Body weight change in aged rats on either chow (solid triangles) or HF diet (open triangles). Data represent mean s.e.m. of chow-fed (n=5) and HF-fed (n=7). All aged rats were susceptible to the detrimental effects of the HF diet. By day 1 on the diet, the HF-fed aged animals had experienced a weight gain significantly greater than their chow -fed counterparts (P < 0.05 by ANOVA).

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0 25 50 75 100 125 -25 0 25 50 75 1006 Months HF 24 Months Chow 24 Months HF 30 Months HF DayChange in Body Weight (g) Figure 3-2. Body weight change in rats of differing ages on chow (dotted line) or HF (solid lines) diet. Data represent mean s.e.m. of 6-month old HF-fed rats (n=5, circles), 24-month old HF-fed rats (n=7, triangles), 30-month old HF-fed rats (n=5, diamonds), and 24-month old Chow-fed rats (n=7, squares). The 30-month-old rats fed a HF diet experi enced a body weight gain greater than the 6 and 24-month-old rats fed a HF diet by day 3 (P < 0.01, one-way ANOVA) and 5 (P < 0.01), respectively The change in body weight was no longer significantly different between t he 24and 30-month-old rats fed a HF diet by day 69 (P > 0.05). The change in body weight in the 3-, 24-, and 30month-old rats fed a HF diet was no longer significantly different by day 89 (P > 0.05) and for the durat ion of the experiment. 68

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0 10 20 30 40 50 0 25 50 75 100 125 150 Phase 2 Phase 1 30 Months HF 6 Months HF 3 Months HF ADayDaily Caloric Intake (kcal) 3 Mos. 6 Mos. 12 Mos. 18 Mos. 24 Mos. 30 Mos. 100 125 150** *BPeak Caloric Intake (kcal) 3 Months 6 Months 12 Months 18 Months 24 Months 30 Months 0 5 10 15 20 25 30 35** ** ** **CPeriod of Elevated Caloric Intake (Days) Figure 3-3. A) Daily caloric intake of 3-m onth-old (circles), 6-month-old (squares), and 30-month old (triangles) rats on a HF die t. Data represent mean s.e.m. of 3 Months HF (n=9), 6 Months HF (n=5), and 30 Months HF (n=5). B)The peak caloric intake after the initiation of HF feeding. Data repr esent mean s.e.m. of 3-month old HF-fed (n= 24), 6-month old HF-fed (n=5), 12-month old HF-fed (n=5), 18-month old HF-fed (n=5), 24 -month old HF-fed (n=7), and 30-month old HF-fed (n=5) rats. P < 0.0001 for difference with age by one-way ANOVA. *P < 0.05 for the difference bet ween 3-month old rats and all other ages by post-hoc analysis. **P-value < 0.001 for the difference between the 30-month-old rats and all other ages by post-hoc analysis. C) The days required to normalize the elevated caloric intake following HF feeding. Data represent mean s.e.m. of 3-month old HF-fed (n=11), 6-month old HF-fed (n=5), 12-month old HF-fed (n=5), 18 -month old HF-fed (n=4), 24-month old HF-fed (n=7), and 30-month old HF-fed (n=5) rats. P < 0.0001 for difference with HF feeding by one-way ANOVA. **P-value < 0.001 for the difference compared to 3-month-old rats by one-way ANOVA and post-hoc. 69

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0 10 20 30 40 50 20 30 403 Months Chow-fed 3 Months HF-fed 30 Months Chow-fed 30 Months HF-fed ADayPercent Fat Mass (%) 0 10 20 30 40 60 70BDayPercent Lean Mass (%) 0 10 20 30 40 50 0.3 0.4 0.5 0.6 0.7CDayFat Mass/Lean Mass Figure 3-4. Percent fat mass (A), percent lean mass (B), and the ratio of fat-to-lean mass (C) over time in 3(circles) and 30-month-old (squares) rats on either a HF (open symbols) or a standard chow (c losed symbols) diet. Statistical analysis performed by comparing t he data collected on Day 38. Data represent mean s.e.m. of 3-month old chow-fed (n=1 0), 3-month old HF-fed (n=12-32), 30-month old chow-fed (n=4 -6), and 30-month old HF-fed (n=5) rats. A) P < 0.0001 for t he interaction; P < 0.001 for he difference with HF feeding and the difference with age by two-way ANOVA. B) P = 0.0003 for the interaction; P < 0.01 for the di fference with age, regardless of dietary treatment; P < 0.001 for the difference with HF feeding in aged rats only by two-way ANOVA. C) P = 0.0003 for the interaction; P < 0.0001 for the difference with HF feeding and differ ence with age by two-way ANOVA. 70

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Day 60 Chow Day 60 High Fat 0 10 20 30 40 503 Months 30 Months A ** Serum Leptin (ng/mL) Chow High Fat 0 10 20 30 40 50B* ** White Adipose Tissue (g) Figure 3-5. A) Serum leptin at day 60 in 3and 30-month-old rats on chow or HF diets. Ages represent the age of the animal when HF or chow feeding was begun. Assessments were determined 60 days late r. Data represen t mean s.e.m. of 3-month old rats (n = 9-10) and 30-month old rats (n = 9-13). P < 0.0001 for the difference with HF feeding and difference with age by two-way ANOVA; P = 0.0041 for the interaction. *P < 0.05 for the difference between chow-fed young adult and aged rats by post-hoc analysis. **P < 0.001 for the difference between HF fed young adult and aged rats by post-hoc analysis. P < 0.05 for the difference between c how-fed and HF fed young adult rats by post-hoc analysis. P < 0.001 for the difference between chow-fed and HF fed aged rats by post-hoc analysis. B) White adipose tissue mass at sacrifice from threeand thirty-month-old rats fo llowing chow or HF feeding. Ages represent the age of the animal when HF or chow feeding was begun. Assessments were determined 60 days later. Data represent mean s.e.m. of 3-month old (n=10-12) and 30-month old (n=8-14) rats. P < 0.0001 for the difference with HF feeding and difference with age by two-way ANOVA. *P < 0.001 for the difference between chow -fed young adult and aged rats by posthoc analysis. **P < 0.001 for the diffe rence between HF fed young adult and aged rats by post-hoc analysis. P < 0.001 for the difference between chowfed and HF fed young adult rats by posthoc analysis. P < 0.001 for the difference between chow-fed and HF fed aged rats by post-hoc analysis. 71

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5 Mos. Ch ow 3 2 Mos. Cho w 5 M o s. HF 11 Mos. HF 17 Mos HF 23 M o s. HF 32 Mos. H F 0 250 500 750 1000 1250** Wheel Running (m/day) Figure 3-6. Voluntary wheel running over a 4-day period. WR activity was determined 2 months after HF or chow feeding for rats initially of 3 or 30 months of age and after 5 months of HF or chow feeding in rats initially of 6, 12 or 18 month of age. Ages represent the age at the ti me of WR. Data represent mean s.e.m. *P < 0.001 for the difference between fiveand thirty-two-month-old chow-fed rats. P < 0.001 for the diffe rence between five-month-old chow-fed and five-month-old HF fed ra ts. **P < 0.01 for t he difference between fivemonth-old HF fed and all other HF fed groups by post-hoc analysis. 72

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Table 3-1. Hypothalamic PTP1B protein le vels, hypothalamic SOCS-3 mRNA levels, BAT UCP-1 protein levels NPY mRNA levels, and POMC mRNA levels at sacrifice from 3and 30-month old rats following chow or HF feeding. Young Adult (3-month old) Aged (30-month old) Chow-Fed HF Fed Chow-Fed HF Fed PTP1B protein (Arbitrary units) 1.00 0.04 1.25 0.06 1. 31 0.04* 1.27 0.09 SOCS-3 mRNA (Arbitrary units) 1.00 0.04 1.27 0.09** 1. 20 0.04 1.41 0.08 BAT UCP-1 protein (Arbitrary units) 1.00 0.14 2.53 0.40** 1. 30 0.25 3.19 0.48** NPY mRNA 1.00 0.04 1.01 0.05 1. 29 0.07* 1.02 0.04 POMC mRNA 1.00 0.07* 0.93 0.06 0. 80 .12 0.84 0.09* Data represent mean s.e.m. of 3-9 rats per group. Levels in young adult chow fed rats are set to 1.0 and s.e.m adjusted accordingly. PTP1B protein measured by Western analysis : P = 0.017 for difference with HF feeding and P = 0.038 for difference with age by two-way ANOVA. *P < 0.05 for the difference between chow-fed threeand thirtymonth-old rats by post-hoc analysis. SOCS-3 mRNA measured by RT-PCR : P = 0.006 for difference with HF feeding and P = 0.022 for difference with age by two-way ANOVA. **P < 0.05 for the difference between three-month-old HF-fed and threemonth-old chow-fed. BAT UCP-1 protein measure d by Western analysis : P < 0.0001 for the difference with HF feeding by two-way ANOVA. **P < 0.01 for the difference with HF feeding in both threeand thirty-month-old rats by post-hoc analysis. NPY mRNA measured by RT-PCR : P = 0.02 for the interaction; P < 0.01 for the difference with age in chow-fed rats by two-way ANOVA. POMC mRNA levels measured by RT-PCR : P < 0.0001 for the interaction; P < 0.001 for the difference with age on chow diet; P < 0.001 for the difference with HF feeding in aged rats by two-way ANOVA. 73

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Table 3-2. Serum leptin levels in young adult and aged rats on a chow diet after 7-day saline or leptin infusion. Dose (mg/day) Young Adult Rats (ng/ml) Aged Rats (ng/ml) Control 6.91 1.12 13.25 .00 0.03 10.10 0.47 0.05 14.05 0.86* 19.56 .34 0.07 19.75 1.38* 0.1 20.16 .08* 25.38 2.86** 0.5 22.23 2.15* 24.62 2.26** Data represent mean s.e.m. of 7-9 rats per group. Young Adult Rats: P < 0.0001 for the difference with leptin infusion. *P < 0. 01 for the increase in serum leptin levels compared with controls. Aged rats: P=0.0008 for the difference with leptin infusion. **P < 0.01 for the increase in serum leptin leve ls compared to control values by post-hoc analysis. 74

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0 1 2 3 4 5 6 7 -30 -25 -20 -15 -10 -5 0 5Control 0.03 mg/day 0.05 mg/day 0.07 mg/day 0.1 mg/day 0.5 mg/day ADayChange in Body Weight (g) 0 1 2 3 4 5 6 7 -15 -10 -5 0 5 BDayChange in Food Intake (g) Figure 3-7. A) Change in body weight during a 7-day peripher al leptin infusion in 3month-old chow-fed rats. Data represent mean s.e.m. of 7 rats per group. P < 0.0001 for the difference with leptin treatment by one-way ANOVA. Each individual dose is significantly diffe rent from control (P < 0.0002 for the difference in slope). B) Change in food intake in young adult rats during a 7day peripheral leptin infusion. Data represent mean s.e. m. of three-monthold chow-fed (n=7 per group) rats. P < 0.0001 for the difference with leptin treatment by one-way ANOVA. P < 0. 01 for the difference between control and each dose at Day 7 and between control and each dose for cumulative food intake. 75

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0 1 2 3 4 5 6 7 -30 -25 -20 -15 -10 -5 0 5Control 0.05 mg/day 0.1 mg/day 0.5 mg/day ADayChange in Body Weight (g) 0 1 2 3 4 5 6 7 -15 -10 -5 0 5 BDayChange in Food Intake (g) Figure 3-8 A) Change in body weight during a 7-day peripheral leptin infusion in 30month-old chow-fed rats. Data represent mean s.e.m. of 7-8 per group. P = 0.9975 for the difference with leptin treatment by one-way ANOVA. B) Change in food intake in aged rats during a 7-day peripheral leptin infusion. Data represent mean s.e. m. of 30-month-old chow -fed (n=7-8 per group) rats. There was no difference (P = 0. 087) in cumulative food intake with leptin treatment. 76

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CHAPTER 4 A NOVEL STUDY OF HIGH-FAT DIET -INDUCED HYPERPHAGIA AND RESPONSES TO CB1 ANTAGONIST, AM251, IN YOUNG AND AGED RATS Introduction When laboratory rats are introduced to a HF diet with ad libitum access, they experience an immediate and spontaneous incr ease in caloric intake (77). As discussed in Chapter 3, the peak and durati on of this increased hyperphagia escalates with age. For example, 3-month-old male F 344xBN rats were previously found to have a peak caloric intake of approximately 100 kcal and required one week to return to basal levels while 30-month-old rats had a peak ca loric intake of 140 kcal and required at least one month to return to pre-HF diet leve ls (174). Normalization of this HF dietinduced increased caloric intake is dependent upon leptin receptor signaling. When young adult rats are infused with a lept in receptor antagonist, they are unable to normalize this elevated caloric intake leve l (35, 51, 98). Leptin is a peptide hormone that is produced primarily in white adipose tissue and circulates in proportion to whole body adiposity. In a normal, leptin respons ive animal, leptin tr avels across the blood brain barrier and acts on hypothalamic lept in receptors to decrease food intake and increase energy expenditure (51). Lept in resistance, however, is generally characterized by the lack of resp onsiveness to endogenously or exogenously administered leptin (51). The mechanisms for developing or treating leptin resistance are not fully understood, but t he condition is often associ ated with genetic, diet-induced, and adult-onset obesity (9, 15, 37, 38). Another dysregulated pathway that is becoming increasingly associated with obesity is the ECS (117). EC action on ene rgy homeostasis is mediated through central CB1 receptor activation, which results in increased food intake and decreased energy 77

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expenditure (38). Hypothalamic EC levels are el evated in db/db mice which lack functional leptin receptors and in obese Zuck er rats and ob/ob mice which lack leptin (38). Leptin action in t he hypothalamus down-regulates EC activity, although the mechanism is still unclear (38). In fact, w hen leptin is administered intravenously in rats, a 40-50% reduction in hypothalamic EC levels is observed (15, 37, 38). Presumably, rats with leptin resistance or dysfunctional leptin signaling may lack the leptin-mediated down-regulation of hypothalamic EC levels (5, 64, 144, 160). This interaction has been examined through limit ed studies in genetically obese rodent models that are either complete ly leptin deficient or are c haracterized by impaired leptin signaling. To date, no studies have been reported comparing young adult, leptin responsive and aged-obese, leptin resistant rats, especially with respect to HF dietinduced hyperphagia. Administration of a CB1 antagonist in nor mal animals has been shown to decrease food intake, especially on a highly palatable diet, and body weight in many different experimental designs (162). However, t he responsiveness to a CB1 antagonist during the period immediately after introducing a HF diet (the HF-die t-induced hyperphagic period) has not been examined in aged rats compared with young adult rats. To this end, we administered two doses of AM251 by da ily i.p. injection in young adult, leptin responsive and aged, leptin resistant rats duri ng chow or acute HF feeding periods. We hypothesize that, similar to genetically obes e rodent models (77), our age-related obese rats will be hyper-responsive to the anorectic effects of the CB1 antagonist. Additionally, we sought to determine if the CB1 antagonist changes the rodents preference for the chow vs. a highly palatable HF diet. 78

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Experimental Design Experiment 1 Young adult (n = 24, 4 mont hs of age) and aged (n = 30, 29 months of age) rats initially weighing 305. 85 6.1 g and 551.70 7. 3 g, respectively, were provided a chow (15% fat; 3.3 kcal/g diet 2018; Harlan Teklad Madison, WI, USA) diet ad libitum until the experiment began. Approximat ely one week prior to the in itiation of the experiment, the rats were all given mock injections over a 2-day protocol during which they were introduced to both the i.p. in jection procedures and HF diet (60% fat; 5.2 kcal/g D12492; Research Diets, New Brunswick, NJ, USA). After the mock injections, rats were fed chow diet ad libitum and allowed to rest 1 week before experimentation began. On the first day of the expe riment, all rats were acutel y fasted for 2 hours prior to the onset of the dark cycle. Just before the dark cycl e, the rats receiv ed an i.p. injection of either vehicle (7.7% DMSO, 4.6% Tw een 80, 87.7% saline) or 0.83 mg/kg AM251 (dissolved in 7.7% DMSO, 4.6% Tween 80, 87.7% saline; Cayman Chemical, Ann Arbor, MI; half-life of 22 hours). Injection solutions were prepared fresh each day. At the time of the injection, ei ther chow or HF diet was gi ven to the rats and they were allowed ad libitum access to the food for t he remainder of the week-long experiment. The rats were given the i.p. injection of vehicle or AM251 for 6 consecutive days an hour before the onset of the dark cycle. Body weight and food intake were recorded daily. At the conclusion of the injection period, serum was drawn by tail nicking, and the rats were allowed to rest for approximately 1 week. For the next phase of the experiment, a crossover design was employed such that the rats previously receiving vehicle now received 2.78 mg/kg AM251 and t he rats previously receiving AM251 now 79

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received vehicle using the same experimenta l design. Again, serum was collected by tail nicking at the conclusion of this portion of the experiment. Experiment 2 Young adult (n = 15, 6 months of age) rats that were maintained on chow food were then provided both chow and HF diets, simultaneously, ad libitum. These rats experienced the immediate incr ease in food intake, and the ra ts were allowed to eat ad libitum until food intake (measured in grams) values had retur ned to pre-HF diet levels. At this point (Day 5), daily i.p. injecti ons of 0.83 mg/kg AM251 were administered as described in Experiment 1, while still allo wing the rats free access to both diets. In a second paradigm, aged (n=15, 30 months of age) rats that were maintained on chow food were provided the choice of chow or HF diet and simultaneously administered daily i.p. 0.83 mg/kg AM251. Results Experiment 1 When first introduced to a HF diet, rats display an immediate hyperphagia accompanied by an increase in body weight (77, 132). With ei ther age or leptin resistance, this HF diet-induced hyperphagia is further exacerbated (77). In this experiment, we tested responsiveness to two doses, 0.83 and 2.78 mg/kg/day, of 80

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AM251 in young adult and aged rats with and wi thout simultaneous introduction of a HF diet. As expected, chow-fed, vehicle-treat ed young adult rats maintained a steady caloric intake and body weight throughout the study, whereas HF-fed, vehicle-treated rats demonstrated a transient hyperphagia wi th a peak caloric intake of approximately 100 kcal, a 38% increase in cumulative caloric consumption, and a steady gain in body weight (Figure 4-1, open sym bols, open bars). While the lo w dose (0.83mg/kg/day) of AM251 in young adult rats did not significantly reduce either caloric intake (Figure 4-1, A) or body weight (Fi gure 4-1, B) in chow-fed rats, th is dose combined with HF feeding resulted in a 22% reduction in peak caloric inta ke (Figure 4-1, A) and a prevention of HF diet-induced weight gain (Fi gure 4-1, B). Similarly, cumu lative food consumption was unchanged in the AM251 chow-fed but dimini shed with AM251 in the HF-fed animals (Figure 4-1, A inset). Adip osity levels, lean mass, and t he fat-to-lean mass ratio were not affected by diet or this lower dose of drug treatment in these young adult rats (Table 4-1). However, despite no overall increase in adiposity with this short-term HF feeding, there was a nearly 50% increase in serum l eptin levels in the young adult rats, which was unchanged by AM251 treatment (Table 4-1). The higher dose (2.78 mg/kg/day) of AM 251 caused a complete inhibition of the HF diet-induced hyperphagia in young adult rats (Figure 4-1, C). Caloric intake was reduced on both diets, with the reduction in cumulative caloric intake during the 5-day treatment period being 21% in chow-fed and 27% in HF-fed young adult rats with AM251 treatment (Figure 41, C inset). None of the vehicl e-treated, but all of the rats treated with the high dose of AM251 immediat ely lost body weight compared to diet81

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matched controls by Day 1, regardless of di etary treatment (Figur e 4-1, D). Moreover, the AM251-mediated body weight loss was greater with HF feeding beginning on day 1 with an overall reduction in body weight of approximately 10 grams in chowand 16 grams in HF-fed young adult rats by day 5 co mpared with diet-matched controls (Figure 4-1, D). Body composition was unchanged in the young adult rats with this dose of AM251 (Table 4-1). In the aged rats, the vehicletreated, c how-fed animals maintained a stable body weight and caloric intake while the HF-fed, vehicle-treated ra ts demonstrated a hyperphagia of approximately 150 kcal, nearly 50% greater than that observed in the young adult rats (Figure 4-2, open symbols). Unlike treatm ent in the young adult rats, the low dose (0.83 mg/kg) of AM251 caused a 16% reduction in the HF-induced peak in caloric intake, and reduced the normal peri od of hyperphagia from 30 days (77) to 6 days (Figure 4-2, A). Similarl y, the five-day cumulative caloric intake was reduced by 24% in chow-fed and 20% in HF-fed aged rats with AM251 treatment (Figure 4-2, A inset). HF feeding resulted in a considerable gain in body weight in the vehicle treated rats (Figure 4-2, B). Peripheral AM251 tr eatment reduced body weight beginning at day one in chow-fed and partially prevented body wei ght gain by day 1 in the HF-fed rats. By the end of the 5-day period, body weig ht differed by 17 grams in chow-fed and 22 grams in HF-fed aged rats compared to diet-m atched controls (Figure 4-1, B). In addition, AM251 treatment with HF feeding improved the fat-to-lean mass ratio by 14% compared to diet-matched controls, while whole body adiposity and lean mass were unchanged (Table 4-2). Intere stingly, this low dose of AM251 significantly blunted the HF-diet-induced increase in serum leptin levels in the aged rats (Table 4-2). 82

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The high dose of AM251 in aged rats completely inhibited HF diet-induced hyperphagia. Interestingly, t he absolute level of caloric intake in both chowand HF-fed aged rats was nearly equivalent by Day 5 (Fi gure 4-2, C). However, when compared to their diet-matched controls, the response to AM251-treatment in the HF-fed aged rats was considerably greater than that in the young adult rats with a reduced cumulative caloric intake of 76% compared with 29%, res pectively (Figure 4-2, C inset). This higher dose of AM251 caused a parallel reducti on in body weight in chowand HF-fed aged rats. However, if these rats are co mpared to their diet-matched controls, the AM251 responsiveness was dramatically a ugmented in the HF-fed aged rats, with body weight differing by approximately 27 and 58 grams in chow-fed and HF-fed aged rats, respectively (Figure 4-2, D). Body com position analysis revealed that adiposity, lean mass, and serum leptin were not significantly different with this high-dose AM251 treatment in chow-fed, whereas the fat-to -lean mass ratio was improved by 9% (Table 4-2). In contrast, drug tr eatment coupled with HF f eeding reduced adiposity by 23%, the fat-to-lean mass ratio by 19%, and serum leptin levels by 73%, whereas lean mass was unchanged (Table 4-2). In essence, t he HF diet-induced elevation in adiposity and serum leptin levels and the decrease in fa t-to-lean mass ratio were reversed with highdose AM251 treatment in aged rats (Table 4-2). Experiment 2 Data regarding CB1 receptor-mediated actions on food palat ability are limited and conflicting. To examine if CB1 rec eptor antagonistm is able to change the food palatability or pref erence of a HF diet relative to chow, we administered AM251 in young adult and aged rats while providing both HF and chow food in two different protocols. 83

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In the young adult, HF food was first introduced along with the chow and AM251 injected after the HF-induced hyperphagic period normalized. Prior to addition of the second diet (60% HF food), the young adult rats consumed approximately 20 grams of chow food each day (Figure 4-3, A inset). U pon initiation of the HF diet (in addition to the chow), total food intake spiked at appr oximately 28-29 grams/da y, primarily due to HF diet intake and equivalent to appr oximately 140 kcal/day. The hyperphagia was gradually normalized over the next few days. During this phase of the experiment, the rats ate almost exclusively of the HF diet even though they were given the free choice between the diets (Figure 4-3, A). When food consumption, measured in grams/day, returned to pre-choice level, approxim ately 20 grams/day, we began daily AM251 treatment. On day 5, we began AM251 treatment and observed an immediate decrease in caloric intake and body weight similar to that seen in Experiment 1 ( Figure 4-3, A & B). Interestingly, CB1 antagonist tr eatment did not change the preference for chow vs. HF food in the young adult rats. Throughout the experiment and even after termination of drug treatment (data not shown), the rats continued to eat ex clusively HF diet (Figure 43, A). In a second paradigm, the effect of da ily AM251 treatment wa s examined on diet selection in aged rats. The aged rats were ma intained on chow diet and were given the choice between the chow and HF diets simu ltaneous with daily i. p. AM251 treatment. Thus, we administered i.p. vehicle or AM251 simultaneously with the introduction of the diet choice on Day 0. Similar to the results in aged rats in Experiment 1, AM251 treatment significantly reduced HF-diet in take on Days 2 and 3, and caused a reduction 84

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in body weight, which began on Day 1 and lasted throughout the treatment period (Figure 4-4, A & B). Moreover, as seen in the y oung adult animals, AM251 treatment did not change the preference fo r the aged rats to consume al most exclusively HF diet (Figure 4-4, A). Discussion Ad libitum access to a highly palatable diet stimulates rodents to immediately consume more calories for a period of time, the peak and duration of which is dependent upon the animals state of leptin responsiveness (77). Aged-obese, leptin resistant rats display a heightened sensitiv ity to this HF-diet-induced hyperphagia and experience a prolonged elevation of calori c intake and exacerbated body weight gain (15, 37, 81). Obesity is also associated with elevated EC levels and increased CB1 receptor activity (18, 38). In fact, leptin administrat ion significantly reduces both peripheral and central EC levels, s uggesting that endogenous leptin negatively modulates EC levels and that this downregulat ion may be blunted or absent in the leptin resistant state (38, 162). Genetically obese rodents, in cluding models with impaired leptin signaling, display enhanced anorectic responses to CB1 antagonist administration (5, 144), predicting that our aged-obese, leptin resistant rats will also display enhanced decreases in body weight and caloric intake during AM251 treatme nt. Indeed, the data described here demonstrate t hat CB1 receptor antagonist responsiveness is enhanced with both age-related obesity and short-term HF feeding. However, for the dose and diets tested, AM251 was unable to increase t he preference of t he chow diet when compared to the highly palatable HF diet. Conflicting evidence exists regarding the ability of a CB1 receptor antagonist to reduce the intake of chow and/or highly pal atable diets. Some studies have suggested 85

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that CB1 antagonists preferentially reduce caloric intake on a highly palatable diet versus a bland chow diet (5, 144). These researchers showed t hat the CB1 antagonist SR141716 selectively reduced intake of a sweet diet, in both solid and liquid forms, without affecting chow or water intake. Their data suggest that the endogenous EC activity may act by increasing the preference of the available diets or drinking solutions (103, 161). This is partially consistent with the present study in that AM251-mediated CB1 antagonism, regardless of dose or age of animal tested, reduced HF-diet intake to a much greater extent than chow intake. However, our larger dose of AM251 in Exper iment 1 was also able to significantly, albeit to a lesser degree, reduce caloric intake on the chow diet. While these data appear to conflict with that mentioned above, it is consistent with McLaughlin, et al. and Verty, et al., who showed that AM251 equally suppressed feeding on chow, highcarbohydrate, and HF diets in Wistar and Sprague-Dawley rats (74). These authors suggest that data interpretation has a large impact on the conclusions reached. For example, when rats are provided a HF or high carbohydrate diet ad-libitum, they consume more of these di ets than they do of chow, creating higher baseline consumptions for these highly pa latable diets. If the AM251-induced reduction of intake of these diets is compared to their own bas elines, the results show a more dramatic anorectic effect in the highly palatable di ets when compared to normal chow, whereas analysis using grams of food consumed after AM251 treatment did not reach significant differences among groups. Another study by Jarrett, et al. showed that AM251 was able to reduce the intake of both sweet and mildly bitter solutions, suggesting that the feeding behaviors after CB1 receptor ant agonist treatment may be mediated by a 86

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change in perceived food palatab ility (77). Thus, possibl e explanations for these contradictions include dietary composition, data interpretation, and dose and type of CB1 receptor antagonist used. The present study showed that AM251 re sponsiveness is enhanced in aged rats and even further increased with HF f eeding. We propose that this hyperresponsiveness is due to the state of leptin resistance in the aged rats. If endogenous leptin is unable to down-regulate brain EC levels, the EC levels may be elevated and create a situation in which CB1 receptor s may be hyper-responsive to antagonism. HF feeding has been shown to increase serum leptin levels (64, 153) so this may explain the enhanced responsiveness with HF feeding, ra ther than the palatabi lity of the food. Interestingly, AM251 treatment was abl e to reverse both age-related and dietinduced elevations in adiposity and serum leptin levels, which has also been seen in studies by other investigators (146). More experiments are required to determine if this is a cause-effect relationship or if they ar e merely parallel responses. Regardless of the mechanism, the lower leptin levels may at least partially restore leptin responsiveness. Experiment 2, examining AM251-induced ch anges in preference of the chow vs. HF diets, showed that 0.83 mg /kg/day AM251 reduced total caloric intake by causing a reduction in only the HF-diet intake. T hus, AM251 treatment did not increase the preference of the chow diet versus the HF diet because both young adult and aged rats continued to consume only HF diet when gi ven the choice. Very few data regarding CB1 antagonist treatment during a food choice test are ava ilable, but our results are consistent with South, et al. These in vestigators gave C57BL/6 mice access to highand low-fat diets ad libitum and then administer ed 5 mg/kg/day AM251, i.p., for 4 days. 87

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This dose is considerably higher than the dose tested here, but t hese researchers found that AM251 treatment reduced intake of only t he HF-diet (5). Similarly, a study with Wistar rats showed that CB 1 receptor antagonism preferentially reduced intake of palatable sucrose pellets over regular chow (8, 117, 156). Ther efore, CB1 antagonism dose not appear to reduce the pref erence of a 60% HF diet rela tive to the regular chow consisting of 15% fat. In summary, we showed that there is a dose-dependent reduction in HF dietinduced hyperphagia and caloric intake in young adult and aged rats with AM251 administration. The aged rats displayed enhanced responsivenes s to AM251 treatment, and the anorectic responses were further amplified during HF feeding in both ages. In the second experiment, we showed that AM251 treatment does not change the preference for chow vs. a 60% HF diet in either young adult or aged rats. Examination of AM251 responses during the HF diet-indu ced hyperphagic period is a novel approach and one that may provide new insights into both age-related and diet-induced obesity and leptin resistance. 88

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89 -1 0 1 2 3 4 5 -25 0 25 50 75 100 125 150 A DayCaloric Intake (kcal) 5-Day Cumulative Caloric Intake Chow HF 0 50 100 150 200 250 300 350 400 450Vehicle AM251 kcal 0 1 2 3 4 5 -50 -40 -30 -20 -10 0 10 20 30 40 50HF Vehicle Chow Vehicle Chow AM251 HF AM251 B *DayChange in Body Weight (g) -1 0 1 2 3 4 5 -25 0 25 50 75 100 125 150 C DayCaloric Intake (kcal) 5-Day Cumula tive Caloric Intake Chow HF 0 100 200 300 400 500 600Vehicle AM251 *kcal ** 0 1 2 3 4 5 -50 -40 -30 -20 -10 0 10 20 30 40 50 D ** **Chow-Ve **hicle Chow-AM 251 HF-Vehic le HF-AM25 1 Day Change in Body Weight (g) Figure 4-1. Change in caloric intake (A & C) and body weight (B & D) in young adult rats during daily i.p. 0.83 mg /kg or 2.78 mg/kg AM251 administration. Data represent mean s.e.m. of chow-fed with vehicl e treatment (n = 6, open triangles), chow-fed with AM251 treatment (n = 6, closed triangles), HF-fed with vehicle treatment (n = 6, open ci rcles), and HF-fed with AM251-treatment (n = 6, closed circles). A) AM251 treat ment did not reduce caloric intake in young adult rats, regardless of dietary treatment. Inset: HF diet stimulated an increase in cumulative caloric intake (P-value < 0.001) that was unchanged by AM251. B) Only the HF-fed v ehicle-treated young adult rats gained weight during the experimental period. *P-value < 0.01 for the gain in body weight in HF-fed vehicle-treated compared to all other groups starting at day 2 by t-test. C) AM251 treatment inhi bited the HF diet-induced hyperphagia. Inset: 5-day cumulative caloric intake was significantly reduced by AM251 treatment in chow-fed (*P < 0.05) and HF-fed (**P < 0.01) rats. D) AM251 treatment caused body weight loss in y oung adult rats on both diets (P < 0.05 for the difference between AM251-tr eated rats and their diet-matched controls, starting on Day 1). **P<0. 05 for the difference between HF-fed AM251-treated and Chow-fed AM251treaded rats on Days 1-3.

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90 Table 4-1. Adiposity, lean mass, and serum leptin levels after respective AM251 doses in young adult rats during hyperphagia. Experimental Group Adiposity (g) Lean Mass (g) Fat/Lean Mass Ratio Serum Leptin (ng/mL) Chow-Vehicle 70.0 3.5 191.6 9.3 0. 37 0.01 6.0 1.1 Chow-AM251 71.7 2.7 190.6 5.1 0. 38 0.01 6.1 1.2 HF-Vehicle 73.2 4.5 200.3 10.0 0. 36 0.01 12.5 1.6 Young Adult Rats, 0.83mg/kg AM251 HF-AM251 70.7 1.7 196.2 6.5 0. 36 0.00 11.5 1.5 Chow-Vehicle 93.2 5.6 230.0 10.4 0.40 0.01 No data available Chow-AM251 89.7 2.9 222.6 5.3 0.40 0.01 No data available HF-Vehicle 79.4 3.9 211.9 7.2 0. 37 0.01 13.1 3.1 Young Adult Rats, 2.78mg/kg AM251 HF-AM251 73.7 4.2 200.2 11.9 0.37 0.01 8.4 1.5 Data represent mean s.e.m. of 6 rats per group analyzed by 2-way ANOVA. indicates the difference with HF feeding from corresponding chow-fed rats.

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91 -1 0 1 2 3 4 5 -25 0 25 50 75 100 125 150 A *DayCaloric Intake (kcal) Chow HF 0 100 200 300 400 500 600 700Vehicle AM251 5-Day Cumulative Caloric Intake ** Cumulative Caloric Intake (kcal) 0 1 2 3 4 5 -50 -40 -30 -20 -10 0 10 20 30 40 50Chow-Vehicle Chow-AM251 HF-Vehicle HF-AM251 B* ** * *DayChange in Body Weight (g) -1 0 1 2 3 4 5 -25 0 25 50 75 100 125 150 Ce (kcal) DayCaloric Intak 5-Day Cumulative Caloric Intake Chow HF 0 100 200 300 400 500 600 700Vehicle AM251 Cumulative Caloric Intake (kcal) 0 1 2 3 4 5 -50 -40 -30 -20 -10 0 10 20 30 40 50 DChow-Vehicle Chow-AM251 HF-Vehicle HF-AM251 DayChange in Bo dy Weight (g) Figure 4-2. Change in caloric intake (A & C) and body weight (B & D) in aged rats during daily i.p. 0.83 mg /kg or 2.78 mg/kg AM251 administration. Data represent mean s.e.m. of chow-fed and vehicle-treated (n =6, open triangles), chow-fed and AM251-treated (n = 7, closed triangles), HF-fed and vehicle-treated (n = 7, open squares) and HF-fed and AM251-treated (n = 8, closed squares). A) AM251 treatment si gnificantly reduced caloric intake in HF-fed rats beginning on Day 2. *P-value < 0.01 for the difference in caloric intake between HF-fed vehicle-treat ed and AM251-treated aged rats. Inset: 5-day cumulative caloric intake is reduced in all AM251treated aged rats (*P < 0.05, **P < 0.001) P-val ue < 0.001 for the increase in cumulative caloric intake with HF-feeding compared to tr eatment-matched chow-fed rats. B) AM251 treatment caused a reduction in body weight compared to dietmatched, vehicle-treated aged rats (*P-val ue < 0.01). C) AM251 completely inhibited HF diet-induced hy perphagia and inhibited chow intake in aged rats. Inset: 5-day cumulative caloric intake is reduced with AM251 treatment on both diets (*P < 0.001, P-value < 0.001 fo r the increase in cumulative caloric intake with HF-feeding compared to tr eatment-matched chow-f ed rats.) D) AM251 reduced body weight of both c howand HF-fed aged rats beginning at Day 1 (P < 0.001 for difference between AM251-treated and diet-matched vehicle-treated rats at all days during experimental period, by t-test).

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92 Table 4-2. Adiposity, lean ma ss, and serum leptin levels after respective AM251 doses in aged rats during hyperphagia Experimental Group Adiposity (g) Lean Mass (g) Fat/Lean Mass Ratio Serum Leptin (ng/mL) Chow-Vehicle 154.7 5.5 305.7 5.3 0. 51 0.01 24.2 1.9 Chow-AM251 139.1 7.4 304.0 7.7 0. 46 0.02 20.7 2.2 HF-Vehicle 184.4 6.3 318.8 4.3 0. 55 0.02 70.0 9.8 Aged Rats, 0.83 mg/kg AM251 HF-AM251 149.8 9.8 321.6 11.6 0. 47 .03 40.8 5.9 Chow-Vehicle 148.8 5.6 295.7 5.3 0. 50 0.01 18.0 2.4 Chow-AM251 136.1 4.8 296.9 5.8 0. 46 0.01 14.6 1.8 HF-Vehicle 163.9 10.4 307.8 9.0 0. 54 0.02 50.8 10.5 Aged Rats, 2.78mg/kg AM251 HF-AM251 126.3 8.5 292.5 10.0 0.43 0.02 13.9 3.9 Data represent mean s.e.m. of 6-8 rats per group analyzed by 2-way ANOVA. indicates the difference with HF feeding from corresponding chow-fed rats ; indicates the difference with AM251 treat ment from corresponding diet-matched rats.

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-3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 0 20 40 60 80 100 120 140 160Chow-Vehicle HF-Vehicle Chow-AM251 HF-AM251 i.p. AM251 A ** Chow-Before Treatment HF-Before Treatment DayCaloric Intake (kcal) -3 -2 -1 0 1 2 3 4 5 6 15 20 25 30 DayFood Intake (g) -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 -10 0 10 20 30 40 i.p. AM251Vehicle AM251 B * Before Treatment DayChange in Body Weight (g) Figure 4-3. Caloric intake and change in bo dy weight in young adult rats during food choice and i.p. vehicle or 0.83 mg/kg AM251 treatment. Da ta represent mean s.e.m. of vehicle-treated (n = 8, circles) chow (solid line) or HF (dotted line) intake and AM251-treated (n = 7, triangles ) chow (solid line) or HF (dotted line) intake. A) Young adult rats exclusively consum e the HF diet both before and during AM251 treatment. *P-value < 0. 05 for the difference in HF-diet intake between vehicleand AM251-treated ra ts by t-test. In set: Food intake in young adult rats prior to AM251 treat ment. B) Peripheral AM251 treatment immediately prevented HF-diet-induced body weight gain in young adult rats. *P-value < 0.05 for the difference betw een vehicle and AM251 treated rats by t-test. 93

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-3 -2 -1 0 1 2 3 4 5 6 0 20 40 60 80 100 120 140 160Vehicle-Chow Vehicle-HF AM251-Chow AM251-HF A *Chow-Before Treatment DayCaloric Intake (kcal) -3 -2 -1 0 1 2 3 4 5 6 -10 0 10 20 30 40Vehicle AM251 B * *Before Treatment DayChange in Body Weight (g) Figure 4-4. Caloric intake and change in bo dy weight in aged rats during food choice and vehicle or 0.83 mg/kg AM251 treatment Data represent mean s.e.m. of vehicle-treated (n = 7, squares) chow (s olid line) or HF (dotted line) intake and AM251-treated (n = 8, triangles) chow (solid line) or HF (dotted line) intake. A) AM251 treatment reduc ed the HF-diet-induced hyperphagia beginning on Day 2. *P-value < 0.05 on Day 2 and 3 for the difference in HFdiet intake between vehicl eand AM251-treated rats by t-test. B) Peripheral AM251 treatment prevented the HF-diet-induced body weight gain normally seen in aged rats. *P-value < 0.05 fo r the difference between vehicle and AM251 treated rats by t-test. Data repr esent mean s.e.m. of 6-8 rats per group analyzed by 2-way ANOVA. indica tes the difference with HF feeding from corresponding chow-f ed rats; indicates the difference with AM251 treatment from corresponding diet-matched rats. 94

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CHAPTER 5 RESPONSES TO THE CANNABINOID RECEPTOR-1 ANTAGONIST, AM251, ARE MORE ROBUST WITH AGE, WITH ESTABLISHED HIGH-FAT FEEDING-INDUCED OBESITY, AND WITH LEPTIN RESISTANCE Introduction Endocannabinoids (ECs), such as anadamide (AEA) and 2-arachidonoylglycerol (2-AG), act through G-protein-coupled cannabin oid-1 (CB1) receptors in the brain and in various peripheral organs to increase bot h energy intake and adipogenesis (162). CB1 receptor knockout mice, compared to wild-type littermates, consume less food after being fasted (101). These knockout mice also display reduced adiposity levels and resistance to HF diet-induced obesity, ev en when they consume the same amount of food as controls (38, 162). Obese animals with established obesit y and leptin resistance demonstrate enhanced responsiveness to CB1 receptor antagonists compared with lean animals (8, 60, 62, 101). Other evidence suggests that both a highly palatable diet and obesity, which can both induce leptin resistance in the long term, raise central and peripheral EC levels (100). Conversely, CB1 antagonist anorectic responses can be enhanced in lean animals after EC tone is increased, for exam ple, when animals are exposed to a highly palatable diet or acutely fast ed (90). Collectively, this s uggests that there is higher EC tone in the obese state. Similarly, this predicts that the proposed leptin negative modulation of ECs may be absent or blunted in t he obese, leptin resistant state. If so, then CB1 receptor blockade in obese rodents will result in greater physiological responses, particularly in long-term HF fed young adult and more so in long-term HF fed aged-obese rats. To this end, we tested the AM251 responsiveness in young adult and aged rats with established HF diet-induced obesity. 95

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Experimental Design Young adult (n=29, 4 mont hs of age) and aged (n=32, 29 months of age) rats initially weighing 311. 5 5.4 g and 559.5 6.3 g, respectively, were provided a chow (15% fat, 3.3 kcal/g diet 2018; Harlan Teklad Madison, WI, USA) or HF (60% fat, 5.2 kcal/g D12492; Research Diets, New Brunswick, NJ, USA) di et ad libitum for 60 days. Body weight and food intake were recorded daily. On Day 60, a 14-day osmotic minipump (model 2002, Durect, Cupertino, CA ), administering either saline or 0.1 mg/day leptin, i.p. was implanted in a s ubcutaneous pocket on the dorsal surface of the rat. After the infusi on period, the rats were allowed to recover for approximately 2 weeks before AM251 injections began. A crosso ver design was employed, in that rats previously receiving saline now received 0.45 mg/day AM251 (dissolved in 7.7% DMSO, 4.6% Tween 80, 87.7% saline; Caym an Chemical, Ann Arbor, MI), and those rats previously receiving leptin now rece ived vehicle (7.7% DMSO, 4.6% Tween 80, 87.7% saline) with all groups maintained on thei r respective diets, ad libitum. Rats were administered either vehicle or AM251, daily, by i.p. injection for 6 consecutive days, and afterwards, the rats were killed for tiss ue analysis. Blood was collected by cardiac puncture for serum leptin measurements. 96

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Results The responsiveness to AM251 was examined in young adult and aged rats with established obesity as a result of 60 days of HF feeding as compared with chow-fed animals. Prior to examining AM251 responsiveness, but after 60 days of chow or HF feeding, leptin responsiveness was examined in both age groups by peripheral leptin infusion. The HF-fed young adult rats demonst rated a partial leptin resistance and all the aged rats displayed a complete leptin resistance (Figure 5-1). The absolute reduction in body weight on Day 10, on which the rats displayed the greatest response to the leptin treatment, was reduced by both HF feeding and age (young adult chow-fed: 15.5 3.1g, young adult HF-fed: 11.8 1.2g, aged chow-fed: 9.7 2.3g, and aged HFfed: 4.3 1.5g). After a two week recovery period, responsiveness to AM251 was examined. The young adult, chow-fed rats didnt show a decr ease in cumulative caloric intake during the 7-day AM251 treatment (0. 45 mg/day), although they did lose body weight (Figure 5-2, A & B), suggesting that an increase in energy expenditure accounts for the decrease in body weight. The anorectic re sponses to AM251 were more marked with HF feeding compared to chow feeding. Cumulative caloric intake was reduced by 23% in HF-fed AM251-treated rats, but there was no effect on cumu lative caloric intake in chow-fed rats (Figure 5-2, A) Initially, the young adult rats on both the chow and HF diets responded to the 7-day AM251 treatment (1 .2 mg/kg/day or 0.45 mg/rat/day) with similar decreases in body weight. Beginning at day 5, the chow-f ed animals started to regain the lost weight, such that by the end of the treatment per iod, the decrease in body weight was nearly double in the HF-fed co mpared with the chow-fed rats. This is indicated by the positive slope of the body weight change in chow-fed compared with 97

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the negative slope in the HF-fed rats (Figure 5-2, B dotted boxes). The reduction in body weight in AM251-treated versus vehicl e-treated on Day 7 was 60% greater in HFfed compared with chow-fed young adult rats ( Figure 5-2, B). In addition, there was a decrease in adiposity level in AM251-treat ed young adult rats on both chowand HFdiets compared with an increase in adiposit y in the vehicle-treated young adult rats (Table 5-1). Similar to the young adult rats, the responses of AM 251 were more pronounced in aged rats with HF feeding compared with chow feeding. In contrast to the young adult, the aged rats also displayed a robust respons es to daily AM251 (0.8 mg/kg/day or 0.45 mg/rat/day) treatment in the chow-fed rats The cumulative caloric intake was significantly reduced by AM251 treatment by 30% in chow-fed young adult rats, with an even greater 45% reduction in HF-fed aged rats of (Figure 5-3, A). Similarly, AM251 treatment reduced body weight by Day 7 by 21 and 34 grams in chowand HF-fed aged rats, respectively, when compared to vehicletreated, diet-matched controls (Figure 5-3, B). Both adiposity and lean body mass levels were also reduced in aged rats treated with AM251 compared to their diet-matc hed, vehicle-treated controls (Table 5-2). The degree of AM251 responsiv eness is directly correlated to the degree of peripheral leptin responsiveness in these young adult and aged rats (Figure 5-4). The young adult, chow-fed rats, which display the greatest body weight loss in response to peripheral leptin treatment, display the least anorectic response to daily AM251 treatment. Conversely, the aged, HF-fed rats, which are co mpletely leptin resistant, display the greatest anorectic responses to AM251. The responses in young adult, HF98

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fed and aged, chow-fed fall between these two extremes such that the correlation is significant, with an R squared value of 0.9649. Biochemical Indicators In rats with established obesity, several indicators of energy homeostasis were assessed. Several neuropeptides, including leptin, signal through phosphorylation of Signal Transducer and Activator of Tran scription-3 (STAT3) (131, 170). At death, hypothalamic pSTAT-3 levels were elevated with age and with HF feeding in both young adult and aged rats (Table 5-1 & 5-2). This is consistent with previous reports where HF feeding increased hypothalamic P-STAT3 leve ls compared to chow-fed controls (1). More interestingly, whereas AM251 treatme nt raised pSTAT-3 levels in both young adult and aged chow-fed rats, it only reache d significance in aged rats (Table 5-1 & 52). Phosphorylation of Acetyl Co-A Carboxyl ase leads to fatty acid oxidation and the breakdown of adipose tissue to increase available substrate (77). Both HF feeding and age decreased pACC levels in PWAT (Table 5-1 & 5-2), consistent with the positive energy balance under both of these conditions. Surprisingly, AM251 treatment also caused a significant reduction in pACC levels in aged rats, despite the state of negative energy balance (Table 5-2). The elevation of UCP1 protein levels in BAT is often used as a marker of energy expenditure (77, 170, 174). In accordance with previ ous reports, in t he present study HF feeding increases UCP-1 levels in bot h young adult and aged rats (5, 64, 144). In addition, the current study found that AM251 t ended to increase UCP-1 levels in BAT in chow-fed young adult and aged rats, although these increases did not reach significance (Table 5-1 & 5-2). 99

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Comparisons of Young Adult and Aged Rats with Chow and HF Feeding In addition to the comparisons with die t, the responsiveness of AM251 with age was compared using the data from Chapters 4 and 5. Fi gure 5-5 summarizes the loss in body weight in AM251-tr eated compared to vehicle-tr eated rats for each diet and dose of AM251 across ages. Based on the dat a presented in Figures 5-2 and 5-3, it appears that the 0.83 mg /kg/day dose is submaximal w hereas the 2.78 mg/kg/day of AM251 is maximal. With chow feeding, only the aged rats responded to the low dose of AM251 suggesting increased sensitivity to AM251 with age (Figure 55, A). Second, the responsiveness to this dose within HF-fed rats of both ages is increased, indicating increased sensitivity with HF feeding (Figure 5-5, A). Comparison of the maximal dose (Figure 5-5, B), indicates that maximum efficacy is increased with HF feeding and further augmented with age. Comparisons with age were made in rats with established obesity, as described in Chapter 5. In this case, comparisons across age were complicated by the dosing regime. In this experimen t, the same dose of AM251 per animal (0.45 mg/rat/day) was administered to rats of both ages. Because of the considerable differences in body weights between young adult and aged rats, the dose if recalculated based on rodent body weight yields 1.20 mg/kg/day and 0.80 mg/kg/day, respectively for young adult and aged rats. Thus, direct comparisons cannot be made between the young adult and aged rats. However, the more robust body weight reduction was observed in the aged rats, which received the smaller dose per kilogram of body weight. With AM251 treatment, the HF fed aged rats with established obesity lost 65% greater body weight compared with the corresponding chow-fed rats (Figure 5-5, C). Lastly comparison among aged rats between those first introduced to the HF diet (C hapter 4) and those 100

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HF fed rats with established obesity (Chapt er 5) indicates that low-dose AM251 treatment evoked more robust body weight r eduction in latter (Figures 5-5, A and C, right pairs of bars). Discussion When the data from this chapter are evaluated with the data from Chapter 4, they demonstrates that the res ponsiveness of the CB1 re ceptor antagonist was enhanced with both HF diet-induced obesity and age, with both increased sensitivity and maximum efficacy. These enhanced responses were observed with respect to both body weight reduction and the anorectic respons e. The latter includes the time course of food consumption as well as the overall reduction in cumulative caloric intake during HF feeding compared to chow feeding. In addition, the HF diet-induced increas e in AM251 sensitivity appears to be additive with age, with the greatest degree of responsiveness observ ed in aged, HF fed rats. In HF fed young adult rats, enhancem ent in AM251 antagonism is modest. These data are consistent with previous reports that CB1 receptor ant agonists preferentially block consumption of a more palatable diet as compared with standard chow diet (38). With age, the AM251 antagonist response is gr eatly magnified, with this antagonistic response further exacerbated in HF-fed aged rats. Because both age and HF dietinduced obesity are associated with leptin re sistance, these data are consistent with a positive relationship between CB1 receptor antagonist efficacy and leptin resistance. Leptin is believed to down regulate EC levels in the brain. Thus, in leptin resistant animals, the EC system may be fr ee from leptin down regulati on, leading to hyperactive CB1 receptors (38, 44, 96). Under these circumstances, leptin resistance would expect to be associated with enhanced AM251 responsiv eness. Moreover, the present study 101

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provides a direct relationship between lept in resistance and blocka de of CB1 receptor activity. The increase in AM251 efficacy with HF feeding appears to be influenced by the duration of the diet and resulting established obesity as well as the presence of leptin resistance. Young adult rats with longterm HF feeding displayed both reduced perhipheral leptin and enhanced AM251 res ponsiveness when compared to chow-fed young adult rats. However, in aged rats the efficacy of AM251 was enhanced to an even greater degree with longterm HF feeding and establis hed obesity. Moreover, there is a strong corre lation between enhanced AM251 responsiveness and leptin resistance in young adult and aged chowand HF -fed rats. Similarly, these responses in long-term chowor HF-fed rats were enhanced when compared to rats given shortterm exposure to the respective diets. This suggests that t he duration of dietary exposure and resulting obesity as well as the development of diet-induced obesity significantly contribute to the enhanced AM251 responsiveness observed in long-term fed rats. The present data directly link the presence of leptin resistance to the increase in AM251 responses. Recent data indicate that some obese states lacking normal leptin function are associated with elevated EC leve ls (38). In fact, hy pothalamic EC levels are significantly elevated in Zucker rats which have defective leptin receptors, db/db mice which lack leptin receptors; and ob/ob mi ce which lack leptin (38). In ob/ob mice, exogenous administration of leptin is effe ctive in reducing EC levels back to those matching lean littermates (38, 112). Other evidence also s uggests that EC levels are elevated in animals with both diet-induced and genetic obesity, but status of the CB1 102

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receptor activity with obesity is unclear (4 ). In some cases, receptor levels are reciprocally related to EC levels, thus di minished with HF feeding. These data do not support our findings of enhanced AM251 efficacy in the presence of a HF diet. While the mechanism underlying the in creased efficacy with a HF diet or with age remains speculative, leptin resistance appears to play a significant role. One interesting observation is that the HF fed rats displayed elevated UCP1 protein levels in BAT, even in the aged, HF fed rats. Perhaps there is a component of BAT thermogenesis that is contributing to energy homeostasis in the aged HF-fed rats that is absent in aged chow-fed rats, and blockade of this component by AM251 contributes to the increased efficacy wi th age, though additional experiments are necessary to solidify these speculations. In summary, both age and a HF diet are associated with enhanced CB1 receptor antagonist efficacy, with the greater re sponse in the aged rats, and the greatest enhancement in HF fed aged rats. The enhanced efficacy with age and a HF diet appears to be more related to the diet, than the presence of leptin resistance, and the degree of obesity may play a role. In addi tion, CB1 receptor activity appears to contribute to the hyperphagia observed wi th the introduction of a HF diet. However, the complete underlying me chanism of the enhanc ed CB1 receptor antagonist responsiveness remains speculative. 103

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Young Chow Young HF Aged Chow Aged HF 0 1 2 3 4 5 6 ** **Body Weight Lost (%) Figure 5-1. Decrease in body weight at day 10 following peripheral vehicle or leptin infusion in chow or HF f ed rats. Data represent t he difference in body weight from pretreatment values and is the mean s.e.m. of chow-fed young adult (n = 8), HF-fed young adult (n = 8), c how-fed aged (n = 6), and HF-fed aged (n = 8). HF feeding and to a great er extent, age caused a reduced responsiveness to peripheral leptin infusion (*P < 0.05 for the difference with HF feeding in young adult rats; **P < 0.01 for the difference with age, regardless of dietary treatment. Delt a body weight in all groups were significantly different from pretreat ment (Day 0) by paired t-test). 104

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Chow HF 0 100 200 300 400Vehicle AM251 ** Akcal 0 1 2 3 4 5 6 7 -45 -35 -25 -15 -5 5 BChow-Vehicle Chow-AM251 HF-Vehicle HF-AM251 ** DayChange in Body Weight (g) Figure 5-2. Change in cumulative caloric intake and body weight in young adult rats during 7-day daily i.p. administration of AM251 (0.45 mg/rat/day; 1.2mg/kg/day). Data represent m ean s.e.m. of chowor HF-fed and vehicle-treated (n = 8, closed symbol s), chowor HF-fed and AM251-treated (n = 7, open symbols). A) The co mbination of HF feeding and AM251 treatment caused a reduction in cumulati ve caloric intake. **P-value < 0.01 for the difference with AM251 treatment in HF-fed rats. B) AM251-induced body weight reduction was similar in chowand HF-fed young adult rats. 105

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Table 5-1. Change in body composition and biochemical markers of young adult rats during daily i.p. vehicle or AM251 injections. Chow HF Vehicle AM251 Vehicle AM251 Change in Adiposity (g) 3.8 + 1.2 -2.1 + 1.8 3.5 + 0.9 -3.6 + 1.6 Change in Lean Mass (g) -0.4 + 1.6 -4.6 + 2.2 0.7 + 1.3 -2.2 + 3.4 Hypo P-STAT3 1.00 0.08 1.32 0.07 1.79 0.11* 1.72 0.09* PWAT pACC 1.00 0.19 1.31 0.29 0.47 0.16 0.29 0.13* BAT UCP-1 1.00 0.08 1.19 0.07 1.42 0.05* 1.53 0.10* NPT mRNA 1.00 0.06 1.00 0.02 0.96 0.04 0.96 .04 PTP1B mRNA 1.00 0.11 1.30 0.11 1.00 0.05 1.20 0.26 POMC mRNA 1.00 0.08 1.05 0.09 0. 97 0.07 0.98 0.13 Data represent mean s.e. m. of 4-8 rats per group analyzed by 2-way ANOVA. Adiposity and lean mass were determined prior to and after 7-day vehicle or AM251 daily injections; biomolecular marker prot ein levels, measured by Western analysis, and mRNA levels, measured by RT-PCR, of all young adult, chow-fed & vehicle-treated groups are set to 1.00 and s.e. m. adjusted accordingly. P < 0.05, indicates the difference with AM251 treatment from corresponding vehicle-treated and diet-matched rats. *P < 0.05, indicates the difference with HF feeding from corresponding chow-fed rats. 106

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Chow HF 0 100 200 300 400Vehicle AM251 ** ** Akcal 0 1 2 3 4 5 6 7 -45 -35 -25 -15 -5 5 BChow-Vehicle Chow-AM251 HF-Vehicle HF-AM251 ** *DayChange in Body Weight (g) Figure 5-3. Change in cumulative caloric intake and body weight in aged rats during 7day daily i.p. administration of AM251 (0.45 mg/rat/day; 0.8 mg/kg/day). Data represent mean s.e.m. of chowor HF-fed and vehicl e-treated (n = 8, closed symbols), chowor HF-fed and AM251-treat ed (n = 7, open symbols). A) Cumulative caloric intake was reduc ed by AM251 administration, regardless of dietary treatment (**P -value < 0.001). B) AM251 treatment induced a loss of body weight in aged rats that was enhanced with prolonged HF feeding. *P-value < 0.001 for the difference wi th diet and AM251 treatment in aged rats. **P-value < 0,001 fo r the difference in change in body weight in both dietary groups. 107

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Table 5-2. Change in body composition and biochemical markers of aged rats during daily i.p. vehicle or AM251 injections. Chow HF Vehicle AM251 Vehicle AM251 Change in Adiposity (g) -4.9 + 1.6 -18.1 + 2.2 -7.2 + 1.7 -25.0 + 1.9 Change in Lean Mass (g) -0.7 + 1.3 -7.6 + 1.3 -0.4 + 1.8 -10.3 + 1.8 Hypo P-STAT3 1.26 0.10 1.87 0.25 2.35 0.24 2.08 0.10 PWAT pACC 0.42 0.06 0.20 0.03 0.22 0.04 0.11 0.02 BAT UCP-1 0.91 0.04 1.29 0.12 1.79 .15 1.78 0.09 NPY mRNA 0.74 0.03 0.91 0.03 0.85 0.03 0.82 .05 POMC mRNA 0.76 0.11 1.27 0.12 1.10 0.09 0.95 0.14 PTP1B mRNA 0.78 0.04 0.81 0.01 0.90 0.10 0.70 0.05 Data represent mean s.e. m. of 4-8 rats per group analyzed by 2-way ANOVA. Adiposity and lean mass were determined prior to and after 7-day vehicle or AM251 daily injections; biomolecular marker prot ein levels, measured by Western analysis, and mRNA levels, measured by RT-PCR, of all young adult, chow-fed & vehicle-treated groups are set to 1.00 and s.e. m. adjusted accordingly. P < 0.05, indicates the difference with AM251 treatment from corresponding vehicle-treated and diet-matched rats. *P < 0.05, indicates the difference with HF feeding from corresponding chow-fed rats. 108

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0 1 2 3 4 5 6 7 8 9 10 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Young HF Aged Chow Aged HF Young Chow Response to AM251 (% Decrease in Body Weight)Response to Leptin (% Decrease in Body Weight) Figure 5-4. Percent change in body weight in response to peripheral leptin infusion versus peripheral AM251 injections in young adult and aged rats with and without HF feeding. Data represent m ean s.e.m. of young adult chow-fed (n = 5, squares), young adult HF-fed (n = 8, triangles), aged chow-fed (n = 7, circles) and aged HF-fed (n = 7, diamonds). P-value = 0.0177 and R 2 = 0.9649. 109

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Young Aged -70 -60 -50 -40 -30 -20 -10 0Chow HF 0.83 mg/kg/day with Introduction of HFD A ** **Grams Young Aged -70 -60 -50 -40 -30 -20 -10 02.78 mg/kg/day with Introduction of HFDChow HF B **Grams Established Obesity Young: 1.2 mg/kg/day Aged: 0.8 mg/kg/day Young Aged -70 -60 -50 -40 -30 -20 -10 0Chow HF C **Grams Figure 5-5. Change in body weight in AM 251-treated rats compar ed to vehicle-treated rats on Day 5 of the respective experiments. A) 0.83 mg/kg AM251 treatment during hyperphagia reduced body weight in young adult HF-fed and aged chowand HF-fed rats. *P-value < 0.05 for the difference in HF-fed compared to chow-fed young adult rats **P-value < 0.001 for the in aged rats versus diet-matched young adult ra ts. B) 2.78 mg/kg AM251 treatment during hyperphagia caused an increase in s ensitivity with HF feeding in aged rats. *P-value < 0.001 for the differenc e with HF feeding in aged rats. **Pvalue < 0.001 for the difference wi th age during HF-feeding. C) 0.45mg/rat/day during prol onged chow or HF feedi ng caused a significant reduction in body weight in aged rats, which was enhanced with HF feeding. *P-value < 0.001 for the difference with HF feeding in aged rats. **P-value < 0.001 for the difference in age on a HF diet. 110

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CHAPTER 6 GENERAL DISCUSSION AND CONCLUSION Obesity is a major health issue, and the ra tes of obesity are continually on the rise in both adults and children ar ound the world. Obese indi viduals often experience a reduced quality of life and are at higher risk for many other diseases, including type 2 diabetes, heart disease, stroke, and certain types of cancer (2). There are many hormones and signaling molecules involved in the modulation of energy homeostasis, but one of the key regulators is leptin. Lept in is produced in adipose tissue and acts in the central nervous system to suppress food intake while increasing energy expenditure (2, 47, 105, 148). Upon its discovery, leptin was believed to be the magic pill for curing obesity, but it was soon apparent that the situation wa s far more complex than researchers first thought. While leptin was e ffective at treating pat ients with congenital leptin deficiencies, it had little impact in obese rodents and humans with normal genetic profiles (109). Circulating leptin levels rise in proportion to adiposity, but this elevated leptin over time becomes unable to proper ly regulate energy homeostasis in obese individuals (18, 75, 97). This phenomenon is termed leptin resistance, and is common in many obese humans and rodents. Leptin signaling opposes the activity of the ECS, primarily through downregulating ECs and/or inhibiting their biosynthesis ( 101, 102, 112, 147). Ac tivation of the CB1 receptor stimulates energy intake and storage. This signaling system is hyperactivated during HF feeding, and data suggest it is chr onically overactivated in obese individuals (38). Studies suggest that in states of leptin resistance, leptin is unable to downregulate EC levels, thus allowing the ECS to become overactivated (77). 111

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The increasing obesity rate in humans is often attributed to the consumption of a HF diet. These diets are readily available, energy dense, and highly palatable. In fact, when rodents are provided acce ss to a HF diet ad libitum, they respond with immediate hyperphagia, which normalizes to pre-HF diet levels over time (174). This normalization is dependent upon leptin receptor activity (139, 174). In this dissertation, the major objective wa s to further understand the role of leptin and the ECS in energy regul ation and the changes associated with both aging and HF feeding. We put forth thr ee major hypotheses during our studies. First, the leptin resistance associated with age-related obes ity results in a prolonged hyperphagia during HF feeding, contributing to exacerbat ed weight gain. Second, the overactive ECS associated with obesity and likely wi th age-related obesity contributes to the exacerbated hyperphagia and weight gain observed in aged-obese rats during HF feeding. Third, long-term HF feeding or aging leads to leptin resistance, therefore it is likely that the leptin negative modulation of CB1 receptor acti vity is blunted or absent in obese rodent models. Thus, we predicted that CB1 re ceptor antagonism would be enhanced in young adult rats with diet-induc ed obesity due to long-term HF feeding and that this exacerbated CB1 receptor antagonism would be further enhanced in long-term HF fed aged rats. The goals of this dissertation were to, first, characterize physiological responses to a HF diet in rats of varying ages; second, to examine the effects of a CB1 receptor antagonist on body weight and caloric intake with or without HF feeding both shortand long-term. Major Finding and Conclusions In Chapter 3, we demonstrat ed that as male F344xBN rats age, they are more susceptible to the detrimental effects of a HF diet. When rodents are provided a highly 112

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palatable caloric dense diet ad libitum, they immediately consume an elevated level of calories that gradually normalizes to control levels. However, leptin resistant animals display impaired normalization of this hy perphagia (57, 135, 175). Because aged obese rats are leptin resistant, I hypothesized that they would fail to rapidly normalize the elevated caloric intake after HF diet exposur e. Indeed, the 30-mont h-old rats, the oldest tested, displayed the most exaggerated hyperphagia, reaching the highest peak intake level and requiring the longest amount of time to normalize. Investigation of this phenomenon at multiple ages proved that both t he peak of caloric intake up on initiation of HF feeding and the time to normalizati on increased with age. Moreover, this hyperphagia was a result specific to HF feeding because all of the ages consumed the same amount on the chow diet prior to initia tion of the HF diet, c onfirming earlier studies (152). This failure to normalize caloric intake after initiation of HF feeding appears to result in greater rate of body weight gain in the aged rats during the first few days of HF feeding. In addition, the rats continued to gain weight even after caloric intake normalization. Together, this suggests that responses to HF feeding are dependent upon the degree of leptin resistance, and the aged rats might have an accompanying decrease in energy expenditure. However, both the young adult and aged HF fed rats responded equally with an increase in UCP1 protein level in BAT. Thus, any changes in energy expenditure do not appear to be related to the thermic effect of food. Research has shown that body weight and physical acti vity are inversely related so we tested voluntary wheel running, one measure of voliti onal activity that involves motivational, exploratory, muscular, age, and body size components (152). Because locomotor 113

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activity tends to decline with both age and obes ity (22, 30, 41), I pr edicted that both age and HF feeding would negatively impact volunt ary wheel running. In Chapter 3, I showed that the aged rats ran significantly less than the young adu lt rats on a chow diet, and HF feeding also suppressed the vol untary wheel running activity in the young adult rats. This is consistent with previous reports that aged rats or rats on a HF diet run significantly less than controls (70, 71) Interestingly, the aging and HF feeding suppressive effect on wheel running do not appear to be independent. For example, the oldest group tested ran the least, but HF feeding had little additional suppressive effect. Collectively, these data suggest that the tendency for inactivity with age may be one contributing factor in age-related obesity. In fact, the i nactivity with HF feeding may accelerate the rate of di et-induced obesity in all ages. Using body composition m easurements, I showed that aging also increases the susceptibility for fat storage with HF feeding. Studies have shown that rats that are naturally growing to maturity have the ability to store energy as both proteins and lipids. However, as the rats age, protein deposition becomes almost nonexistent and, eventually, all energy consumed is stored as fat (137). Supporting this, the data in Chapter 3 demonstrated that only some young adult rats are susceptible to diet-induced weight gain, whereas all aged rats are susc eptible to this negative effect upon initiation of the HF diet. Body composition anal ysis proved that the aged rats gained a disproportionate amount of body fat compared with young adult counterparts during HF feeding. These data indicate that all aged rats, as opposed to only some young adult rats, are prone to develop diet-induced obesity on a HF diet. 114

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We then verified that 3-month-old male F344xBN rats ar e leptin responsive while 30-month-olds are completely leptin resi stant. The young adult rats displayed dosedependent anorectic responses, measured by reductions in both food intake and body weight, to a peripheral leptin infusion, but the aged rats we re completely resistant to leptins anorectic effects. Aged rats hav e reduced numbers of leptin receptors and a parallel decrease in leptin signaling (5, 144). In Chapter 3, I also found increases with age in SOCS-3, a negative regulator of leptin signaling, and PTP1 B, a phosphatase that dephosphorylates activated components in the leptin signaling cascade. These changes, along with evidence of leptin resi stance in the aged animals are likely to impair the native responses to the endogenous l eptin elevation triggered by HF feeding. However, it should be noted that leptin re sponsiveness was only examined in the 3and 30-month-old rats. While they demonstrated leptin resist ance by 30 months of age, we cannot dismiss the possibility that this resi stance may be fully manifested prior to this age. If this is the case, the leptin resi stance may only be one factor in the progressive susceptibility to the adipogenic e ffects of a HF diet with age. In Chapter 4, I set out to prove that dysregulation of the ECS is at least one contributor in the exacer bated hyperphagia observed in HF fed aged rats. Because these aged rats are leptin resist ant, the endogenous leptin may be unable to downregulate ECs. This pr edicts that CB1 receptors ar e overactive and that CB1 antagonist administration will have increased e fficacy in these leptin resistant rats. I first demonstrated that daily i.p. AM 251 administration in both young adult and aged rats was able to reduce HF-diet intake to a greater extent than chow intake. This is consistent with other studies that show ed CB1 antagonist adminis tration preferentially 115

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reduced intake of a sweet diet, in both solid and liquid forms, without affecting chow or water intake (103, 161). However, the la rger dose of AM251 test ed in Chapter 4 was also able to significantly reduce caloric inta ke on the chow diet, albeit to a lesser degree than the HF diet. While other researc hers have demonstrated that AM251 suppresses feeding equally on chow, high-ca rbohydrate, and HF diets in rats, they suggest that data interpretation has a large impact on the c onclusions reached (5, 144). For example, when rats are provided a highly palatable di et ad libitum, they consume more of these diets than of chow, creating a higher basel ine consumption. Thus, if the AM251induced reduction in caloric intake is compar ed to the baseline of the respective diet, the results show a more dramatic anorectic effect in the highly palatable diets compared to normal chow. This aforementioned method of data analysis was em ployed in Chapter 4, confirming that CB1 rec eptor antagonism produced grea ter reductions in the consumption of the highly palat able HF diet when compared to the chow diet. Taken together, these data suggest t hat the endogenous EC activity may act to increase the preference of the palatable f ood (77). One possible expl anation for the results in Chapter 4 is that the lar gest dose of AM251 tested (2.78 mg/kg/day) was able to override the majority of the endogenous orexi genic signals, causing the rodents to consume fewer calories overall. In contrast, the lower dose may only override some of endogenous orexigenic signals, explaining wh y the rats experienced only a minor decrease in caloric intake during low-dose AM251 treatment. Second, the data in Chapter 4 verified that AM251 responsiveness is enhanced in aged rats and further increased with HF feeding. One possibility, although it is untested 116

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in this dissertation, is that this hype r-responsiveness is due to the state of leptin resistance in the aged rats. When the endogenous leptin is unable to downregulate brain EC levels, the ECs may become elev ated and allow the CB1 receptors to become hypersensitive to antagonism. In addition, HF feeding stimulates serum leptin levels. This elevation of leptin levels may induce or exacerbate leptin re sistance. If so, the state of leptin resistance may explain the enhanced AM251 responsiveness with HF feeding (64, 153), rather than the palability of the food. Third, AM251 administration was able to reverse both age-related and dietinduced elevations in adiposity and serum leptin levels. This is consistent with other studies (5, 146), but more experiments are needed to determine if this reversal is a direct effect of AM251 treatment or if it is merely a parallel response. Either way, the lower leptin levels may help to restore leptin responsiveness in these aged rats. Lastly, I showed that, at the dose test ed (0.83 mg/kg/day), AM251 treatment did not alter the preference of the chow and HF diets. When given the choice between the chow and HF diet during daily AM251 adminis tration, the rats both young adult and aged continued to consume the HF diet exclus ively. Very few other researchers have investigated this phenomenon, but our data are consistent with South, et al. and Arnone, et al. who demonstrat ed that CB1 receptor antagoni st treatment reduced the intake of only the highly pal atable HF diet (38, 162). After examining AM251 responsiveness dur ing the HF diet-induced hyperphagic period in Chapter 4, I sought to investigate AM251 responsiveness after long-term chow or HF feeding in both young adult and aged rats in Chapter 5. Obese animals display enhanced responses to CB1 receptor antagonist s compared with lean controls (8, 60, 117

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62, 101). In addition, obesity and consumption of a highly palatable diet raise both central and peripheral EC levels (100). Moreover, anorectic responses to CB1 antagonists are enhanced in lean animals afte r EC tone is increased, which can be done by exposure to a highly palatable diet or acute fasting (38). These data suggest that there is higher EC t one in obese rodents, and predicts that long-term HF feeding will further enhance AM251 responsiven ess in both young adult and aged rats. Indeed, the data in Chapter 5 verify t hat the AM251 responsive was enhanced with long-term HF feeding in young adult and aged rats, as measured by an increase in sensitivity and maximum efficacy. These res ponses were observed with respect to both body weight reduction and the anorectic response, which includes the time course of food consumption as well as the overall re duction in cumulative caloric intake. Moreover, the HF diet-induced increase in AM 251 sensitivity is additive with age, with the greatest degree of respons iveness in the aged HF-fed rats. When compared with the aged rats, the HF diet-induced enhancem ent of AM251 antagonism is modest. The enhanced efficacy with age and HF feedi ng appears to be both related to the duration of the diet and the associated established obesity as well as the presence of leptin resistance. In Chapters 4 and 5, the young adult rats displayed similar responsiveness to AM251 regardless of the duration of feeding. However, the AM251 efficacy in the aged rats was enhanced to a greater degree with long-term HF feeding and established obesity (Chapter 5) when compared to the in troduction of the HF diet (Chapter 4). In fact, the aged, HF-fed rats were less responsive to peripheral leptin infusion and more responsive to AM251 admi nistration when compared with chow-fed aged rats, although the difference in leptin responses did not reach statistical 118

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significance. This suggests that the duratio n of dietary exposure and resultant obesity as well as the development of diet-induced obesity significantly contribute to the enhanced AM251 responsiveness observed in long-term HF-fed rats. Because both age and HF diet-induced obesity are associated with leptin resistance, and there is a strong correlation between enhanced AM251 responsiveness and leptin resistance in young adult and aged chowand HF-fed rats, these data are consistent with the concept that leptin resistance is one cont ributor to enhanced CB1 receptor antagonist efficacy with HF feeding and age (38, 44, 96). Obese rodents lacking normal leptin func tion have higher EC levels than normal controls (38). For example, hypothalamic EC levels are elevated in Zucker rats with defective leptin receptors, db/db mice with no leptin rec eptors, and ob/ob mice with no leptin (38, 112). However, exogenous adminis tration of leptin in ob/ob mice and normal rodents reduces EC levels back to those of l ean control animals. In addition, it is wellknown that EC levels are elevated in animals with diet-induced and genetic obesity, but the status of the CB1 receptor activity is still unclear (65). In fact, some data suggest that receptor levels are downregulated during HF feeding. These data do not support my findings of enhanced AM251 efficacy with HF feeding. Thus, the mechanism behind the increased efficacy with HF f eeding and age remains speculative. Interestingly, all the HF-fed rats display ed elevated UCP1 protein levels in BAT, suggesting that there is a co mponent of BAT thermogenesis that is contributing to energy homeostasis in the aged HF-fed rats t hat is absent in the aged chow-fed rats. Perhaps blockade of this component by the CB1 receptor antagonist contributes to the increased efficacy with age. 119

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In summary, this dissertation com pared young adult and aged for to their responses to chow or HF feeding, peripher al leptin infusion, and daily i.p. CB1 antagonist treatment. When male F344xBN ra ts are provided access to a HF diet ad libitum, they immediately consume more calori es than chow-fed controls. In fact, as the rats age, the peak and days requi red to normalize the elevated caloric intake increases. This hyperphagia in the aged rats is accompanied by a disproportionate gain in fat tissue, and all the aged rats are susceptible to the adipogenic effect s of the HF diet while some of the young adult rats appear obes ity resistant. Afte r both shortand longterm exposure to the HF diet, AM251 tr eatment in these young adult and aged rats preferentially reduced consumpt ion of the highly palatable HF diet, but was apparently unable to alter the palatability or preference of t he diets when the rats were given a choice. We do not believe that the anorec tic effects of AM251 are due to aversion to the drug. However, we did not test this aversion by re introducing the diets subsequent to the AM251 treatment but in absence of the drug administrati on. Moreover, the anorectic effects, measured by body weight and caloric intake redu ction, of the CB1 antagonist were enhanced with both age and HF feeding. However, this enhanced responsiveness cannot be clearly attributed to the presence of leptin resistance so the underlying mechanism remains speculative. Future Directions and Potential Improvements This dissertation was founded on 3 major hy potheses. First, the leptin resistance associated with age-related obesity result s in a prolonged hy perphagia during HF feeding, contributing to exacerbated weight gain. Second, the overactive ECS associated with obesity and likely with age -related obesity contributes to the exacerbated hyperphagia and body weight ga in observed in aged-obese rats during HF 120

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feeding. Third, long-term HF feeding or aging leads to leptin resistance, and it is likely that the leptin negative modulat ion of CB1 receptor activity is blunted or absent in obese rodent models. This predicts that CB1 receptor antagonism will be enhanced in young adult rats with diet-induced obesity due to long-term HF feeding and this exacerbated CB1 receptor antagonism will be further enhanced in long-term HF fed aged rats. In Chapter 3, I confirmed the first hypothesis by demons trating that as the rats age, both the peak and norma lization period of the HF diet-induced hyperphagia increases. This prolonged hyperphagia contributes to an exaggerated body weight gain and, moreover, a disproportionate gain in fa t versus lean mass in the aged, leptin resistant rats. In Chapters 4 & 5, I partially confir med the second and third hypotheses by demonstrating that adminis tration of a CB1 receptor antagonist produces anorectic effects in young adult, leptin responsive and, to a greater extent, in aged, leptin resistant rats. However, more ex periments are needed to prove whether the HF diet-induced hyperphagia is dependent on both leptin and CB1 receptor activity and whether the enhanced responsiveness observed in aged, l eptin resistant rats is dependent upon the state of leptin resistance. In Chapter 3, we observed that only some young adult rats, but all aged rats, are susceptible to the adipogenic effects of a HF diet. However, we were unable to provide a clear underlying mechanism for this phenomenon. Future studies could compare measures of energy expenditure during rest and activity, locomotor activity levels, and muscular strength in rats during HF feeding with increasing age. In Chapter 3, we demonstrated that HF feedi ng and, moreover, aging dec reased voluntary wheel running, but it is unclear if the gain in body weight associated with HF feeding and aging 121

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is a contributor to this decrease in acti vity. Because wheel running may be dependent on muscle strength, we subsequently subjected the aged rats to a grip strength and inclined plane test. There was no statistical significance between dietary groups so limited conclusions could be drawn. Per haps muscle strength and voluntary activity was already maximally diminished in t hese rats. Thus, measures of energy expenditure, locomotor activity levels, and muscular strength with HF feeding at various ages throughout the rats life may provi de important inform ation regarding the exacerbated hyperphagia and body weight gain observed during HF feeding. If energy expenditure decreased with age, it would suggest that t he exacerbated body weight gain during HF feeding with age, especially that which occurs after the hyperphagia normalizes, may be at least partially due to the decrease in energy expenditure. In Chapter 4, we speculat e that dysregulation of the ECS is at least partially responsible for the exacer bated hyperphagia observed in aged rats on a HF diet. Our data demonstrate that daily administration of a CB1 receptor antagonist reduces both the peak and the days required to normalize the HF diet-induced hy perphagia. At both the low and high doses tested, the CB1 re ceptor antagonist reduced HF intake to a greater extent than chow inta ke and prevented HF diet-induced body weight gain in both young adult and aged rats. However, we were unable to prove beyond a doubt that central CB1 receptors are overactivated during this hyperphagia. This could be tested by administration of a CB1 receptor agonist in young adult rats, which would hypothetically increase both the peak and t he days required to normalize the caloric intake. In the aged rats, CB1 agonist treatment may not further exacerbate the HF diet122

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induced hyperphagia, suggesting that the CB1 receptors ar e maximally stimulated in these completely leptin resistant rats. Thus, we administered the CB1 agonist WIN 55, 212-2 by daily i.p. injection using the same experimental protocol as described in Chapter 4 (data not shown). In contrast to our predictions, WIN 55, 212-2 treatment reduced caloric intake in the young adult rats. We chose to use WIN 55,212-2 becau se, at the time, it was one of the most extensively studied commercially available CB 1 receptor agonists, it produces all the pharmacological effects of tetrahydrocanna binol (THC), and it has been successfully substituted for all other cannabinoids in discrim inative stimulus tests (54, 81). However, because it has high affinity for both CB1 and CB2 receptors and has a moderate selectivity in favor of the CB2 receptor, we feared that AM251 wa s activating peripheral CB2 receptors, which are highly associated with the immune syst em. If so, activation of the immune system could cause a discomfort pr ompting the rats to consume less food. To avoid activating peripheral CB2 recept ors, we next adminis tered WIN 55,212-2 directly into the hypothalamus (data not shown). A brain infusion cannula directed at the ventral medial hypothalamus (VMH) or ventral tegmental area (VTA) and infusion pump were surgically implanted in young adult rats. The rats were allowed to rest for one week before WIN 55,212-2 was loaded into the infusion pumps. However, even central infusion of WIN 55,212-2 caused a reduction in caloric intake and body weight t hat was not seen in control rats. There is no agreement in the liter ature regarding the effect of CB1 agonist administration on food intake. While some investigators report CB1 agonist-mediated decreases in food intake or no change at all (73, 104), others report significant increases in food intake after 123

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central or peripheral administration of va rious CB1 agonists (174) In fact, it appears that many factors contribute to apparent controversial results in the literature, including the hunger status of the rodent; the location, time, and method of agonist delivery; and the dose of the agonist. However, future studies could examine CB1-mediated feeding behavior using another agonist. An ideal agonist is one that preferentially binds to the CB1 receptor, is stable at r oom temperature, is more wa ter soluble, and produces the full range of pharmacological effects observed with THC. Another way to investigate the interactions of the l eptin and ECS during HF dietinduced hyperphagia is to examine the rela tionship between maximal leptin signaling and the hyperphagia. Studies in the Scar pace lab have demonstr ated that maximal leptin signaling capacity is blunted in y oung adult rats after only 2 days of HF feeding, suggesting that leptin receptor desensitization occurs rapidly (56). We propose that this desensitization may play an impor tant role in the duration of caloric normalization during HF feeding. Future studies co uld compare the maximal leptin signaling level at different points along the caloric normalization curve in both young adult and aged rats. Increased desensitization may increase both the peak and the days required to normalize the hyperphagia, whereas maximal leptin signaling may be restored by the time caloric intake is fully restored. In addition, this may shed some light on the interactions of the leptin and CB1 signa ling systems during this HF diet-induced hyperphagia. If leptin recept ors are desensitized to a great er extent in the aged rats, the leptin negative modulation of the ECS will be absent or blunted for a longer period of time. Because of this, the CB1 recept ors may be allowed to become even further overactivated, causing t he rats to consume more food for a longer duration. 124

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In order to directly implicate the dysregulation of t he ECS during the HF dietinduced hyperphagic period, future studies c ould also measure CB 1 receptor protein level by Western and/or message level by RT -PCR. However, to date, commercially and personally produced CB1 receptor anti bodies have been variable, unreliable and nonspecific for the CB1 receptor (18). If specific, reliable antibodies were available, an increase in hypothalamic CB1 receptor num ber with age and HF feeding may indicate hyperactivation of the ECS. Alternatively, a decrease in hypothalamic CB1 receptors with age and HF feeding would support our dat a that the same dose of AM251 was able to produce greater anorectic effects in t he aged obese rats. In addition, measurements of EC levels in white adipose tissue would pr ovide an important piece of the puzzle (64). AM251 administration produces body weight reductions long after the food intake response has normalized (81). This suggests that a peripheral activation of CB1 receptors, particularly on adi pose tissue, may be involved. Similarly, future studies could include m easurements of brain EC levels with age and HF feeding. EC levels have been shown to vary in different brain regions, but the changes most related to feeding behaviors are t hose in regions of the brain dealing with feeding, satiety, and reward ( 38, 67). Of particular intere st are those regions that contain both leptin and CB1 re ceptors, such as the hypot halamus and the VTA (140). If EC levels in these regions are elevated wi th both age and HF feeding, both of which are associated with leptin resistance, this would support the hypothesis that leptin in a leptin resistant state is unable to downregulate EC levels. Thus, these EC levels may become elevated, creating hyperactive CB1 receptor activity, and further inducing food intake in these leptin resistant animals. 125

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In Chapter 4, we demonstrat ed that daily i.p. adminis tration of AM251 in young adult and aged rats was unable to alter the pr eference of the chow diet when compared to the HF diet. However, future studi es may examine the effect of AM251 on food intake of two diets closer in palatability. For example, if the rats were given a choice between 60% and 32% HF diets, there may be a decrease and increase in the respective intake of diets with AM251 treat ment. In addition, studies suggest that experience with the diets before the choice is important. Unpublished studies in the Scarpace lab indicate that when rats are conditioned on one diet and then introduced to another, they will initially consume elevat ed amounts of the second diet. Thus, our choice experiments in Chapter 4 barely scratch the surface in this area of research because there are many other diet combinations and experimental designs that can be studied in the future. In Chapter 5, we suggested that the leptin negative modulation of CB 1 receptor activity is impaired with age. We conf irmed that CB1 antagonist administration produced greater anorectic effects, measured by a decrease in caloric intake and an inhibition in HF diet-induced body weight gain, in aged rats compared with young adult rats. In fa ct, this enhanced efficacy was further emphasized with HF feeding, producing the greatest anorectic effects in the aged, HF-fed rats. Moreover we were able to verify that these results were directly correlated to the leptin resistant stat e in these rats. One possible future study to provide further conf irmation is to create leptin resistant young adult rats using leptin gene delivery to induce central leptin overexpression. A previous study in the Scarpace lab show ed that central lept in gene delivery in young adult rats induced a 40% increase in cerebrospinal fluid leptin levels nearly 50 days after the third 126

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ventricle injection (130). Moreover, the leptin -treated young adult rats did not become obese as is typically associated with increas ed leptin levels. After the development of leptin-induced leptin resi stance, these animals disp lay exacerbated hyperphagia and body weight gain, similar to what has been de scribed in this dissertation in aged rats, when exposed to a HF diet I predict that daily i.p. CB1 antagonist treatment in these rodents will be highly efficacious and produce dramatic anorectic effects, like those described here in aged, leptin resistant rats. Therefore, this animal model may prove to be an ideal situation for study ing CB1 antagonist responsiveness in leptin resistant, but not obese, animals. If hypothalamic EC and CB1 receptor levels are elevated in these rats, it would provide a direct connection between leptin resistanc e and the overactive ECS. Conclusions Obesity is a serious, chronic disease that increases the risk of developing various secondary diseases including type 2 diabetes, uterine cancer, gallbladder disease, osteoarthritis, stroke, hyper tension, coronary heart diseas e, breast cancer, and colon cancer. Caloric dense, highly palatable HF diets are often implicated as a primary contributor to the prevalence of obesity today. T he leptin and endocannabinoid signaling systems induce opposing effects on feeding behavior and fa t storage, and the dysregulation of these syst ems may be important in age-related and diet-induced obesity. In the research described in this dissert ation, we demonstrated that all aged rats, while only some young adult rats, are susceptible to the adipogenic effects of a HF diet. These aged, leptin resistant rats, displa y increased and prolonged caloric consumption during HF feeding when compared to young adul t controls. We revealed that the CB1 127

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antagonist-mediated anorectic effects are greater in these aged rats and further enhanced with both shortand long-term HF f eeding. These results contributed to the understanding of the intera ctions between the leptin and endocannabinoid signaling systems as well as how these interactions change with both age and HF-feeding. I hope these findings will contribute to t he prevention and treat ment of obesity. 128

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BIOGRAPHICAL SKETCH Melanie Kae Judge was born in Sarasota, Florida in 1983 to Robert & Susette Mitchell. Four years later, the Mitchells were expecting again. They were crazy enough to give her the opportunity to name her new baby sister, already k nowing that Melanie had a female doll which she called Tim. T hankfully, one of Melanies best friends at the time had a unisex name so Robin Lynn Mi tchell soon became a shining star in the Mitchell family. Robin, being blessed with many talents, dabbled in horseback riding, playing the violin, gymnastics, dance, oil painting, and other various forms of art. On the other hand, Melanie excelled in scienc e and math from an early age. Throughout elementary and middle school, she won first place in many school science fairs and even won first place in the Martin County Sc ience Fair in second grade. Melanie used sports (soccer, basketball, softball, track & field, swimming) as a means of staying in shape, meeting new people, and keeping a competitive edge. She attended the prestigious Florida State University and was awarded many leadership and community service honors, including induction into the Seminole Torchbearers, Omicron Delta Kappa, and the National Society of Collegiate Scholars. Mel anie received a Bachelor of Science degree in biochemistry in 2005 with the hopes of joining a Federal Bureau of Investigation forensic science team. W hen these plans changed ra ther abruptly, she enrolled in the Interdisciplinary Program in Biomedical Research at the University of Florida in 2005. She has spent the last several years working with Dr. Philip J. Scarpace studying the molecular basis of obes ity. In the future Melanie plans to continue her career in obesity research while raising at least 3 children and continuing to do a lot of baking.