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The metabolic effects of leptin therapy in a mouse model of type 1 diabetes. Denroche, Heather Courtney 2014

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THE METABOLIC EFFECTS OF LEPTIN THERAPY IN A MOUSE MODEL OF TYPE 1 DIABETES  by  HEATHER COURTNEY DENROCHE  B.Sc., The University of British Columbia, 2008    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Cell and Developmental Biology)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    April 2014  © Heather Courtney Denroche, 2014  ii ABSTRACT Type 1 diabetes is a life-long disease, with devastating consequences and no cure. While the hormone insulin has been the only effective treatment to date for type 1 diabetes, emerging evidence has revealed that the fat-derived hormone leptin can also exert profound glucose lowering effects, and reduce mortality in type 1 diabetes. This has generated interest in the therapeutic potential of leptin as an anti-diabetic treatment, and propelled leptin into clinical trials for type 1 diabetes. The fact that leptin monotherapy (without insulin administration) can so potently lower blood glucose in insulin-deficient diabetes is surprising, given that for almost a century, insulin has been assumed to be the only hormone that can lower blood glucose in type 1 diabetes. The overarching goal of this thesis was to perform pre-clinical studies to elucidate the mechanism of the anti-diabetic effects of leptin in type 1 diabetes. To this end, we thoroughly assessed the changes in metabolic and energy homeostasis that occur in a mouse model of type 1 diabetes receiving leptin therapy. The roles of hepatic and neuronal leptin receptor signalling in the anti-diabetic action of leptin were also investigated, through tissue specific disruption of leptin signalling using the Cre-lox method. In addition, we assessed whether leptin therapy can serve as an adjuvant to islet transplantation therapy in type 1 diabetes. This thesis revealed that leptin therapy lowers blood glucose in a mouse model of type 1 diabetes, correlating with decreased hepatic glucose production and enhanced insulin sensitivity. The anti-diabetic action of leptin is not blunted in mice with disrupted neuronal leptin signalling outside of the arcuate and ventromedial hypothalamus, or in mice with disrupted hepatic leptin signalling, suggesting that leptin acts through alternate or redundant pathways to lower blood glucose. Finally, low-dose leptin administration dramatically enhanced the ability of islet transplants to restore euglycemia, suggesting that leptin and islet co-therapy could be a successful therapeutic strategy for type 1 diabetes. Collectively the findings in this thesis contribute insight into the mechanism of action, and the therapeutic potential of leptin as a treatment for type 1 diabetes.  iii PREFACE All studies in this thesis were conceived and designed by HC Denroche and TJ Kieffer. Portions of Chapter 1 are from the following published review: Denroche HC, Huynh FK, Kieffer TJ. (2012) The role of leptin in glucose homeostasis. Journal of Diabetes Investigation 3(2):115-129. Most studies in Chapter 3 are published in the following article: Denroche HC, Levi J, Wideman RD, Sequeira RM, Huynh FK, Covey SD, Kieffer TJ. (2011) Leptin therapy reverses hyperglycemia in mice with streptozotocin-induced diabetes, independent of hepatic leptin signalling. Diabetes 60(5):1414-23. This publication was written by HC Denroche with editing by SD Covey and TJ Kieffer. Studies in Chapter 3 were performed by HC Denroche, with assistance from J Levi, RD Wideman, FK Huynh, and SD Covey. J Levi, RD Wideman and RM Sequeira, performed the experiment in Figure 4. Studies described in Chapter 4 were performed by HC Denroche with assistance from M Philippe, WL Quong, MM Glavas, JK Fox, H Streicek, S O’Dwyer, and MJ Kwon. Metabolic cage measurements and body composition measurements in Chapter 4 and 6 were performed in collaboration with the laboratory of SM Clee. S Karunakaran oversaw the running of metabolic caging equipment. SC Chua kindly provided the Leprflox/flox mice, which were used in Chapters 4 and 5. Studies pertaining to Leprflox/flox Albcre mice in Chapter 5 were published in the following article: Denroche HC, Levi J, Wideman RD, Sequeira RM, Huynh FK, Covey SD, Kieffer TJ. (2011) Leptin therapy reverses hyperglycemia in mice with streptozotocin-induced diabetes, independent of hepatic leptin signalling. Diabetes 60(5):1414-23. All other studies in Chapter 5 are unpublished. Studies described in Chapter 5 were performed by HC Denroche with assistance from WL Quong, M Philippe, JK Fox, E Tudurí, and MM Glavas. Studies described in Chapter 6 were performed by HC Denroche with assistance from WL Quong, MJ Kwon, JK Fox, and UH Neumann. Measurements of short CoA esters were performed in collaboration with the laboratory of RW Brownsey by JE Kulpa. HC Denroche collected and provided the liver tissues for these measurements. Chapter 7 is published in the following article: Denroche HC, Quong WL, Bruin JE, Tudurí E, Asadi A, Glavas MM, Fox JK, Kieffer TJ. (2013) Diabetes 62(8):2738-46. Studies in Chapter 7 were performed by HC Denroche with assistance from WL Quong. E Tudurí assisted with islet isolations and JE Bruin and JK Fox assisted with islet transplantation surgery. A Asadi performed quantitative analysis of immunofluorescent area from pancreata collected by HC Denroche and WL Quong. The publication was written by HC Denroche with editing by WL Quong and TJ Kieffer.  This thesis was written by HC Denroche with editing provided by WL Quong, WT Gibson, SM Clee, JD Johnson, CB Verchere, SD Covey and TJ Kieffer. Animal studies described in this thesis were approved by the University of British Columbia Animal Care Committee (Certificate # A06-0105, A10-0275, and A10-0059).  iv TABLE OF CONTENTS Abstract ....................................................................................................................................... ii Preface........................................................................................................................................ iii Table of Contents ...................................................................................................................... iv List of Tables.............................................................................................................................. vi List of Figures ........................................................................................................................... vii List of Symbols and Abbreviations ........................................................................................... x Acknowledgements ................................................................................................................. xiv Dedication ................................................................................................................................ xvi CHAPTER  1: INTRODUCTION ........................................................................................................... 1 1.1 Diabetes Mellitus ............................................................................................................................ 1 1.2 The Hormone Leptin ...................................................................................................................... 4 1.3 Leptin Therapy for Type 1 Diabetes............................................................................................. 19 1.4 Thesis Investigation ..................................................................................................................... 24 CHAPTER  2: MATERIALS AND METHODS ........................................................................................ 26 2.1 Animals ........................................................................................................................................ 26 2.2 Blood and Plasma Analytes ......................................................................................................... 31 2.3 Chemical Manipulations ............................................................................................................... 33 2.4 Surgical Manipulations ................................................................................................................. 33 2.5 Body Composition ........................................................................................................................ 36 2.6 Indirect Calorimetry and Metabolic Cages ................................................................................... 36 2.7 In Vivo Assays.............................................................................................................................. 37 2.8 Liver Metabolites .......................................................................................................................... 39 2.9 Gastric Distension ........................................................................................................................ 40 2.10 PCR .............................................................................................................................................. 40 2.11 Histology ...................................................................................................................................... 42 2.12 Data Analysis ............................................................................................................................... 45 CHAPTER  3: LEPTIN THERAPY REVERSES METABOLIC DISTURBANCES IN STZ-DIABETIC MICE ........... 46 3.1 Introduction .................................................................................................................................. 46 3.2 Results ......................................................................................................................................... 47 3.3 Discussion .................................................................................................................................... 55 CHAPTER  4: GENERATION AND CHARACTERIZATION OF MICE WITH DISRUPTED NEURONAL LEPTIN SIGNALLING ................................................................................................................................. 59 4.1 Introduction .................................................................................................................................. 59  v 4.2 Results ......................................................................................................................................... 60 4.3 Discussion .................................................................................................................................... 81 CHAPTER  5: THE ROLE OF NEURONAL AND HEPATIC LEPTIN SIGNALLING IN LEPTIN-MEDIATED GLUCOSE LOWERING IN DIABETES ................................................................................................. 84 5.1 Introduction .................................................................................................................................. 84 5.2 Results ......................................................................................................................................... 86 5.3 Discussion .................................................................................................................................... 99 CHAPTER  6: LEPTIN DIMINISHES SUBSTRATES FOR HEPATIC GLUCOSE PRODUCTION IN DIABETIC MICE ................................................................................................................................................. 103 6.1 Introduction ................................................................................................................................ 103 6.2 Results ....................................................................................................................................... 104 6.3 Discussion .................................................................................................................................. 120 CHAPTER  7: THE THERAPEUTIC POTENTIAL OF LEPTIN CO-THERAPY AS AN ADJUNCT TO ISLET TRANSPLANTATION ..................................................................................................................... 123 7.1 Introduction ................................................................................................................................ 123 7.2 Results ....................................................................................................................................... 124 7.3 Discussion .................................................................................................................................. 136 CHAPTER  8: CONCLUSIONS AND FUTURE DIRECTIONS ................................................................. 139 8.1 Conclusions ................................................................................................................................ 139 8.2 Future Directions ........................................................................................................................ 148 Bibliography ............................................................................................................................ 152     vi LIST OF TABLES  Table 1. Direct actions of leptin on peripheral Lepr-b expressing tissues. ............................. 15 Table 2. Composition of 45% and 10% fat diets. .................................................................. 30 Table 3. Primer sequences for qPCR ................................................................................... 42 Table 4. Plasma analytes in female Leprflox/flox Syncre mice .................................................. 70   vii LIST OF FIGURES Figure 1. Schematic of Cre-induced recombination of the Leprflox allele. ................................... 27 Figure 2. Generation of Leprflox/flox Syncre mice.. ....................................................................... 29 Figure 3. Leptin therapy reverses hyperglycemia and hyperketonemia in STZ-diabetic mice. ... 48 Figure 4. The effect of leptin therapy on STZ-induced hyperglycemia does not persist beyond leptin therapy cessation. ............................................................................................................ 49 Figure 5. Leptin therapy does not acutely lower blood glucose in STZ-diabetic mice.. .............. 50 Figure 6. Leptin improves post-prandial glucose metabolism in STZ-diabetic mice. .................. 51 Figure 7. Leptin reduces plasma lipids in STZ-diabetic mice.. ................................................... 52 Figure 8. Leptin reduces plasma glucagon and growth hormone levels in STZ-diabetic mice.. . 53 Figure 9. Leptin enhances insulin action in STZ-diabetic mice. ................................................. 54 Figure 10. Glucose uptake in the soleus muscle cannot account for the anti-diabetic effect of leptin therapy. ............................................................................................................................ 55 Figure 11. The Syncre transgene is active throughout the brain. ............................................... 61 Figure 12. The Syncre transgene is not active in pancreatic islets. ........................................... 61 Figure 13. Leprflox/flox Syncre mice have attenuated Lepr-b expression in the CNS. ................... 62 Figure 14. Functional leptin signalling is disrupted in the DMH, LHA and PMV of Leprflox/flox Syncre mice.. ............................................................................................................................. 64 Figure 15. Male and female Leprflox/flox Syncre mice have normal body weight and fasting blood glucose. ..................................................................................................................................... 65 Figure 16. Age-related body weight variability of Leprflox/flox Syncre mice is independent of Syncre genotype. ................................................................................................................................... 66 Figure 17. Leprflox/flox Syncre mice do not have altered food intake or energy expenditure.  ....... 68 Figure 18. Leprflox/flox Syncre mice have decreased lipid oxidation. ............................................. 68 Figure 19. Leprflox/flox Syncre male mice are hyperinsulinemic and hyperleptinemic. .................. 69 Figure 20. Leprflox/flox Syncre male mice do not have significantly altered fasting lipids. ............. 69 Figure 21. Leprflox/flox Syncre male mice have elevated glucose-stimulated insulin levels. .......... 71 Figure 22. The Syncre transgene has no effect on basal or glucose-stimulated plasma insulin levels.. ....................................................................................................................................... 72  viii Figure 23. Pancreatic insulin and glucagon postive area are unaltered in Leprflox/flox Syncre mice. .................................................................................................................................................. 72 Figure 24. Leprflox/flox Syncre male mice have an increased counter-regulatory response. ......... 74 Figure 25. Leprflox/flox Syncre mice are protected from weight gain on an HFD. .......................... 75 Figure 26. Leprflox/flox Syncre mice are protected from HFD-induced glucose intolerance. .......... 76 Figure 27. Leprflox/flox Syncre mice gain similar relative fat and lean mass to Leprflox/flox mice on HFD. .......................................................................................................................................... 77 Figure 28. Leprflox/flox Syncre mice are protected from HFD-induced weight gain (cohort 2). ...... 78 Figure 29. Leprflox/flox Syncre mice do not have significantly altered energy homeostasis on HFD. .................................................................................................................................................. 79 Figure 30. Leprflox/flox Syncre mice do not develop HFD-induced hyperinsulinemia. ................... 80 Figure 31. Leprflox/flox Syncre mice do not develop HFD-induced hyperinsulinemia (cohort 2). ... 81 Figure 32. Attenuated neuronal leptin receptor signalling does not block therapeutic leptin action in STZ-diabetic mice. ................................................................................................................. 87 Figure 33. Leptin responsivity in STZ-diabetic mice negatively correlates with pre-STZ leptin levels. ........................................................................................................................................ 88 Figure 34. Subdiaphragmatic vagotomy does not attenuate leptin action in STZ-diabetic mice. 90 Figure 35. Validation of subdiaphragmatic vagotomy. ............................................................... 91 Figure 36. Injection of 6OHDA to chemically sympathectomize mice does not attenuate therapeutic leptin action in STZ-diabetes. .................................................................................. 93 Figure 37. Verification of reduced sympathetic neurons in 6OHDA injected mice. ..................... 94 Figure 38. The long form of the leptin receptor is truncated in livers of Leprflox/flox Albcre mice. .. 95 Figure 39. Leptin reverses STZ-induced hyperglycemia in Leprflox/flox Albcre mice. .................... 97 Figure 40. Leptin therapy enhances insulin sensitivity in Leprflox/flox Albcre mice. ....................... 99 Figure 41. Leptin therapy reverses hyperglycemia and the symptoms of insulin-deficient diabetes. .................................................................................................................................. 106 Figure 42. Leptin therapy reverses the symptoms and energy disturbances of STZ-induced diabetes.. ................................................................................................................................. 107 Figure 43. Leptin treated STZ-diabetic mice are sensitive to prolonged fasting.. ..................... 109  ix Figure 44. Leptin therapy depletes hepatic energy substrates, but does not inhibit gluconeogenesis. ..................................................................................................................... 111 Figure 45. Gluconeogenic substrate availability is altered during prolonged fasting in leptin treated mice. ............................................................................................................................ 114 Figure 46. Diminished plasma glycerol and hepatic glycogen coincide with amelioration of hyperglycemia by leptin. .......................................................................................................... 116 Figure 47. Leptin therapy reduces fasting plasma lipids with time. .......................................... 117 Figure 48. Glycerol injection can raise blood glucose in STZ-leptin treated mice. ................... 118 Figure 49. The effects of leptin in STZ-diabetic mice are consistent with the activation of an energy sink.. ............................................................................................................................ 119 Figure 50. Leptin reverses STZ-diabetes in a dose-dependent manner. ................................. 125 Figure 51. Leptin improves glucose tolerance in STZ-diabetic mice in a dose dependent manner. ................................................................................................................................................ 127 Figure 52. Leptin administration enhances the efficacy of islet transplantation for treatment of STZ-diabetes. .......................................................................................................................... 129 Figure 53. Leptin co-therapy does not alter circulating insulin levels or β-cell recovery. .......... 133 Figure 54. Leptin and islet co-therapy improves glucose tolerance. ........................................ 134 Figure 55. Low dose leptin administration normalizes lipid levels in STZ-diabetic mice. .......... 136 Figure 56. Putative targets and neural circuits that mediate leptin-induced glucose lowering in type 1 diabetes. ....................................................................................................................... 141 Figure 57. Leptin therapy lowers glycemia in type 1 diabetes by depleting glycerol and glycogen for hepatic glucose production. ................................................................................................ 145     x LIST OF SYMBOLS AND ABBREVIATIONS 6OHDA 6-hydroxydopamine AAV Adeno-associated virus AgRP agouti-related peptide AMPK 5’ adenosine monophosphate (AMP)-activated protein kinase ANCOVA analysis of covariance ANOVA analysis of variance AQP aquaglyceroporin (protein) Aqp9 aquaglyceroporin 9 (gene/transcript) ARH arcuate hypothalamic nucleus ATP adenosine triphosphate AUC area under the curve BAT brown adipose tissue BB Bio Breeding CamKII calcium/calmodulin protein kinase II CCK cholecystokinin CDM Centre for Disease Modelling cDNA complementary deoxyribonucleic acid CNS central nervous system CoA coenzyme A Cre Cre recombinase CSF cerebrospinal fluid DMH dorsomedial hypothalamic nucleus DNA deoxyribonucleic acid  xi EDTA ethylenediaminetetraacetic acid EGFP enhanced green fluorescent protein ERK extracellular signal-regulated kinase GABA gamma-aminobutyric acid Glut2 glucose transporter 2 Glut4 glucose transporter 4 HbA1c hemoglobin A1c HBSS Hank’s balanced salt solution HEPES hydroxyethyl piperazineethanesulfonic acid HFD high fat diet HMG CoA 3-hydroxy-3-methylglutaryl coenzyme A HPLC high performance liquid chromatography Hprt hypoxanthine phosphoribosyltransferase HSL hormone sensitive lipase ICV intracerebroventricular IGF1 insulin-like growth factor 1 IGFBP2 insulin-like growth factor 1 binding protein 2 IL-2 interleukin-2 i.p. intraperitoneal i.p.GTT intraperitoneal glucose tolerance test ITT insulin tolerance test JAK janus kinase Lep leptin gene Lepr leptin receptor (Lepr is the leptin receptor gene) LFD low fat diet  xii LH leutenizing hormone LHA lateral hypothalamic area mTOR mammalian target of rapamycin NHPP National Hormone and Peptide Program NOD non-obese diabetic NPY neuropeptide Y NTS nucleus of the solitary tract NZO New Zealand obese OGTT oral glucose tolerance test PBS phosphate buffered saline PCR polymerase chain reaction Pdx1 pancreatic and duodenal homeobox-1 Pepck phosphoenolpyruvate carboxykinase Pgk1 phosphoglycerate kinase PI3K phosphoinositide 3-kinase PMV ventral premammillary nucleus POMC proopiomelanocortin Ppia peptidylprolyl isomerase A P-STAT3 phosphorylated signal transducer and activator of transcription 3 PTP1B protein tyrosine phosphatase 1B qPCR quantitative polymerase chain reaction RER respiratory exchange ratio RIP rat insulin promoter RNA ribonucleic acid RT-PCR reverse transcription-polymerase chain reaction  xiii RT-qPCR reverse transcription-quantitative polymerase chain reaction SEM standard error of the mean SF1 steroidogenic factor 1 SH2 Src Homology 2 SOCS3 suppressor of cytokine signalling 3 STAT signal transducer and activator of transcription STZ streptozotocin TH tyrosine hydroxylase UCP1 uncoupling protein 1 VMH ventromedial hypothalamic nucleus WAT white adipose tissue    xiv ACKNOWLEDGEMENTS  My doctoral training has been one of the most challenging and most rewarding experiences of my life. This experience was shaped by the many talented people I have been fortunate to work and interact with throughout my training, all of whom I owe gratitude to for contributing to the researcher, and the person I am today.  First and foremost I want to thank my PhD supervisor, Dr. Timothy Kieffer. Every graduate student should be lucky enough to train under a supervisor such as Tim, who is both a brilliant scientist and mentor. I am deeply grateful to him for his time, patience, support, for providing me with the perfect balance of guidance and freedom in my research project, and for pushing me to my full potential.  I also want to thank the other mentors I have had along the way. I am grateful to Dr. George Mackie, who provided me with the encouragement and training I needed as an undergraduate to start along this path; it was in his lab I first realized my passion for research. I want to thank to Dr. Scott Covey, who introduced me to the Kieffer lab, and taught me many of the essentials of good science. I also want to thank to Dr. Susanne Clee, Dr. William Gibson, Dr. Jim Johnson, and Dr. Bruce Verchere; their support, mentorship, and advice both in and outside of committee meetings have been truly invaluable to me, and I am extremely fortunate to have had their input along the way.  Working with a group of talented and supportive people has made my experience in the Kieffer lab unforgettable, and for that I thank all members of the Kieffer lab, both past and present. Our times together both in, and outside of the lab got me through grad school, and I will be forever grateful for the friendships that have formed from our time together. I want to thank the post-docs and fellow grad students for their comradery, advice and support: Blair Gage, Jasna Levi, Dr. Frank Huynh, Dr. Gary Yang, Mike Juan, Cathy Merchant, Irene Yu, Anna D’souza, Ursula Neumann, Chiara Toselli, Michelle Kwon, Dr. Rhonda Wideman, Dr. Michael Riedel, Dr. Young Min Cho, Dr. Majid Mojibian, Dr. Jenny Bruin, Dr. Maria Glavas, Dr. Suheda Erener, Dr. Sandra Pereira and Dr. Eva Tudurí. I particularly am grateful to Eva, my long term office mate, whose friendship got me through the ups and downs of grad student life. I want to thank Ali Asadi, for his time spent teaching me the ins and outs of immunofluorescence. I thank Dr. Rob Baker for designing primers for me, and for teaching me some of his elegant tricks for cloning and PCR. I owe a debt of gratitude to Travis Webber, who keeps everything organized, and for his countless hours of genotyping. I owe special thanks to Shannon O’Dwyer and Jessica Fox, who spent many long days helping me with surgeries and in vivo experiments. I owe special thanks to the talented students of whom I had the pleasure of co-mentoring with Tim. Whitney Quong worked tirelessly, and her dedication and input were critical to many of the studies in this  xv thesis. I will always be grateful to Michelle Kwon, Marion Philippe, and Heidi Streicek for their hard work, and contribution to the data in this thesis. I also thank the Kieffer lab members who contributed to both the published and unpublished work in this thesis. In addition, I want to sincerely thank our collaborators who offered their time and expertise to perform experiments and provide critical input to the data in this thesis: Dr. Roger Brownsey, Dr. Jerzey Kulpa, Dr. Susanne Clee, and Shuba Karunakaran. I also want to thank the staff at the CDM whose hard work was critical to the success of my studies, in particular: Teresa Calla, Brian Ryomoto, Naomi Winckler, and Dr. Shelly McErlane.  Finally, I want to thank my family and friends for their love and support. Mom and Dad, you are the reason I have been able to do this. Through example you taught me to work hard, to pursue my dreams, and to give something back to the world. Rachel and Eryn, you have always brought me love and laughter, especially in times when I need it most. Mark, you have supported me all the way through grad school, never complaining about the endless hours of work and the late nights and weekends spent in the lab, listening to me and encouraging me, and giving me strength and support. I am truly lucky to have such a supportive, loving family.    xvi  DEDICATION      For my parents: They have been my strength from the start,  and have never stopped believing in me.  And in loving memory of my nan, Joan Denroche Carter, who is my inspiration. 1 CHAPTER  1: INTRODUCTION 1.1 DIABETES MELLITUS Background Diabetes mellitus (herein referred to as diabetes) is a debilitating disease characterized by elevated blood glucose levels resulting from insulin insufficiency. An estimated 382 million people globally have diabetes, and this number is expected to rise to 592 million by the year 2035 (1). In Canada alone, 2.6 million Canadians aged 20-79 are estimated to have diabetes, and in 2013, deaths totaling 17,239 in this age range were attributed to diabetes (1). The most prevalent form of diabetes is type 2 diabetes, accounting for an estimated 85-95% of global cases. The second most common form of diabetes is type 1 diabetes, accounting for 5-10% of cases, with an incidence that rises by 3% each year (2). Type 1 diabetes is a particular concern for Canadians, with 300,000 people living with type 1 diabetes (3). As a country, Canada has the sixth highest incidence of type 1 diabetes in children under 14 years of age (1,4). Although current treatments have improved the quality and length of life for patients, these treatments are inadequate. People with diabetes are hospitalized 4 times more often with heart failure, 6 times more often with chronic kidney disease and 19 times more often for limb amputation than individuals without diabetes (3). Moreover, mortality rates are 2-6 times higher in people with diabetes (3),  life expectancy is decreased by as much as 15 years (5), and more than half of the people who die from diabetes are under the age of 60 (1). In addition to the devastating human cost of diabetes, the economic cost is formidable. In 2013, an estimated $564 billion (CAD) was spent globally on health expenditure related to diabetes (10.8% of global health expenditure), and in Canada, the average economic cost per person with diabetes is approximately $6300 per annum (CAD) (1). If treatments are not improved or a cure is not found, the rising prevalence of diabetes will have disastrous consequences for human life, health care systems, and the economy. The precise causes of diabetes are unknown, and both forms have complex aetiologies. Type 1 diabetes results from cellular-mediated autoimmune destruction of insulin-producing pancreatic β-cells, resulting in extreme insulin insufficiency. Type 1 diabetes is believed to initiate in genetically predisposed individuals following the onset of an environmental trigger, and is marked by the appearance of circulating islet auto-antibodies (6). T-cell mediated autoimmune attack on β-cells results in a progressive decline in insulin  2 secretion, and clinical onset of diabetes is proposed to occur when 80-90% of β-cells have been destroyed (6). The resulting insulin insufficiency is so severe, that patients immediately require insulin therapy, and must remain on insulin therapy for their entire lives. Type 2 diabetes occurs when impaired insulin secretion fails to overcome insulin resistance. It is debated whether impaired insulin secretion or insulin resistance is the primary defect, but both are present at diagnosis (7,8). Type 2 diabetes has a strong genetic component and is associated with obesity. Approximately 90% of type 2 diabetic patients are either overweight or obese (BMI=25-29.9 or >30 respectively) (9), and the rising prevalence of type 2 diabetes correlates with the rising rates of obesity. Treatments for type 2 diabetes include several anti-diabetic agents, injectable insulin, and improved diet and exercise habits to reduce positive energy balance. Insulin and metabolism  In response to elevated blood glucose levels, such as those incurred from a meal, insulin is secreted into the circulation from the pancreatic β-cells and acts on multiple tissues to promote glucose utilization, and the storage of glucose as glycogen or triglyceride (10). Insulin stimulates glucose uptake and utilization by tissues including skeletal muscle and adipose, decreases glucose production by the liver through the inhibition of gluconeogenesis, and stimulates glycogen synthesis in skeletal muscle and liver. Through these actions, insulin lowers blood glucose levels, restoring euglycemia after a meal. Opposing the glucose lowering action of insulin, the hormone glucagon, secreted from pancreatic α-cells in response to low blood glucose, potently elevates blood glucose by stimulating hepatic glucose production from gluconeogenesis and glycogenolysis. Glucagon secretion is dysregulated in diabetes, resulting in hyperglucagonemia (11). Thus in diabetes, hyperglycemia results from diminished insulin-stimulated glucose uptake, and heightened hepatic glucose production due to elevated signals from glucagon relative to insulin. When blood glucose levels rise above the renal threshold for glucose, the concentration of glucose in the glomerular filtrate exceeds the capacity for glucose reabsorption in the proximal tubule of the kidney resulting in glucose spillover into the urine, termed glycosuria. Several symptoms of diabetes are directly due to glycosuria, including frequent urination, dehydration, and increased thirst. While stimulating the utilization of glucose for fuel, insulin promotes the storage of excess energy as lipid, and promotes anabolic processes in lipid and protein metabolism. The action of insulin on lipid metabolism includes the stimulation of lipid synthesis and  3 storage of lipids as triglyceride, and the inhibition of lipolysis and lipid oxidation (10). In uncontrolled type 1 diabetes, the loss of insulin action in white adipose tissue (WAT) results in unmitigated lipolysis, diminishing adipose tissue stores, and increasing circulating free fatty acids (12). Insulin stimulates the catabolism of triglyceride rich lipoproteins, and thus in uncontrolled diabetes, hypertriglyceridemia results (13). With a diminished signal for glucose uptake and utilization, lipid oxidation increases, and circulating ketone bodies are also dramatically upregulated in uncontrolled type 1 diabetes as a result of substantial hepatic lipid oxidation (14), which can lead to the deadly syndrome diabetic ketoacidosis. The loss of insulin-induced lipogenesis and lipid storage, along with protein catabolism contributes to reduced fat and lean mass, and thereby rapid weight loss. In type 2 diabetes, while glucose metabolic pathways become resistant to the action of insulin, there appears to be a bifurcation in insulin signalling such that some pathways of insulin-stimulated lipid metabolism are enhanced (15-17). Circulating free fatty acids (18) and triglycerides (19) are elevated, but ectopic lipid accumulation is also enhanced due to increased lipogenesis (15,17,20). Ketoacidosis is less common than in type 1 diabetes, but can occur during illness (21).  Insulin therapy The Nobel prize winning discovery of insulin published in 1922, changed type 1 diabetes from an acutely fatal disease, into a life-long manageable disease (22). Despite this significant advance, insulin therapy is not a cure, but rather a treatment for diabetes. As stated by Frederick Banting, in his Nobel Lecture for the discovery of insulin, “[Insulin] enables the diabetic to burn sufficient carbohydrates, so that proteins and fats may be added to the diet in sufficient quantities to provide energy for the economic burdens of life” (23). As a treatment, insulin injection therapy has several limitations. Accurate insulin dosing is extremely difficult, and patients with type 1 diabetes have frequent episodes of hyperglycemia which lead to long term complications, and hypoglycemia which is immediately life-threatening. Insulin does not prevent the long term complications of hyperglycemia, and the risk for major complications is substantial (47% for retinopathy, 17% for nephropathy, and 14% for cardiovascular disease) (24). Despite these clear limitations, insulin has been assumed to be the only hormone that can lower blood glucose in patients with type 1 diabetes, and insulin therapy (or insulin replacement) is the only effective treatment to date for this disease.  4 1.2 THE HORMONE LEPTIN Leptin and the leptin receptor Several decades ago, an inheritable obese phenotype spontaneously arose in two separate inbred mouse strains in Jackson laboratories; the strains were named obese (ob) and diabetes (db). Through cross circulation experiments between these two strains, Coleman postulated that ob/ob mice lacked a circulating satiety factor, while db/db mice lacked a functional response to this factor (25). The identity of this satiety factor was finally uncovered in 1994, when the obese gene was identified through positional cloning (26); the protein product had weight reducing effects when administered to ob/ob and wildtype mice, and thus was named leptin, after the Greek word Leptos, meaning thin (27). The ob mutation (herein referred to as Lepob) is a single missense mutation in the Leptin gene that produces an early stop codon at position 105, resulting in the production of a truncated protein product (26). Consequently, Lepob/ob mice have undetectable circulating leptin levels, and exogenous leptin administration effectively reduces their obesity (27,28). Several rare homozygous loss-of function mutations in Leptin (29-34), one of which is identical to the mouse Lepob mutation (29), have been identified in morbidly obese humans, resulting in congenital leptin deficiency. In addition to obesity, both mice and humans with congenital leptin deficiency have a plethora of neuroendocrine, reproductive and immune dysfunctions. Similar to Lepob/ob mice, leptin administration in humans with congenital leptin deficiency is effective in inducing weight loss and correcting many of the neuroendocrine disturbances in these patients (29,31-38). Leptin is expressed in many tissues, but white adipose tissue (WAT) is likely the major source of circulating leptin. This is supported by several lines of evidence: leptin is expressed in white adipocytes (27), and circulates in proportion to body fat mass (39,40); humans and rodents with general lipodystrophy have markedly reduced circulating leptin levels (41-43); and transplantation of white adipose tissue into Lepob/ob mice, or mice with congenital lipodystrophy, raises circulating leptin levels and mimics the effects of leptin replacement in these animals (41,44). Leptin is secreted from WAT largely through the constitutive pathway (45), and plasma leptin levels reflect intracellular leptin levels in human and rodent adipocytes. Leptin transcript is markedly upregulated in obese rodents and humans (40,46), and is increased in response to food intake (39,47), and insulin (48); similarly, leptin synthesis decreases during fasting (39,47,49)  and weight loss (46). This  5 regulation of leptin contributed to the proposal that leptin acts as a signal of adipocyte energy stores. The discovery of the leptin receptor gene (Lepr), followed soon after leptin was identified, through expression cloning from a mouse choroid plexus cDNA library (50). Lepr was subsequently localized to the db mutation (herein referred to as Leprdb) (51). The Lepr gene encodes an alternately spliced transcript, capable of producing at 6 isoforms (Lepr-a to Lepr-f) (51,52), and belongs to the class I cytokine receptor family (50). The Lepr-b isoform contains the longest intracellular domain (51,52). Mice carrying the Leprdb mutation have an insertion mutation in Lepr that prevents the normal splicing of the Lepr-b isoform, resulting in a truncated intracellular signalling domain, whereas all other Lepr isoforms remain intact (51,52). The nearly identical phenotypes of Lepob/ob and Leprdb/db mice when backcrossed to the same genetic background (53,54) indicate that leptin action is primarily conducted through Lepr-b signalling. Humans carrying rare mutations that affect all transmembrane containing Lepr isoforms have been subsequently identified, and similar to Leprdb/db mice are obese, have neuroendocrine abnormalities, and high circulating leptin levels (55,56). Little is known about the role of Lepr isoforms other than Lepr-b. The Lepr-a isoform has the most abundant and broad tissue expression (57), and the short leptin receptor isoforms have been suggested to be involved in leptin transport and clearance (51,58). The Lepr-e isoform, which is the only isoform that does not contain a transmembrane domain, is believed to encode the soluble leptin receptor in mice (51). However, Lepr-e expression has not been identified in humans, and thus potentially does not contribute to soluble leptin receptor in human plasma (59,60). Alternatively, ectodomain shedding of transmembrane containing Lepr isoforms may be the major source of soluble leptin receptor (61,62), but the relative contributions of Lepr shedding and Lepr-e have not been clarified. Soluble leptin receptor levels are inversely correlated with adiposity in humans (63), are believed to be predominantly produced by the liver (64), and have been shown to stabilize circulating leptin (65,66), while sequestering it from signal transducing interactions with Lepr-b (67).  The Lepr-b isoform is expressed throughout the central nervous system (CNS), including the hypothalamus and brainstem (50,51,68,69), and is also expressed in many peripheral tissues (70) (summarized in Table 1). Lepr-b signals through Janus Kinase (JAK)/Signal Transactivators of Transcription (STAT) pathways (70). Upon leptin binding to Lepr-b, a conformational change in the receptor homodimer induces the tyrosine kinase activity of constitutively associated JAK2, resulting in JAK2 auto-phosphorylation, and JAK2-mediated phosphorylation of 3 intracellular tyrosine residues of Lepr-b (Y985, Y1077,  6 Y1138) (71-74). Each phosphorylated residue creates a binding motif for SH2-containing proteins to induce distinct signalling cascades. Phosphorylated Y985 binds to SH2-containing tyrosine phosphatase-2 (SHP2) initiating the extracellular signal-regulated kinase (ERK) signalling pathway (71). STAT5 binds to phosphorylated Y1077, and STAT3 binds to phosphorylated Y1138, resulting in phosphorylation, and nuclear translocation of both STATs (71,72,75-77), promoting the transcription of target genes. Activated STAT3 induces the expression of suppressor of cytokine signalling-3 (SOCS3) (71), which can form a negative feedback loop by binding to phosphorylated Y985 and inhibiting leptin signal transduction (78). Additional signalling cascades are regulated by leptin, notably the phosphoinositide 3-kinase (PI3K) pathway. This pathway and the relative roles of PI3K and STAT signalling in leptin action are discussed in further detail below.  Leptin and energy homeostasis By circulating at levels proportional to adipose stores, leptin is believed to act as a signal of energy stores to the brain. Humans and rodents with congenital leptin deficiency are extremely hyperphagic and obese, and leptin administration in both species can reduce body weight by inhibiting food intake and increasing energy expenditure (26,31,35,79). Unlike caloric restriction, resultant weight loss from leptin administration is due to the specific loss of adipose tissue, while lean mass is unaffected (27,79). Leptin administration in normal rodents also reduces food intake, and stimulates energy expenditure, resulting in the marked depletion of adipose stores, albeit at higher doses than required for Lepob/ob mice (27,79,80). A large body of evidence supports that the actions of leptin on energy homeostasis occur through the brain. Lepr-b is expressed throughout neurons of the CNS, and is particularly concentrated in the hypothalamus (51,68). Hypothalamic nuclei, including the arcuate hypothalamic nucleus (ARH), ventromedial hypothalamic nucleus (VMH), and dorsomedial hypothalamic nucleus (DMH), lateral hypothalamic area (LHA), along with brainstem neuronal circuits, express Lepr-b (68), and are activated by peripheral leptin administration (81,82). Intracerebroventricular (ICV) administration of low dose leptin mimics the effects of systemic leptin on body weight, food intake and energy expenditure (79,83,84). Deletion of Lepr-b broadly in the hypothalamus induces obesity and hyperphagia (85), and furthermore reconstitution of Lepr-b throughout the CNS (86) largely reverses the obese phenotype of Leprdb/db mice. Cohen et al. reported that deletion of the leptin receptor gene in neurons induced marked obesity in mice, (although there are issues with the interpretation  7 of these results, which will be discussed in Chapter 4) (87). Furthermore, reconstitution of Lepr-b in the hypothalamus, or transplantation of wildtype hypothalamic neural progenitors, can reduce body weight and food intake in leptin receptor deficient rodents (88-90). Circulating leptin is transported across the blood brain barrier into the cerebrospinal fluid (CSF) via a saturable mechanism (91,92) that may be partially mediated by leptin receptors (93). Lepr-a is highly expressed in the choroid plexus (50), and the choroid plexus contains binding sites for circulating leptin (94). Thus, Lepr-a has been suggested to mediate leptin transport across the blood brain barrier, however this remains to be definitively tested. Although leptin is present in the CSF, several lines of evidence suggest that this is unlikely to be a major source of leptin for neuronal targets. Firstly, leptin concentration in the CSF is lower than the Kd for Lepr under normal conditions (50,95). Secondly, ICV leptin administration produces a different binding pattern than peripheral leptin administration (96). Alternatively, leptin may gain access to brain regions through endothelial transcytosis, since brain microvessels have been shown to bind and internalize leptin (97). In addition, leptin responsive neuronal circuits in the ARH likely have direct access to circulating leptin through their proximity to the median eminence, a circumventricular organ in which the blood brain barrier is diminished (98). For targets including the DMH, the LHA, and brainstem, transcytosis through nearby vasculature is likely the major access route for leptin. The ARH contains the most well characterized leptin responsive neurons, namely a subset neurons that express and release the orexigenic peptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) (AgRP neurons), and another subset of neurons that express pro-opiomelanocortin (POMC), which is post-transcriptionally processed into anorectic peptides including α-melanocyte stimulating hormone (α-MSH) and β-melanocyte stimulating hormone (β-MSH). Activation of AgRP neurons results in AgRP and NPY release, which stimulates feeding, whereas activation of POMC neurons results in α-MSH release and inhibits feeding. α-MSH and β-MSH activate melanocortin 3 and 4 receptors (MC3/4R) in second-order neurons to induce satiety (99-101), whereas AgRP is an endogenous antagonist of MC3/4R. NPY release stimulates feeding by acting primarily on Y1 receptors (102,103). Both POMC and AgRP neurons express Lepr-b, and leptin inhibits AgRP neurons, and stimulates POMC neurons, thereby inhibiting food intake (83,104). Leptin action in the ARH partially mediates leptin’s effects on body weight, since reconstitution of Lepr-b in the ARH in leptin receptor deficient rodents is capable of reducing obesity and food intake (88,90). However, selective disruption of leptin receptor signalling in  8 POMC (105), or AgRP neurons, or both (106), only results in mild hyperphagia and obesity, suggesting that additional leptin responsive neurons mediate the anorectic action of leptin. Likewise, restoration of leptin signalling only in POMC neurons of leptin receptor deficient mice only modestly ameliorates obesity (107,108). Studies dissecting the role of leptin signalling in other neuron populations have shown that disruption of leptin receptor signalling in steroidogenic factor-1 (SF1) containing neurons of the VMH (109), hypothalamic nitric oxide synthase expressing neurons (110), or hindbrain neurons (111), leads to a mild obese phenotype. As none of these models, nor widespread hypothalamic or neuronal deletion of leptin receptors recapitulate the obesity of Leprdb/db mice (85,87), additional or redundant neuronal circuitsare likely to mediate leptin’s anorectic actions. In support of this, combined deletion of leptin receptors in POMC and SF1 neurons has an additive effect on body weight and adiposity (109) The role of leptin signalling in other hypothalamic regions including the DMH, LHA and ventral premammillary nucleus (PMV) is not well-defined, partly due to the lack of specific Cre driver lines for neurons in these regions. Leptin resistance The discovery of leptin’s ability to inhibit food intake, stimulate energy expenditure, and induce weight loss, initially generated excitement regarding its potential as a treatment for obesity. However, unlike obesity resulting from congenital leptin deficiency, common forms of obesity (and diet-induced obesity in rodents) are associated with high circulating leptin (39,40,46). The failure of hyperleptinemia to limit food intake and adiposity in obese individuals is considered evidence for leptin resistance. Therefore, resistance to leptin action, rather than leptin deficiency, may play a causal role in the development of common forms of obesity. Indeed, rodent models of diet-induced or age-related obesity are resistant to the weight reducing effects of leptin administration compared to lean controls (112,113), requiring higher doses of leptin to lose a defined amount of weight relative to lean controls (113). The cause of leptin resistance is unclear, but is believed to involve both impaired leptin transport across the blood brain barrier, and post-receptor defects in leptin receptor signalling. Obese individuals have an increased plasma:CSF leptin ratio suggesting that leptin transport may be impaired in obesity (95,114). This is supported by the fact that initially animals with diet-induced obesity show resistance to peripherally but not centrally administered leptin (79,112), as do New Zealand obese (NZO) mice (79). However, as obesity increases, diet-induced obese rodents become resistant to both peripheral and  9 central leptin action (115,116), indicative of post-receptor signalling defects. One factor that may be impair leptin signal transduction in obesity is SOCS3, which is induced in hypothalamic neurons by leptin administration and diet-induced obesity (117-119). Protein tyrosine phosphatase 1B (PTP1B) can dephosphorylate JAK2, and is another factor implicated in post-receptor leptin resistance (120-122). Interestingly, neuronal leptin resistance primarily occurs in the ARH of diet-induced obese rodents, whereas leptin signalling is maintained in other hypothalamic nuclei (123,124). Therefore, the ARH likely plays a key role in resistance to the anorectic action of leptin in obesity. Another interesting possibility arising from selective ARH leptin resistance, is that the maintenance of leptin signalling in other neurons could actually contribute to diet-induced obesity or its ensuing consequences. Indeed, the maintenance of leptin signalling in the DMH has been implicated in the causation of hypertension and sympathetic overactivation in obesity (124-127). The physiological role of leptin in energy homeostasis Based on the ability of leptin to inhibit food intake and stimulate energy expenditure resulting in adipose specific weight loss, and the marked hyperphagia and obesity of genetic leptin deficiency, a predominant view of the physiological role of leptin is that it acts as a satiety factor to limit adipose tissue accumulation. However, this action of leptin has to be reconciled with the rapid development of leptin resistance in obesity, and inability of high levels of leptin to induce weight loss in most obese individuals. Furthermore, the development of a satiety factor can be argued from an evolutionary standpoint. Since food scarcity rather than food abundance is the prevailing condition in nature, it is expected that there would be little selection pressure for a satiety factor, but strong selection pressure for traits that promote food seeking behaviour. Therefore, an alternative view is that leptin developed as a starvation cue, such that hypoleptinemia in individuals experiencing low food supply would promote behaviours and physiological responses to increase energy stores (49). Indeed, preventing the starvation-induced fall in leptin levels prevents the alterations in neuroendocrine pathways that occur to increase energy efficiency (49). Moreover, experimentally induced hypoleptinemia results in voracious food intake and reduced energy expenditure until initial weight is regained (80). The view of leptin as a starvation signal is also consistent with the fact that many of leptin’s actions are most effective at low or moderate leptin concentrations, and less effective in hyperleptinemic states (128). The view of leptin as a starvation signal is also supported by the plethora of neuroendocrine actions of leptin. Starvation, weight loss or severely depleted adipose stores due to exercise or  10 lipodystrophy result in decreased neuroendocrine, immune, and reproductive function, which can be effectively restored by replacing diminished leptin levels through low dose leptin administration (reviewed in (129,130). Leptin and metabolic homeostasis Accumulating evidence has revealed that leptin plays a primary role in glucose and lipid homeostasis. In addition to obesity, Lepob/ob and Leprdb/db mice have a phenotype similar to human type 2 diabetes, including hyperglycemia, hyperinsulinemia and insulin resistance (28,131-136). It can be postulated that the perturbed glucose metabolism that accompanies leptin or leptin receptor deficiency is secondary to obesity and hyperphagia, however evidence suggests that leptin plays a primary, and body-weight independent role in regulating metabolism. Firstly, hyperinsulinemia develops prior to obesity in Lepob/ob mice (133,135-137) and in rodents with disrupted leptin receptor function, including Leprdb/db mice, Zucker fatty (fa/fa) rats, and obese Koletsky (fak/fak) rats (134,138-141). To disect the chronology of events in acute leptin deficiency in adult mice, our laboratory previously examined the effect of disrupting endogenous leptin action in wildtype mice using a leptin antagonist (142). Mice developed hyperinsulinemia, and hepatic insulin resistance within 3 days of antagonist treatment, prior to development of obesity. Further supporting a primary role of leptin in metabolic homeostasis, the metabolic actions of leptin are beyond those that can be achieved by reduced food intake alone. Leptin reduces circulating insulin and glucose levels in Lepob/ob mice, to a greater extent than pair-feeding (143-145), and leptin can lower circulating insulin and glucose levels in Lepob/ob mice prior to changes in body weight (146,147). Notably, even leptin-induced weight loss seems to involve a metabolic shift that is distinct from the anorectic actions of leptin. While weight loss induced by caloric restriction involves the loss of both fat and lean mass and can induce ketonogenesis, leptin specifically reduces fat mass, and results in lower plasma lipids and ketones (79,113). We also demonstrated that the glucose lowering effect outlasts the anorectic effect of leptin in Lepob/ob mice (148). Finally, low-dose leptin administration can normalize circulating insulin and glucose levels in Lepob/ob mice even when the dose is insufficient to reduce food intake or body weight (28,149). Further evidence that the metabolic actions of leptin are independent of weight reduction, rodents and humans with low circulating leptin due to lipodystrophy have hyperinsulinemia, insulin resistance, hyperglycemia, and dyslipidemia, which are improved following leptin replacement (43,150). Thus, in conditions of extreme obesity and the  11 absence of adipose tissue, leptin deficiency results in perturbed glucose homeostasis, which can be corrected by leptin replacement. These studies firmly establish that leptin replacement has a more potent effect on metabolism than body weight in leptin deficient animals. Metabolic actions of leptin Leptin modulates metabolism through distinct and coordinated mechanisms in multiple tissues. One way in which leptin can regulate glucose homeostasis is through the modulation of circulating insulin and glucagon levels. Leptin has a potent suppressive effect on insulin secretion. Leptin administration in Lepob/ob mice rapidly lowers circulating insulin, and decreases preproinsulin transcription in pancreatic islets (146,147,151,152). This effect can occur within minutes, and simultaneously increases blood glucose levels, revealing that this effect is not secondary to improved insulin sensitivity (152). Leptin also has been shown to inhibit insulin levels in wildtype rodents (153), albeit with decreased potency. Insulin promotes lipid storage and leptin synthesis in adipocytes, and thus leptin creates a negative feed-back loop between adipogenesis and insulin secretion, previously termed the adipoinsular axis (154,155). Similar to insulin, circulating glucagon levels are elevated in Lepob/ob mice (156,157), and  are reduced by the administration of leptin (157). As glucagon is a major contributing factor to hyperglycemia, the suppressive effect on glucagon could play a key role in the anti-diabetic action of leptin. Leptin can also modulate glucose and lipid metabolism through changes in nutrient flux in peripheral tissues and changes to insulin sensitivity. Short term leptin administration in normal rats inhibits glycogenolysis, but stimulates gluconeogenesis, resulting in no net change in hepatic glucose output (158). However, during hyperinsulinemic-euglycemic clamps, leptin infusion enhances insulin-mediated suppression of hepatic glucose production (158,159), as the inhibition of glycogenolysis is greater than the stimulation of gluconeogenesis (158). Therefore, during fasting leptin appears to modulate hepatic glucose flux, but has no net effect on glucose output due to hepatic autoregulation, whereas under hyperinsulinemic conditions, leptin promotes insulin-mediated suppression of glucose production. Furthermore, our laboratory previously found that leptin administration improves hepatic insulin sensitivity in Lepob/ob mice (147), and that acute global disruption of leptin signalling in wildtype mice leads to hepatic insulin resistance (142). In addition to modulating glucose entry into the blood via the liver, leptin can also influence glucose uptake and utilization in tissues. Leptin has been reported to improve  12 insulin-stimulated glucose metabolism in skeletal muscle of lean, and diet-induced obese rodents (160-162). Leptin regulates lipid metabolism in skeletal muscle by stimulating lipid oxidation through the activation of AMPK (163), which may in part contribute to enhanced skeletal muscle insulin sensitivity. Although leptin appears to enhance skeletal muscle insulin sensitivity, there are conflicting results as to whether leptin stimulates glucose uptake in skeletal muscle. Some studies show enhanced glucose uptake in response to acute leptin infusion, (164), whereas others show no effect (158,163,165). In contrast, studies appear to uniformly report that leptin stimulates a robust induction of glucose uptake in brown adipose tissue (BAT) (161,164,165). Interestingly, leptin does not stimulate glucose uptake in WAT (161,164,165), and therefore WAT glucose utilization does not likely contribute to the stimulatory effects of leptin on glucose metabolism. The lack of a stimulatory effect on WAT glucose uptake is consistent with leptin’s role to limit lipid stores, since glucose can be converted to triglyceride for storage. As evidenced by the massive reduction in adipose tissue mass that is responsible for leptin-induced weight loss (79,113), leptin has a potent lipolytic effect on WAT. Interestingly, the lipolytic action of leptin on adipocytes is distinct from other lipolytic factors, in that lipolysis is stimulated but free fatty acids are not released (166). This has been attributed to the simultaneous activation of lipolysis and lipid oxidation internally within adipocytes by leptin (167). Leptin also promotes lipid oxidation in non-adipocytes, resulting in widespread depletion of intracellular triglycerides in tissues including the liver, skeletal muscle and the pancreas, and diminished circulating lipids (163,168). Since glucolipotoxicity can contribute to impairements in glucose metabolism (169,170), the lipopenic action of leptin likely plays a major role in leptin-stimulated glucose metabolism. This is particularly evident in lipodystrophic patients, in which leptin replacement diminishes ectopic lipid accumulation, and robustly improves insulin sensitivity and glucose metabolism (150,171-173). Neuronal mechanisms of leptin’s metabolic actions Similar to the regulation of energy homeostasis by leptin, the CNS is believed to play a major role leptin’s metabolic actions. Primary evidence for this is provided by studies showing that ICV leptin administration has a similar effect on glucose uptake and hepatic glucose production to peripheral leptin administration (164,174). Moreover, ICV leptin administration corrects insulin resistance and diabetes in Lepob/ob and lipodystrophic mice (175). The effect of leptin on both BAT and skeletal muscle is diminished by denervation and is thought to involve activation of the sympathetic nervous system (124,163,164,176-178).  13 ICV leptin administration or central leptin gene therapy can lower insulin levels in non-diabetic rats and Lepob/ob mice (179,180), and has been shown to suppress glucose-stimulated insulin secretion through a sympathetic dependent mechanism (181). One proposed sympathetic pathway that mediates this effect is the sympathetic inhibition of osteocalcin secretion from osteoblasts by leptin, which in turn reduces β-cell insulin secretion (182).  Further evidence supporting a role for neuronal leptin action in glucose metabolism is provided by studies that have either genetically disrupted or rescued receptor signalling specifically in neurons. Mice with Cre-mediated disruption of leptin receptor signalling in neurons are hyperinsulinemic, with or without accompanying obesity (87,182). Similarly Cre-mediated disruption of neuronal leptin signalling in the hypothalamus through the use of calcium/calmodulin protein kinase II (CamKII)-cre results in robust hyperinsulinemia (85). Transgenic Lepr-b expression throughout neurons of the CNS, achieved by the combination of two neuron-specific promoters, largely corrects obesity, hyperglycemia, and hyperinsulinemia in Leprdb/db mice (86). Studies dissecting the precise neuronal leptin targets involved in regulating glucose metabolism, have focused on the hypothalamus, and particularly the ARH. Cre-mediated disruption of leptin signalling specifically in either POMC neurons, AgRP neurons, or both, induces obesity and perturbs glucose metabolism (105,106,183). Reconstitution of Lepr-b unilaterally in the ARH, or specifically in POMC neurons of leptin receptor deficient mice results in robust metabolic improvements with only modest reductions in body weight (88,107,108). Remarkably, in all 3 studies, blood glucose levels were completely normalized, and in the study by Berglund et al., female mice had no change in body weight, but a complete restoration of euglycemia. The normalization of blood glucose was accompanied by a marked increase in insulin sensitivity and interestingly, hyperinsulinemia was only partially corrected (88,107,108). Additional studies showed that directing Lepr-b expression to the ARH through microinjections of adenovirus expressing Lepr-b in obese, non-diabetic Koletsky rats (which have a loss of function mutation in all Lepr isoforms) had no effect on body weight but markedly enhanced hepatic insulin sensitivity compared to controls (184,185). Thus, leptin signalling in the ARH, and particularly POMC neurons is sufficient to mediate leptin’s effects on blood glucose but not body weight. However, additional redundant pathways must exist since the specific deletion of leptin action in POMC neurons does not recapitulate the severe diabetic phenotype of Leprdb/db mice (105,183). Disruption of leptin receptor signalling in SF1 neurons, which are located in the VMH, modestly increases body weight (109,186), and induces  14 hyperinsulinemia followed by age-related glucose intolerance and dyslipidemia (186). Supporting a role for the VMH in leptin-stimulated glucose metabolism, microinjection into the VMH of normal rats stimulates glucose uptake in BAT, and skeletal muscle (176,178). Interestingly, this glucose uptake effect appears specific to the VMH, since leptin microinjection into the ARH or DMH does not achieve the substantial increase in glucose uptake in BAT or skeletal muscle (187). Collectively, these studies demonstrate a clear role for neuronal leptin signalling in glucose metabolism. However, since neuron-specific disruption of leptin signalling does not recapitulate the impaired metabolism of leptin receptor deficient rodents, peripheral tissues likely play a critical role in the metabolic actions of leptin. Peripheral mechanisms of leptin action on glucose metabolism Leptin receptors, including the Lepr-b isoform, are expressed in multiple peripheral tissues. The effects of direct leptin action on peripheral tissues, demonstrated through in vitro experiments and studies employing mice with tissue specific leptin receptor disruption, are summarized in Table 1. The direct effects of leptin on peripheral tissues that influence glucose and lipid metabolism are discussed in detail in this section.   15 Table 1. Direct actions of leptin on peripheral Lepr-b expressing tissues. Tissue Effect Model β-cell ↓ insulin synthesis and secretion β-cell lines, primary islets (146,152,188-194), mouse: β-cell  specific* deletion of Lepr signalling domains (195), pancreas specific** deletion of Lepr (196) α-cell ↓ glucagon synthesis and secretion α-cell lines, primary islets (197,198) Liver modulation of hepatic insulin signalling cultured hepatic cells (147,199-201) modulation of gluconeogenesis and glycogen synthesis cultured hepatic cells, perfused rodent livers (199,201-205)  ↓ insulin sensitivity modulation of lipoprotein metabolism mouse: hepatocyte specific deletion of Lepr signalling domains (206,207) Skeletal Muscle  modulation of glucose metabolism and insulin signalling isolated muscle, cultured myocytes(160,162,208-211)  ↑ fatty acid oxidation isolated muscle, myocyte cell line (163) Adipose ↓ insulin action, lipogenesis, glyceroneogenesis ↑ lipolysis, lipid oxidation, thermogenesis cultured adipocytes, isolated WAT/BAT (166,212-216), mouse: adipose specific Lepr antisense RNA (217) Innate Immune Cells ↑ monocyte and macrophage activation and proliferation ↑ inflammation and chemotaxis cultured primary monocytes (218), macrophages (219,220), macrophage cell lines (221), polymorphonuclear neutrophils (222,223) Adaptive Immune Cells ↑ T-cell activation ↑ Th1 polarization primary T-cells (224) Adrenals ↓ corticotropin-induced adrenal aldosterone, cortisol, and dehyrdoepiandrosterone secretion primary adrenal cortex (225) ↑ chatecholamine synthesis ↔ chatecholamine secretion cultured chromaffin cells (225), adrenal medullary cells (226)  Heart ↓ contractile function cultured cardiomyocytes (227) ↓ systolic dysfunction and heart failure in response to primary insult mouse: cardiomyocyte specific leptin receptor deletion (228,229) Endothelium ↑ proliferation and angiogenesis cultured endothelial cells (230,231) ↑ leptin clearance and uptake mouse: endothelial cell specific deletion of Lepr signalling domains (232,233) Intestine modulation of sugar absorption primary small intestinal rings (234), jejunal loops (235) L-cell ↑ glucagon-like peptide 1 secretion cultured intestinal cells, L-cell lines (236) Ovaries ↓ insulin-stimulated and LH-stimulated steroidogenesis ↑ thecal cell proliferation cultured granulosa (237,238), thecal (239) cells * partial hypothalamic deletion of Lepr signalling domains ** possible intestinal and brain deletion of Lepr signalling domains  16 There is a substantial body of evidence supporting that the inhibitory effect of leptin on insulin is through direct β-cell leptin signalling. Leptin binding, Lepr-b expression, and Lepr-b signal transduction have been demonstrated in pancreatic islets or isolated β-cells of mice, rats and humans (154,188-191). In vitro application of leptin to isolated Lepob/ob islets or β-cell lines suppresses insulin synthesis and secretion (146,152,188-194). This is also observed in perfused Lepob/ob mouse pancreas (188,190). Furthermore, mice with a disrupted signalling domain of the Lepr gene in β-cells are hyperinsulinemic, and hypoglycemic during fasting, supporting a role for leptin in the physiological inhibition of insulin secretion (195). Similarly, mice with a pancreatic and duodenal homeobox-1 (Pdx1)- cre mediated deletion of Lepr in the pancreas are also hyperinsulinemic (196). Similar to insulin, leptin-induced glucagon suppression can be mediated by direct leptin action in α-cells. Lepr expression, and immunoreactivity have been demonstrated in a pancreatic α-cell line, and Lepr-b immunoreactivity is present in mouse and human α-cells (197). Application of leptin to isolated mouse α-cells induces STAT3 signalling (198), and leptin application to mouse islets inhibits glucagon secretion, and reduces glucagon content and preproglucagon mRNA (197,198). Thus the inhibitory effect of leptin on glucagon levels in vivo may be due to direct action on α-cells. To assess the role of α-cell leptin signalling in glucose metabolism, our laboratory generated mice with an α-cell-specific disruption of leptin signalling through the Cre-lox method (240). Glucose and glucagon levels were unaltered in these mice, however the glucagon-cre transgene was active in only 43% of α-cells, making it unclear whether the lack of phenotype was due compensation from the remaining α-cells with functional leptin signalling. Thus, determination of the role of α-cell leptin receptor signalling in glucose metabolism requires further investigation.  Interestingly, the direct and indirect actions of leptin on hepatocytes may have opposing effects on glucose metabolism. Lepr-b expression has been demonstrated in hepatocytes of many species (70,199,206,241,242), and leptin signal transduction has been shown in hepatocyte cell lines (147,199,200,241), and rat and mouse liver (201,243). Application of leptin to hepatocytes in culture modulates insulin signal transduction, but both stimulatory and inhibitory effects on the signal cascade have been reported (199-201). Furthermore our laboratory found that administration of leptin to a hepatic cell line, and to Lepob/ob mice in vivo, enhanced hepatic insulin signal transduction, and paradoxically increased PTP1B expression (147), a negative regulator of both insulin and leptin action. Studies also demonstrate that leptin, through application to hepatic cells in vitro or perfusion of rodent livers, promotes glycogen synthesis, inhibits gluconeogenesis (199,201-203), and  17 can inhibit hepatic glucagon action (203,204). Collectively these studies indicate that hepatic leptin signalling modulates insulin signalling and glucose flux, but the effects of leptin are inconsistent. The disparity between outcomes may be due to differences in hepatic nutrient status, as one study found that leptin inhibited glucose production in livers during the postprandial state, but stimulated glucose production during the postabsorptive state (205), reminiscent of the differences seen in acute leptin perfusion during basal and hyperinsulinemic-euglycemic conditions (158). To examine the physiological role of hepatic leptin receptors in vivo, Cohen et al., generated mice with a liver specific deletion of all leptin receptor isoforms using the Cre-lox approach. No resulting phenotype on glucose metabolism was reported (87), but this was only examined at one time point, under free feeding conditions. Our laboratory previously generated similar mice with a deletion of the Lepr-b signalling domain in hepatocytes, and found that these mice were protected from diet- and age-related glucose intolerance, had heightened hepatic insulin sensitivity, increased hepatic triglyceride accumulation (206), and altered lipoprotein metabolism (207). Collectively, these studies suggest that hepatic leptin signalling has a direct inhibitory effect on hepatic insulin sensitivity, and contributes to the regulation of glucose and lipid metabolism by leptin. Direct leptin action on skeletal muscle and adipocytes can also regulate metabolism. Lepr-b is expressed in skeletal muscle (244,245) and leptin application to isolated muscle and muscle cell lines has been shown to enhance glucose uptake, metabolism, and insulin sensitivity (160,208-211). However, other studies have reported either an inhibitory effect or no effect on glucose uptake in muscle (162,163). This again may reflect differences in nutritional status or leptin sensitivity, since leptin application stimulates glycogen synthesis in soleus muscle from Lepob/ob mice, which are very leptin sensitive, but not wildtype controls (162). Leptin can also directly activate AMPK in skeletal muscle, to stimulate lipid oxidation, and thus direct leptin action likely plays a role in regulating skeletal muscle lipid metabolism (163).  Lepr-b and leptin signal transduction have been shown in BAT and WAT (215,246,247). Leptin inhibits insulin action on glucose and lipid metabolism in white and brown adipocytes in culture (213,214), however, leptin can potently stimulate glucose uptake in BAT through the induction of thermogenesis (215,216). Using the Pepck promoter to drive anti-sense RNA against Lepr in white adipocytes, Huan et al. reported that adipocyte specific attenuation of leptin signalling led to obesity and insulin resistance, indicating that direct leptin action on adipocytes plays critical role in adiposity and glucose homeostasis  18 (217). This is consistent with the potent lipopenic effect of leptin on white adipocytes, which stimulates lipolysis without the release of free fatty acids, presumably due to enhanced intracellular lipid oxidation (166,167). Leptin has also been shown to directly inhibit lipid synthesis and glyceroneogenesis in white adipocytes, contributing to the depletion of triglyceride stores in adipocytes (212). The roles of leptin signal transduction pathways As mentioned above, Lepr-b can activate many signal transduction pathways, each of which play a distinct role in collective actions of leptin. Studies using mice in which specific Lepr-b tyrosine residues have been mutated, have revealed that the actions of leptin on energy homeostasis are largely dependent upon Y1138 (248,249), implicating the STAT3 signal transduction pathway. Interestingly, this pathway is not required for the action of leptin on glucose homeostasis, immune function, or fertility (248). Consistent with the inhibitory role of the SOCS3-Y985 interaction on leptin signalling, mice lacking only Y985 are lean, with enhanced leptin sensitivity (250). Mice with phenylalanine substitutions for all 3 tyrosine residues display impaired glucose metabolism, but similar obesity compared to mice with a phenylalanine substitution only on Y1138, suggesting that Y1077 may play a more prominent role in glucose metabolism (251). However, these triple mutant mice do not recapitulate the metabolic phenotype of Leprdb/db mice, suggesting that leptin’s metabolic actions are also mediated through pathways independent of these tyrosine residues and STAT transcriptional activation.  While leptin-induced STAT signalling is critical for leptin’s anorectic actions, there are additional rapid effects of leptin in some cell types that cannot be explained by transcriptional changes in target genes. A primary example of such an effect is the rapid electrophysiological effect of leptin on some neurons. Leptin hyperpolarizes AgRP neurons and a subset of VMH neurons (109), depolarizes POMC neurons (252) and SF1 neurons (109), and inhibits NPY secretion from hypothalamic explants (253) within a time-frame that is not achievable by gene expression. Leptin also inhibits a subset of glucose-sensitive neurons (254) and pancreatic β-cells (190) through activation of ATP-sensitive potassium channels with similar rapidity. The PI3K signalling cascade may mediate these rapid effects of leptin, as leptin activates PI3K within minutes (255), and leptin-induced activation of ATP-sensitive potassium channels in a β-cell line and in ARH neurons is PI3K dependent (256,257). Moreover, pharmacological inhibition of PI3K in the hypothlamus (258), or genetic deletion of PI3K subunits in POMC neurons (259), blocks the anorectic action of leptin.  19 Furthermore, hypothalamic Lepr-b reconstitution in the ARH improves insulin sensitivity in rats, and this effect is blocked by ICV administration of a PI3K inhibitor (184). Leptin has also been shown to modulate nutrient sensing signalling cascades, including AMPK (163,260) and mammalian target of rapamycin (mTOR) (261) pathways. In POMC and AgRP neurons, leptin induces the activation of mTOR, and central inhibition of mTOR by rapamycin blunts the anorectic effect of leptin (261). Given the many similar actions of leptin and insulin on glucose metabolism, PI3K and mTOR provide provocative points of convergence between the two signalling cascades. Clinical utility of leptin Due to the profound ability of leptin to modulate glucose and energy homeostasis, leptin has therapeutic potential for the treatment of metabolic disorders and obesity. While leptin therapy successfully induces weight loss and corrects several neuroendocrine defects in obese individuals with rare leptin deficiency (32,35-38,262,263), leptin monotherapy induces little to no weight loss in obese, hyperleptinemic individuals (264,265). One study has shown promising anti-diabetic effects of leptin in a rodent model of polygenic obesity and type 2 diabetes (266). However, 2 recent studies reported that leptin had little metabolic benefit to obese patients with type 2 diabetes (267,268). Thus the prevalence of leptin resistance in obese individuals is a major hurdle to leptin’s clinical utility. On the other hand, rather than inducing weight loss in obese individuals, accumulating evidence suggests that leptin therapy may help maintain weight loss once leptin levels have decreased (269-274). Additionally, leptin therapy is highly effective in improving glucose metabolism, and dyslipidemia in patients with general lipodystrophy who have extremely low circulating leptin levels (150,171-173). Thus, as previously postulated (275) leptin appears to have its greatest therapeutic action in disorders associated with low leptin levels. 1.3 LEPTIN THERAPY FOR TYPE 1 DIABETES Type 1 diabetes and leptin deficiency Type 1 diabetes is a state of relative leptin deficiency. Following the administration of streptozotocin (STZ, a β-cell toxin that selectively kills approximately 90% of β-cells) in rats, circulating leptin levels rapidly fall (276). This is at least partly due to decreased insulin levels, as insulin potently stimulates leptin synthesis in adipocytes (48,277), and leptin levels can be acutely elevated by injection of exogenous insulin in STZ-diabetic rats (276).  20 Similarly, humans with newly diagnosed type 1 diabetes who have not received insulin treatment have also been reported to have low circulating leptin levels (278,279), and leptin levels are normal in type 1 diabetic patients receiving insulin therapy (278). Thus it follows that type 1 diabetes is another disease that could benefit from the metabolic action of leptin therapy, and that some of the metabolic and energy disturbances in uncontrolled type 1 diabetes, could be due to leptin deficiency rather than insulin deficiency per se. Hyperphagia is a well-known effect of uncontrolled diabetes. Given the role of central leptin and insulin in inhibiting food intake, this could be a direct effect of decreased neuronal leptin and insulin action. Hypothalamic NPY and AgRP expression are upregulated, whereas POMC expression is dramatically blunted in rats with uncontrolled diabetes (280,281); insulin treatment can partially restore normal NPY and POMC expression. Interestingly, although peripheral insulin treatment in STZ-diabetic rats can partially reduce hypothalamic AgRP expression, centrally administered insulin can reduce NPY (282) but not AgRP expression (281). In contrast, centrally administered leptin fully normalizes hypothalamic AgRP expression, indicating that the correction of hyperphagia by peripheral insulin treatment may be due to restoration of leptin levels (281). Levels of ghrelin, an orexigenic peptide, are also increased in uncontrolled STZ-induced diabetes, which could contribute to diabetic hyperphagia; this is corrected by partial restoration of leptin levels through low-dose injections (283). Supporting a direct role of hypoleptinemia in diabetic hyperphagia, partial or full replacement of circulating leptin in STZ-diabetic rats can restore normal food intake levels (284,285). Similarly, some of the neuroendocrine defects in uncontrolled diabetes can be corrected by administration of exogenous leptin to prevent the STZ-induced fall in leptin levels. This includes STZ-induced hyperglucagonemia, insulin resistance and hypercorticosteronemia in rats (286). In contrast, leptin replacement has a very modest glucose lowering impact in STZ-diabetic rats, indicating that hyperglycemia is not secondary to leptin deficiency in type 1 diabetes (286). Leptin as a therapy for type 1 diabetes There are now several studies showing that administration of supraphysiological doses of leptin (doses that result in circulating levels comparable to an obese animal but higher than those of normal animals) can substantially improve glycemic control in rodent models of insulin deficient type 1 diabetes. The profound metabolic effect of leptin therapy in STZ-diabetic rats was first reported by Chinookoswong et al., who had aimed to determine whether the actions of leptin on glucose disposal and glucose production were independent  21 of insulin (287). Remarkably they found that high dose leptin robustly reduced plasma glucose concentrations, without increasing circulating insulin levels. Leptin also reduced plasma lipids and ketones, and improved insulin sensitivity during hyperinsulinemic-euglycemic clamps. This study revealed that leptin treatment alone could completely normalize fasting blood glucose levels in a rodent model of type 1 diabetes. Subsequently, this effect was confirmed by inducing hyperleptinemia through either subcutaneous leptin infusion, adenoviral leptin gene transfer, or transgenic leptin overexpression in STZ-diabetic rats, non-obese diabetic (NOD) mice (288,289), BioBreeding (BB) rats with virally induced β-cell destruction (290), and insulinopenic Akita mice (291). The glucose lowering effect of leptin in insulin-deficient rodent models of type 1 diabetes remains perhaps the most compelling evidence for the profound effect of leptin on glucose homeostasis, and has propelled leptin into clinical trials as an adjunct to insulin therapy for type 1 diabetes. The mechanism through which leptin achieves glycemic control in type 1 diabetes remains unclear.   Similar to leptin replacement, high-dose leptin therapy reverses diabetic hyperphagia in type 1 diabetic rodents; importantly pair-feeding does not mimic the effect of leptin administration on blood glucose, revealing that the metabolic improvements in diabetic leptin treated mice are independent of food  intake (287,288,291). An interesting aspect of leptin therapy in type 1 diabetes is that despite the use of supraphysiological doses to normalize glycemia, no significant body weight reduction is observed relative to untreated diabetic controls (287). This contrasts the potent weight loss that occurs with high leptin doses in normal and Lepob/ob mice (28,79). Furthermore, diabetic controls that are pair-fed to leptin treated diabetic rodents, lose substantially more weight compared to both leptin treated and untreated ad-lib fed animals (287). This reduced weight loss in leptin treated compared to pair-fed untreated diabetic animals is also seen with partial leptin replacement doses that do not improve glycemia in STZ-diabetic rats (284). These findings suggest that there are additional effects of leptin that counter its anorectic action in type 1 diabetic rodents, thereby preventing the weight loss that would otherwise occur with reduced food intake. The lack of weight loss in leptin treated type 1 diabetic rodents may be due to a counter-intuitive decrease in energy expenditure, as has been shown by leptin replacement in STZ-diabetic rats (286). An alternative explanation is that since leptin-induced weight loss is largely due to the loss of adipose tissue mass (27,79), leptin therapy in type 1 diabetic rodents has little effect on weight loss due to the already substantially diminished adipose stores induced by insulin deficiency. Additionally, since both insulin and leptin act centrally to induce negative  22 energy balance (292,293), the lack of a body weight effect may be due to diminished insulin action in the hypothalamus. However, this explanation appears insufficient to explain the lack of weight loss, since some leptin-induced weight loss would be expected, given that leptin and insulin have been shown to reduce body weight additively (294). Although the mechanisms of leptin-mediated glucose lowering in type 1 diabetes have not been defined, several pathways have been suggested or ruled out. Studies in type 1 diabetic rodents report that leptin therapy either decreases or has no effect on insulin levels, or that insulin levels remain undetectable, indicating that the effect is not mediated by a resurgence of circulating insulin (287-289). This observation is consistent with the well-documented inhibitory effect of leptin on β-cell insulin secretion, and further supported by a lack of effect on pancreatic β-cell area in leptin treated type 1 diabetic rodents (288). An alternative mechanism that has been suggested is that leptin could increase the levels of other hormones that have insulin-like actions on glucose metabolism. Yu et al. reported that in STZ-diabetic rats, leptin therapy enhanced circulating insulin-like growth factor 1 (IGF1) and IGF1 signal transduction in skeletal muscle (288). IGF1 binding protein 2 (IGFBP2) is a recently identified target of leptin action; its circulating levels are upregulated by leptin administration, and Hedbacker et al. suggested that IGFBP2 may be responsible for mediating leptin’s anti-diabetic actions (149). They showed that administration of pharmacological levels of IGFBP2 mimicked the effects of leptin in several models, including the reduction of blood glucose in STZ-diabetic mice. However, our laboratory recently found that physiological levels of IGFBP2 did not exert a leptin-like effect in Lepob/ob mice (295). Furthermore, the metabolic effects of leptin were not blunted by inhibition of hepatic IGFBP2 expression through RNAi (considered to be the major source of circulating IGFBP2), suggesting that IGFBP2 is not required for the metabolic actions of leptin. Finally, IGFBP2 levels are already robustly elevated in rodents with uncontrolled STZ-induced diabetes (286), and thus it is unlikely that elevated IGFBP2 levels would mediate the anti-diabetic action of leptin in type 1 diabetes. Another possibility is that leptin itself has insulin-like actions. Indeed, given the ability of leptin to activate the PI3K signalling cascade, as discussed above, this seems plausible. The study by Yu et al., reported that leptin therapy reverses diabetic hyperglucagonemia in NOD mice and STZ-diabetic rats, restoring normal circulating glucagon levels (288). Thus, they proposed that the inhibitory action of leptin on glucagon in type 1 diabetes could mediate glucose lowering. Hyperglucagonemia is a common characteristic of diabetes, and is a requisite for hyperglycemia in several models of insulin  23 deficiency (296). Interestingly, suppression of endogenous glucagon (297), or antagonism of glucagon receptor signalling (298-300), decreases hyperglycemia in STZ-diabetic rats. Subsequently, it was found that global deletion of glucagon receptors in mice prevents STZ-induced hyperglycemia despite the reduction of insulin (289,301). However, German et al. showed that reversal of hyperglucagonemia by leptin in STZ-diabetic rats is not sufficient to normalize blood glucose (286), as low-dose leptin in STZ-diabetic rats resulted in the full reversal of hyperglucagonemia, yet only a modest reduction blood glucose levels. Furthermore, since glucagon receptor knockout mice have extremely elevated levels of glucagon-like peptide 1 (302), and their β-cells are resistant to STZ-induced cell death (303), it is unclear whether the lack of STZ-induced hyperglycemia in glucagon receptor knockout mice is directly due to loss of glucagon action. Another potential pathway through which anti-diabetic agents can lower blood glucose is by enhancing the excretion of glucose in urine. This does not appear to be the case for leptin, since leptin therapy reverses glycosuria in diabetic rodents (288,289).  Aside from the downstream effects that mediate glucose lowering by leptin in type 1 diabetes, the precise leptin target tissues and signalling pathways involved are also unknown. The CNS is believed to play a major role based on recent studies showing that central leptin therapy, can mimic the anti-diabetic action of peripheral leptin therapy. ICV administration of leptin reverses hyperglycemia, hyperketonemia, and dyslipidemia in STZ-diabetic rats and mice (304-306). Similarly, ICV administration of recombinant adeno-associated virus (AAV) vector encoding leptin restored euglycemia in diabetic, insulinopenic Akita mice (307). Notably, both ICV leptin infusion and gene delivery restore euglycemia without a detectable rise in circulating leptin levels suggesting minimal leakage from the CSF to the periphery. Furthermore, ICV leptin can achieve euglycemia at a low dose that is insufficient when administered peripherally (305). These data suggest that the glucose lowering effect of peripheral leptin therapy in type 1 diabetes may be mediated through leptin action in the brain, and that supraphysiological doses may be required when administered peripherally so that sufficient levels of leptin are transported across the blood brain barrier. Several studies have now attempted to identify the precise leptin responsive neuronal subsets involved in the anti-diabetic effects of ICV leptin. Leptin injection into the VMH, and reconstitution of Lepr-b in POMC or GABAergic neurons are sufficient to mediate the anti-diabetic action of ICV leptin in STZ-diabetic rodents, but neither leptin signalling in SF1 neurons nor POMC neurons are required to mediate these effects (308,309). Furthermore, since the Lepr-b reconstitution experiments were performed in leptin receptor  24 deficient mice, which are a model of type 2 diabetes, the distinction between type 1 and type 2 diabetes in these animals is potentially confounding. Therefore it appears that although POMC neurons and neurons of the VMH play a critical role in leptin’s glucose lowering action in type 1 diabetes, there are redundant neural circuits or peripheral tissues that can mediate this action. A target tissue has yet to be identified as a requisite for the anti-diabetic effect of leptin in type 1 diabetes. Leptin is currently being tested clinically as an adjunct to insulin therapy, based on evidence that occasional leptin injections in NOD mice receiving continuous insulin infusion improved glycemia, compared to insulin alone, and at a lower insulin dose (58). However, since high-dose leptin can normalize blood glucose levels in type 1 diabetic rodents without insulin administration, leptin could potentially be used as a monotherapy for type 1 diabetes. Further pre-clinical studies in rodents are needed to better define the potential benefits and pitfalls of leptin therapy for this disease, and its mechanism of action.  1.4 THESIS INVESTIGATION Given the profound effects of leptin on glucose metabolism, and particularly its therapeutic potential for insulin-deficient type 1 diabetes, it is important that the mechanism of leptin’s anti-diabetic actions be elucidated. We hypothesized that leptin therapy corrects glucometabolic disturbances in type 1 diabetes by diminishing substrates for hepatic glucose production, through a mechanism that is independent of neuronal leptin receptor signalling. To address this hypothesis, in Chapter 3 we first performed an in depth characterization of glucose metabolism in response to supraphysiological peripheral leptin therapy in a mouse model of type 1 diabetes. Subsequently, we aimed to determine whether neuronal or hepatic leptin receptor signalling is required for the metabolic effects of leptin therapy in this mouse model of diabetes. To this end, we first generated mice with a selective deletion of the Lepr-b signalling domain in neurons throughout the CNS using the Cre-lox method. We performed a thorough assessment of energy, and glucose homeostasis in these mice which is described in Chapter 4. Subsequently, we examined the efficacy of leptin therapy in these mice after inducing type 1 diabetes in Chapter 5, and assessed the role of parasympathetic and autonomic efferents. To examine the potential role of direct hepatic leptin signalling in mediating the therapeutic action of leptin in type 1 diabetes, the efficacy of leptin therapy in mice with a liver specific deletion of the Lepr-b signalling domain was also examined in Chapter 5. In Chapter 6, we next examined the downstream metabolic pathways that may contribute to leptin-mediated glucose lowering by performing a rigorous examination of  25 hepatic metabolism, and gluconeogenic substrate availability in mice treated with leptin. Finally, in Chapter 7, we explored a novel therapeutic potential for leptin in type 1 diabetes, as an adjunct to islet transplantation. Collectively, these investigations provided novel insight into the mechanism of leptin therapy in type 1 diabetes, and shed further light on the therapeutic potential of leptin for improving metabolic control in type 1 diabetes.  26 CHAPTER  2: MATERIALS AND METHODS 2.1 ANIMALS Housing and facilities All procedures with animals were approved by the University of British Columbia (UBC) Animal Care Committee and carried out in accordance with the Canadian Council on Animal Care guidelines. Mice were housed in a 12-hour light: 12-hour dark cycle, at ambient temperature. Most animal studies were performed at the Centre for Disease Modelling (CDM) at UBC (a specific-pathogen free facility), with the exception of some experiments performed at the D.H. Copp Building at UBC (a conventional animal facility). Studies pertaining to figures 3-4, 6-10, and 38-40 were performed at D.H. Copp, while all other studies were performed at the CDM. Mice housed in D.H. Copp were housed with open cage tops, and had ad libitum access to chow diet (LabDiet, #5015, Richmond, USA) and water. Mice housed at the CDM were housed in filter top cages, and had ad libitum access to irradiated chow diet (#2918, Teklad Diets, Harlan Laboratories, Madison, USA) or irradiated specialty diets where indicated, and autoclaved water.  C57Bl/6 mice Male C57Bl/6 mice were purchased from either Jackson Laboratories (C57Bl/6J #664, Bar Harbor, USA) or directly from the CDM (Vancouver, Canada), which were originally purchased from Jackson Laboratories (#664) and maintained as a colony by the facility. Leprflox/flox Albcre mice  Leprflox/flox mice (50% C57Bl/6, 50% FVB, <1% 129) were kindly provided by Dr. Streamson Chua (Albert Einstein College of Medicine, Bronx, USA). The Leprflox allele contains 2 unidirectional loxP sites flanking exon 17, such that upon Cre-induced recombination, exon 17 is excised (Figure 1) (310). The resultant LeprΔ17allele encodes a Lepr-b transcript in which exon 16 is spliced directly to exon 18b, producing a frame-shift mutation and early stop codon, which results in a truncated Lepr-b isoform that is incapable JAK2 dependent signal transduction. Leprflox/flox mice were mated with mice carrying the Albcre transgene (Albcre mice; B6.Cg-Tg(Alb-cre)21Mgn/J; #3574, Jackson Laboratory) to generate Lepr+/flox Albcre and Leprflox/+ progeny. These progeny were then intermated to generate Leprflox/flox Albcre and Leprflox/flox mice. The line was subsequently maintained  27 through matings between Leprflox/flox Albcre and Leprflox/flox mice to generate litters consisting of 50% Leprflox/flox Albcre and 50% Leprflox/flox mice. Leprflox/flox littermates were used as controls, to control for genetic background which can affect leptin-related phenotypes (311). We previously confirmed that the phenotype of Leprflox/flox Albcre mice is not due to off target effects of the Albcre transgene itself (206).  Figure 1. Schematic of Cre-induced recombination of the Leprflox allele. The Leprflox allele contains 2 unidirectional loxP sites flanking exon 17. Cre-induced excision of exon 17 results in the production of a Lepr-b transcript in which exon 16 is spliced to exon 18b. This produces a frame-shift mutation, altering the amino acid sequence of exon 18b, and producing an early stop codon. The resultant truncated Lepr-b isoform is incapable of JAK2 mediated signal transduction. Primers used for PCR (orange), RT-PCR (green), and RT-qPCR (purple) are indicated.     28 Leprflox/flox Syncre mice Mice homozygous for the Leprflox allele and carrying the rat synapsin I cre (Syncre) transgene (Leprflox/flox Syncre mice) were generated and maintained in the CDM. In order to rederive the Leprflox/flox strain (described above) in the CDM to generate the Leprflox/flox Syncre strain, mice from our original Leprflox/flox strain (50% C57Bl6, 50% FVB, <1% 129) were mated with C57Bl/6 mice to generate Leprflox/+ embryos. Leprflox/+ embryos were rederived in the CDM, and Leprflox/+ offspring were intercrossed to obtain Leprflox/flox mice. Leprflox/flox Syncre mice were generated as follows (Figure 2): Leprflox/flox mice were mated with mice carrying the Syncre transgene (Syncre mice; B6.Cg-Tg(Syn1-cre)671Jxm/J, #3966, Jackson Laboratories, Bar Harbor, ME) resulting in Leprflox/+ Syncre and Leprflox/+ mice; Leprflox/+ Syncre and Leprflox/+ mice were crossed to generate 12.5% Leprflox/flox Syncre and 12.5% Leprflox/flox mice (6.25% for each sex); Leprflox/flox Syncre and Leprflox/flox mice were mated to generate litters consisting of 50% Leprflox/flox Syncre mice and 50% Leprflox/flox littermate controls. Because Syncre is expressed in the testes, which can transmit the deleted target gene through the germline to progeny (312), all experimental mice were generated by crossing Leprflox/flox Syncre females with Leprflox/flox males. All experimental mice were genotyped for Leprflox in ear or tail tissues to identify and exclude mice with partial or complete Leprflox recombination due to early embryonic activation of the Syncre transgene (occurring in approximately 6% of mice) using primers mLepr101 and mLepr102.  29  Figure 2. Generation of Leprflox/flox Syncre mice. A) Schematic depicting the generation of the Leprflox/flox Syncre strain used in Chapters 4 and 5. B) Gel electrophoresis demonstrating genotyping strategy, showing PCR products amplified from mice of each possible genotype that were produced during line generation. For studies examining high fat diet (HFD)-induced obesity, Leprflox/flox Syncre  and Leprflox/flox littermates were switched from chow diet to a diet containing 45% kcal from fat (Research Diets, #D12450i) or 10% kcal from fat as a control diet (Research Diets, #D12451Bi) (Table 1) at 8 weeks of age.   30 Table 2. Composition of 45% and 10% fat diets.  D12451 (45% fat)  D12450B (10% fat) Component g % kcal %  g % kcal % Protein 24 20  19.2 20 Carbohydrate 41 35  67.3 70 Fat 24 45  4.3 10 kcal/g 4.73   3.85  Ingredient (per 100 g diet) g kcal  g kcal Casein, 80 Mesh 23.31 93.2  18.96 75.8 L-Cystine 0.35 1.4  0.28 1.1 Corn Starch 8.48 33.9  29.86 119.4 Maltodextrin 10 11.65 46.6  3.32 13.3 Sucrose 20.14 80.5  33.17 132.7 Cellulose, BW200 5.83 0.0  4.74 0.0 Soybean Oil 2.91 26.2  2.37 21.3 Lard 20.68 186.2  1.90 17.1 Mineral Mix S10026 1.17 0.0  0.95 0.0 DiCalcium Phosphate 1.51 0.0  1.23 0.0 Calcium Carbonate 0.64 0.0  0.52 0.0 Potassium Citrate, 1 H2O 1.92 0.0  1.56 0.0 Vitamin Mix V10001 1.17 4.7  0.95 3.8 Choline Bitartrate 0.23 0.0  0.19 0.0 Dye 0.01 0.0  0.00 0.0 Total 100 472.8  100 384.5  mTmG Syncre mice To assess the specificity of Syncre-mediated target gene recombination, we employed the Cre reporter mouse line (mTmG mice; B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, Jackson Laboratories). These mice harbour a targeted knock-in gene with an open-reading frame for membrane-bound Tomato fluorescent protein (mT) flanked by two unidirectional loxP sites, followed by the open reading frame for membrane-bound enhanced green fluorescent protein (EGFP) (construct referred to as mG), driven by the chicken β-actin promoter (313). The presence of a transcription termination sequence following the mT construct prevents the transcription of the mG construct. Upon Cre-induced recombination of the loxP sites, mT and the transcription termination sequence is excised,  31 resulting in transcription of mG and the production of EGFP. Thus in mTmG reporter mice, cells that contain Cre activity are permanently marked by EGFP fluorescence (313). Mice homozygous for mTmG were crossed with Syncre mice, to generate litters heterozygous for mTmG, 50% of which carry the Syncre transgene (mTmG Syncre mice) and 50% of which lack the Syncre transgene (mTmG mice) for controls. Genotyping DNA was extracted from mouse ear biopsies using a Chelex based strategy. Ear biopsies were incubated in 112.5 mg/mL Chelex 100 (BioRad, Mississauga, Canada), 0.1 mg/mL proteinase K (Fisher Scientific, Ottawa, Canada), and 0.1% Tween-20 (Fisher Scientific) at 55˚C for 45 min, followed by heat inactivation at 95˚C for 15 min. Supernatant (2 μL) was used as template for PCR based genotyping. To genotype Leprflox/flox Albcre and Leprflox/flox littermates, primers for the Cre open reading frame (general Cre primers: forward primer: AGG TGT AGA GAA GGC ACT CAG C; reverse primer: CTA ATC GCC ATC TTC CAG CAG G) were used to amplify the Cre transgene. Reactions contained a second primer set to amplify interleukin-2 (Il-2) as a control gene (forward primer: CTA GGC CAC AGA ATT GAA AGA TCT; reverse primer: GTA GGT GGA AAT TCT AGC ATC ATC C), to ensure that the lack of Cre amplicon was not due to a failed PCR. Mice were genotyped based on the presence of the control amplicon ± Cre amplicon. Mice were genotyped for the Leprflox or Lepr+ allele using a set of primers (forward primer: ACA CCA CAC TGT TGA GAC ACC; reverse primer: CAT TTG ATT CCA CAA AGT GTT CCC TAA AC) that generate an 225 bp product for the Leprflox allele and a 297 bp product for the Lepr+ allele. Leprflox/flox Syncre mice were genotyped for Syncre transgene using the same primers described above for Leprflox/flox Albcre mice. 2.2 BLOOD AND PLASMA ANALYTES All blood samples were collected via the saphenous vein from restrained, conscious mice, unless specified as collected from cardiac puncture, in which case mice were deeply anaesthetized under isoflurane during the procedure. For plasma collection, blood was collected with heparinized capillary tubes into collection tubes containing heparin to achieve a final concentration of 4.0 U heparin/mL whole blood. Plasma was separated by centrifugation at 4600 xg at 4˚C for 9 min. Blood glucose was monitored via a One Touch Ultra Glucometer (Life Scan Inc., Burnaby, Canada) from the saphenous vein. In the event that blood glucose levels exceeded 33.3 mmol/L, either the value was assigned as 33.3  32 mmol/L, plasma samples were collected to measure plasma glucose using a Trinder assay (Genzyme, Charlottetown, Canada), or whole blood was diluted with whole blood collected from a non-diabetic animal and subsequently measured with a One Touch Ultra Glucometer. The use of these particular techniques will be specified in the figure legend where applicable. For hemoglobin A1c (HbA1c) measurements, whole blood was collected with EDTA as an anticoagulant (1.5 mg/mL of whole blood) and frozen prior to determination with a Siemens DCA 2000 Vantage Analyzer (Siemens Healthcare Diagnostics Inc., Tarrytown, USA).  Free fatty acids (HR Series NEFA HR(2) Kit; Wako Chemical USA, Richmond, USA), β-hydroxybutyrate (β-Hydroxybutyrate LiquiColor Test; Stanbio, Boerne, USA), glycerol and triglycerides (Serum Triglyceride Determination kit; Sigma-Aldrich, St. Louis, USA), and cholesterol (Cholesterol E kit; Wako Chemicals USA), were measured in plasma according to manufacturer instructions, but scaled down to a 96-well format. Plasma lactate (Lactate Colorimetric/Fluorometric Assay Kit; BioVision, Milpitas, USA) was measured according to manufacturer instructions. For the determination of plasma alanine concentration, plasma samples were deproteinized by addition of cold Tri-chloro-acetic acid (3.33%) to plasma (3:1 ratio) to final concentration of 2.5%, incubated on ice for 10 min, followed by centrifugation at 4˚C at 21,000 xg for 15 min. Supernatants were assayed for alanine using an alanine determination kit (Alanine Colorimetric/Fluorometric Assay Kit; BioVision). Plasma insulin levels were determined using a Mouse Ultrasensitive Insulin Enzyme-Linked Immunosorbent Assay (ELISA) Kit (ALPCO Diagnostics; Salem, USA) with either the 5 or 25 μL format. Leptin (Mouse Leptin ELISA; Crystal Chem, Inc., Downers Grove, USA), IGFBP2 (Mouse/Rat IGFPB2 ELISA, ALPCO Diagnostics), growth hormone (Rat/Mouse Growth Hormone ELISA; EMD Millipore, Billerica, USA), total corticosterone (Corticosterone ELISA, ALPCO Diagnostics)  and glucagon (Mouse Glucagon RIA, EMD Millipore, Billerica, USA) were measured in plasma according to manufacturer instructions. For glucagon measurements, blood was collected in tubes containing the same concentration of heparin as above, and supplemented with 1.5 μL aprotinin (Sigma-Aldrich) per 100 μL whole blood to achieve a final concentration of approximately 380 KIU/mL.   33 2.3 CHEMICAL MANIPULATIONS Streptozotocin (STZ)-induced diabetes Mice received a single high-dose intraperitoneal (i.p.) injection of STZ (Sigma-Aldrich) prepared in acetate buffer (118 mmol/L sodium acetate, 38.5 mmol/L NaCl, pH 4.5). Studies performed at D.H. Copp used a dose of 200 mg/kg for C57Bl/6 mice and 180 mg/kg for Leprflox/flox and Leprflox/flox Albcre mice. In studies performed at the CDM, STZ doses of 180 mg/kg for C57Bl/6 mice and 170 mg/kg for Leprflox/flox and Leprflox/flox Syncre mice were used. We found that C57Bl/6 mice purchased directly from the Jackson Laboratory had a greater incidence of STZ-induced toxicity in the CDM than in D.H. Copp, which is why the dose was reduced from 200 to 180 mg/kg. Similarly, we found that the Leprflox/flox strains appeared more sensitive to STZ-induced toxicity than C57Bl/6 mice, which is why doses were slightly lower in this strain than in C57Bl/6 mice. Chemically-induced sympathectomy (6-hydroxydopamine) When administered peripherally, 6-hydroxydopamine (6OHDA) causes selective degeneration of noradrenergic nerve terminals, resulting in a chemically-induced sympathectomy (314). C57Bl/6 mice, aged 12 weeks, were injected i.p. with 250 mg/kg 6-hydroxydopamine hydrobromide (6OHDA, Sigma-Aldrich), prepared in sterile saline containing 0.05% L-ascorbic acid (Sigma-Aldrich) (315) 4 weeks prior to STZ administration. 2.4 SURGICAL MANIPULATIONS Leptin delivery via mini-osmotic pump implantation Recombinant murine leptin was delivered via ALZET mini-osmotic pumps (DURECT Corporation; Cupertino, USA) for either 7 days (model 1007D), 14 days (model 1002), 4 weeks (model 2004), or 6 weeks (model 2006) as indicated. Initial studies were performed using leptin from the National Hormone and Peptide Program (NHPP; Torrance, USA) at a dose of 10 μg/day. We later switched to using leptin from Peprotech (PeproTech, Inc., Rocky Hill, USA). In studies performed in the D.H. Copp building with NHPP leptin we found that 10 μg/day leptin was sufficient to normalize hyperglycemia in diabetic C57Bl/6 mice. While performing subsequent experiments in the CDM, we found that 10 μg/day NHPP or 10 μg/day Peprotech leptin were not sufficient to fully normalize blood glucose levels, and thus switched to the use of 20 μg/day Peprotech leptin. Peprotech leptin was used in most  34 experiments, therefore the use of NHPP leptin is indicated in figure legends. NHPP leptin was reconstituted in sterile PBS, and Peprotech leptin was reconstituted in sterile water. Vehicle controls received the same vehicle that was used to reconstitute leptin. Peprotech and NHPP leptin were soluble up to a concentration of 2 mg/mL, and we found that concentrations exceeding this (3.8 mg/mL) resulted in lack of leptin release from osmotic pumps. All experiments were performed using pumps filled with leptin that did not exceed 2 mg/mL in concentration. Following the manufacturer specified equilibration period at 37 °C in sterile PBS, pumps were implanted subcutaneously into mice on day 0. Mice were anesthetized with inhalable isoflurane, and given subcutaneous injections of ketoprofen (5 mg/kg; Merial; Baie d'Urfé, Canada) and bupivicaine (6 mg/kg; Hospira Healthcare Corporation; Montreal, QC, Canada) prior to surgery. Non-diabetic control mice underwent a sham operation, and did not receive osmotic pumps unless otherwise specified. Surgical incisions were closed with wound clips and/or sutures. Implantation of temperature transponders  As a measurement of BAT thermogenesis, interscapular temperature was measured in mice by implanted temperature transponders (Implantable Programmable Temperature Transponder IPTT-300; Bio Medic Data Systems Inc, Seaford, USA). Temperature transponders were implanted at the same time as osmotic pump implants, by inserting a needle loaded with the transponder in the cranial direction through the incision made for the pump implant, and ejecting the transponder subcutaneously between the scapulae. Interscapular temperatures were recorded in non-fasted mice using a hand held Pocket Scanner (DAS-5007; Bio Medice Data Systems Inc). Subdiaphragmatic vagotomy C57Bl/6 mice received subdiaphragmatic vagotomies or sham surgeries via the ventral abdominal approach at 6 weeks of age, performed by Jackson Laboratories (Bar Harbor, ME) as previously described (316), and mice were allowed to recover from surgery for 1 week prior to shipment. Briefly, a 2.0 cm skin incision was made immediately caudal to the xiphoid process in isoflurane-anesthetized mice and the underlying muscle was incised exposing the stomach. Adjacent to the esophagus a section of the dorsal and ventral vagal trunks were excised cranial to the stomach. In sham operated mice the vagus was exposed  35 but not excised. The incision in the abdominal wall and skin were closed separately and the skin closure material was also removed prior to shipment.  Islet isolation for transplantation Twelve week old male C57BL/6J mice were obtained from the CDM for the isolation of islets. The islet isolation procedure was performed with reference to techniques originally described by Lacy and Kostianovsky (317), and a filtration modification previously outlined by Salvalggio et al. (318). Mice were euthanized and pancreatic duct injections were performed with the assistance of Dr. Eva Tudurí, using 1000 U/mL collagenase type XI (Sigma-Aldrich) prepared in filter-sterilized Hank’s balanced salt solution (HBSS – 137 mmol/L NaCl, 5.4 mmol/L KCl, 4.2 mmol/L NaH2PO4, 4.1 mmol/L KH2PO4, 10 mmol/L HEPES, 1 mmol/L MgCl2, and 5 mmol/L glucose, pH 7.2). Following duct injection, and excision of pancreas, digestion of pancreatic exocrine tissue proceeded for 11 min at 37°C, and was stopped with the addition of ice-cold HBSS supplemented with 1 mmol/L calcium chloride. A 70 µm cell strainer was used for the initial filtration of islets, after which, islets were purified from remaining exocrine tissue and pancreatic lymph nodes by a minimum of two passes of hand-picking. Following isolation, islets were cultured at 37°C (humidified air, with 5% CO2), in a complete medium of Ham’s F-10 nutrient mixture containing L-glutamine and sodium bicarbonate (Sigma-Aldrich), supplemented with 7.5% bovine serum albumin (Life Technologies; Burlington, ON, Canada), and 1% penicillin-streptomycin (Life Technologies) for 1-2 days prior to transplantation. Immediately preceding transplantation, islets were aliquotted to ensure that each dose contained the specified number of islets, and a uniform proportion of 1-day-old and 2-day-old islets. Each dose was composed of an equal fraction of small- and medium-sized islets, as it has been previously suggested that smaller islets may be more advantageous for islet transplant efficacy (319). Islet aliquots were washed and prepared in sterile PBS, and drawn up into polyethylene-50 (PE50) tubing with a micromanipulator for transplantation. Islet transplantation Mice were anaesthetized with inhalable isoflurane, and given subcutaneous injections of ketoprofen (5 mg/kg), bupivicaine (6 mg/kg), buprenorphine hydrochloride (0.05 mg/kg; Reckitt Benckiser Healthcare (UK) Ltd., Slough, UK), and baytril (10 mg/kg, Bayer Inc., Toronto, ON, Canada) prior to surgery. Transplantation of islets, and implantation of pumps  36 occurred in the same surgical session 6 days following STZ treatment on day 0. Islets were transplanted beneath the left kidney capsule, as described by Dong-Sheng et al. (320). Briefly, once the mouse was in the surgical plane, an approximately 1.5 cm incision through the skin and muscle layer was made in the region of the left kidney. The kidney was externalized with a saline-wetted cotton-tipped swab, and a superficial nick made in the renal capsule with a 30G needle. A glass probe was used to clear a subcapsular pocket to receive prepared islets, and the deposition of islets contained within PE50 tubing was driven by a micromanipulator. Following transplantation, the kidney nick was cauterized and the kidney was re-internalized. The overlying muscle layer was closed with a continuous internal absorbable suture and the skin was closed with discontinuous sutures (5-0 monocryl sutures; Ethicon, Inc., Markham, ON, Canada). Mice not receiving islet transplantation underwent a sham operation. Islet transplantations were performed by Dr. Jennifer Bruin with assistance from Heather Denroche, Whitney Quong and Jessica Fox.  2.5 BODY COMPOSITION Body composition was measured by Dual Energy X-ray Absorbance (DEXA), using a LunarPIXImus 2.0 Densitometer (Inside Outside Sales, Madison, USA), in collaboration with the laboratory of Dr. S. Clee, and with assistance from S. Karunakaran. Mice were either anesthetized by isoflurane inhalation, (or in Chapter 6 were euthanized with CO2 inhalation) immediately prior measurements. 2.6 INDIRECT CALORIMETRY AND METABOLIC CAGES Indirect calorimetry, activity, and food intake were measured using PhenoMaster metabolic cages (TSE Systems, Chesterfield, USA) in collaboration with Dr. S. Clee and with assistance from S. Karunakaran. Mice were singly housed in metabolic acclimitization cages (same cage set up, without performing indirect calorimetry) for a minimum of 72 hours to allow for acclimitization prior to metabolic measurements. The metabolic run was initiated for a minimum of 72 hours, with measurements collected every every 15-16 min. Data collected from the first light and first dark were discarded. Dark and light cycle values were averaged from 2 cycles, and daily values were averaged from 2 days. Measurements in Chapter 6 were taken from mice housed at 29˚C, and measurements in Chapter 4 were taken from mice housed at 21˚C. Body composition was measured immediately after the metabolic cage run, and energy expenditure was normalized to lean mass by analysis of covariance (ANCOVA; Systat Software, Chicago, USA). Cumulative food intake per light or  37 dark cycle was calculated for mice in Chapter 4. For food intake in Chapter 6, cumulative food intake over the duration of the metabolic run was used, because some leptin treated mice would not eat from the food hopper, and had to have food placed on the cage bottom. Food eaten from the cage bottom was calculated as the difference in weight prior to and after the metabolic cage run, and added to any food eaten from the hopper. Cumulative locomotor activity was calculated as the total beam breaks in the XY plane per light or dark cycle or per day as indicated. Both ambulatory and fine motor movement were included as total activity. Respiratory exchange ratio (RER) was determined and expressed as a function of time. 2.7 IN VIVO ASSAYS Glucose tolerance and glucose-stimulated insulin secretion tests For oral glucose tolerance tests (OGTTs), following a 6-hour fast, mice were given an oral gavage (1.5 or 2 g/kg body weight) of glucose, and blood glucose was measured from the saphenous vein at the indicated time points post gavage. For the i.p. glucose tolerance test (i.p.GTT) in Chapter 3, mice were fasted for 4 hours and given an i.p. injection (1.5 mg glucose/g body weight) of glucose dissolved in sterile PBS, and blood glucose was monitored at the indicated time points post injection. Where glucose-stimulated insulin secretion was investigated, blood was collected at the indicated time points in the figure following a glucose gavage of 1.5 or 2 mg/kg body weight following a 6 hour fast. Neuroglycopenic-induced glucagon secretion test Mice were fasted for 4 hours, and subsequently received 500 mg/kg 2-deoxyglucose (Sigma-Aldrich) dissolved in sterile saline via i.p. injection at time 0. Vehicle injected controls received sterile saline injections. Blood was collected at 15 min post injection for the measurement of plasma glucagon. Insulin tolerance test (ITT) For ITTs, mice were fasted for 4 hours and given an i.p. injection of  0.65 to 0.85 U/kg of human synthetic insulin (Novolin® ge Toronto, Novo Nordisk). Blood glucose was measured from the saphenous vein at the indicated time points post injection. The dose for each experiment is indicated in the figure legend.     38 Pyruvate and glycerol tolerance tests Mice were fasted for 7 hours, the maximum length of time allotted such that STZ-leptin mice would not reach lethal hypoglycemia during the subsequent 2 hour test. In both experiments, half of the mice in each treatment group (non-diabetic, STZ-vehicle, and STZ-leptin) received either glycerol (2.5 g/kg glycerol solution dissolved in sterile water)/pyruvate (2 g/kg dissolved in sterile saline), or corresponding vehicle injection i.p. at time 0, and blood glucose was measured at the indicated times from the saphenous vein. Fasting tolerance test Blood glucose/blood samples were collected prior to fasting (9AM), and subsequently at indicated times following food removal, until STZ-leptin mice became too hypoglycemic to continue fasting (<2 mmol/L). Glucose uptake Four-hour fasted mice were anesthetized with an injectable cocktail of acepromazine (Atravet), midazolam (Versed) and fentanyl (Hynorm). Mice received a tail vein injection of 40 µCi/kg body weight 2-deoxy-D-[14C]glucose (PerkinElmer) in citrate buffer (10 mmol/L sodium citrate, 0.03% BSA) with or without 0.3 U/kg insulin (Novolin). Mice were euthanized 20 min post-injection by cervical dislocation and the soleus muscle was isolated, weighed and flash frozen in N2 (l). 2-deoxy-[14C]glucose-6 phosphate accumulation in soleus muscle was determined as described by Ferré et al. (321) and normalized to tissue mass. Samples for radioactivity measurements were added to scintillation fluid (Ready Safe, Beckman Coulter) and counted using an LS Analyzer (Beckman Coulter). Cholecystokinin (CCK)-induced satiety CCK octapeptide (26-33) (American Peptide, Sunnyvale, CA) was first dissolved in 0.05% w/v ammonium hydroxide (NH4OH) in saline to a concentration of 0.33 mg/mL to facilitate solubilization, and subsequently diluted in sterile saline to an appropriate concentration for injection. Following an overnight fast, mice received an i.p. injection of CCK or vehicle i.p., and were individually placed into a cage with a pre-weighed amount of food, and allowed to feed for 1 hour. Food intake was measured as the difference in food weight prior to and one hour post injection.  39 2.8 LIVER METABOLITES Livers were collected from mice anesthetized with isoflurane, immediately prior to euthanasia, and clamp frozen with N2 (l), and stored at -80˚C until use. For hepatic glycogen content, 10 mg tissue was homogenized in 1 mL of water using a handheld pestle motor, incubated for 5 min at 95˚C, and measured according to manufacturer instructions (Glycogen Colorimetric/Fluorometric Assay Kit; BioVision). Hepatic glucose measurements were determined using this kit without adding glycogen phosphorylase enzyme. For hepatic triglyceride and cholesterol measurements, liver was homogenized in 3 mL of chloroform:methanol (2:1) and extracted with water. Glycerol was quantified from the aqueous phase using Free Glycerol Reagent (Sigma-Aldrich). The organic layer was dried under N2 (g) and 10 μL of Thesit (Sigma-Aldrich) was added and mixed under N2 (g). Water (100 μL) was added and incubated at 37˚C for 1 hour with intermittent vortexing. Triglycerides were quantified using the Serum Triglyceride Determination kit (Sigma-Aldrich) and cholesterol was assayed using the Cholesterol E Kit (Wako Chemicals USA). Hepatic coenzyme A (CoA) esters were measured by reversed phase high performance liquid chromatography (HPLC) as described (322). CoA standards were prepared in 100 mmol/L sodium phosphate buffer, pH 3.0. Samples were prepared by perchloric acid extraction of powdered freeze-clamped mouse liver. Separation of CoA compounds was carried out using a two-pump grandient system performed using a modular liquid chromatograph (Waters 2695 Alliance Separations Module HPLC System) on a Waters Nova Pak C18, 4 μm, 3.9 x 300 mm column. Buffer A (200 mmol/L sodium phosphate, pH 5.0) and Buffer B (3.8 mol/L acetonitrile, 200 mmol/L sodium phosphate, pH 5.0) were prepared from pre-charcoal purified phosphate solution. Samples were injected and separated using a flow rate of 1 mL/min at ambient temperature, with the following conditions: 3% Buffer B 2.5 minutes, increase to 18% Buffer B for 5 minutes, a linear increase to 28% Buffer B at 10 minutes, 28% Buffer B held constant for 2 minutes, linear increase to 37% Buffer B from 12-15 minutes, 37% Buffer B held constant until 17 minutes, linear increase to 90% Buffer B at 34 minutes, and 90% Buffer B maintained for 3 minutes, and then returned to initial conditions at 37.5 minutes. Data were collected for 40 minutues, and amounts of each compound are calculated using a peak height mode of integration, and compared to chromatographic separation of CoA standards.    40 2.9 GASTRIC DISTENSION  Stomachs were harvested from vagotomized, and sham treated mice on day 32 post-pump implant following a 4-hour fast, residual gastric contents were removed, and wet stomach weights were measured. 2.10 PCR PCR analysis of Cre-mediated recombination To assess the tissue specificity of Leprflox recombination, tissues were harvested from 12 week old female Leprflox/flox Syncre and Leprflox/flox mice, and frozen at -20˚C until use. Note that islets were isolated as described under 2.4.3, with the exception that after filtration islets were hand-picked to approximately 98% purity in HBSS supplemented with 1 mmol/L calcium chloride, washed in PBS, and frozen. Frozen tissues were homogenized in salt homogenization buffer (0.4 M NaCl, 10 mmol/L Tris-HCl pH 8.0, 2 mmol/L EDTA, 1% SDS and 0.25 mg/mL proteinase K), and DNA was extracted as previously described (323). PCR was performed with 1 μM each of primers mLepr101 (ATG CTA TCG ACA AGC AGC AGA ATG ACG) and mLepr102 (CAG GCT TGA GAA CAT GAA CAC AAC AAC) (Figure 1). The predicted product size of the Leprflox allele is 1369 bp and the LeprΔ17 allele has an expected product size of 952 bp. RT-PCR  Liver tissue was homogenized in Trizol (Invitrogen, Burlington, Canada) using a tissue homogenizer (Tissue Tearor, Biospec Products). RNA was extracted following manufacturer instructions and 1 g of RNA was used for cDNA synthesis using an iScript cDNA Synthesis kit (Bio-Rad, Mississauga, Canada). Primers G (TAT TCC CAT CGA GAA ATA TCA) and 60 (AGG CTC CAA AAG AAG AGG ACC) (shown in  Figure 1), were used to amplify Lepr-b cDNA are described previously (206). Quantification of wildtype Lepr-b transcript in brain of Leprflox/flox Syncre mice Brain regions were macrodissected and flash frozen in N2 (l) from isoflurane-anesthetized mice immediately following sacrifice. RNA was isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen, Mississauga, Canada) according to manufacturer’s instructions. Briefly, tissue was disrupted and homogenized in 1 mL of QIAzol with a handheld pestle motor, and RNA extracted via chloroform phase separation. On column DNase I digestion  41 was performed using a RNase free DNase kit (Qiagen) for 15 min at room temperature, after which RNA was washed and subsequently eluted in two 30 µL volumes of nuclease free water. RNA was stored at -80˚C. cDNA was synthesized from 310 µg of RNA using a combination of random hexamer and oligo(dT) primers (iScript cDNA Synthesis kit, BioRad). Reverse transciptase negative control (RT-) reactions were prepared with the same conditions, and water instead of RT enzyme. Reactions were incubated as follows: 5 min at 25˚C, 30 min at 42˚C, 5 min at 85˚C. cDNA samples were stored at -80˚C. Quantitative PCR (qPCR) was performed using LeprE17 primers (Table 2) that specifically amplify wildtype Lepr-b (refer to Figure 1) in duplicate using Eva Green SsoFast super mix with Low ROX (BioRad) in a 20 μL reaction containing 2 μL of 1:5 diluted cDNA and 0.2 μM of each primer, using a StepOnePlus Real-Time PCR System (Life Technologies Inc, Burlington, Canada) and the following reaction conditions: 95˚C 10 min, 40 cycles of 95˚C 15s, 60˚C 60s, and melt curve 95˚C 15s, 60˚C 60s, +0.3°C/s. Five reference genes were tested for stability across experimental groups, phosphoglyceratekinase (Pgk1), hypoxanthine phosphoribosyltransferase (Hprt), peptidylprolyl isomerase A (Ppia), beta-actin, and glyceraldehyde 3-phosphate dehydrogenase, bygeNorm analysis using BioGazelle qBase+ software (Zwijnaarde, Belgium) in accordance with MIQE guidelines (324). Relative transcript abundance was determined by the Pfaffl method (325), in which the ratio of the target gene amplification efficiency (raised to the difference in Ct values for the target gene between a calibrator sample and an experimental sample), to the reference gene amplification efficiency (raised to the difference in Ct values for the reference gene between the same calibrator and experimental sample) is calculated.  Quantification of Lepr-b and Socs3 transcripts in HFD fed mice Samples were collected and RNA extracted as above. cDNA was synthesized as above from a total of 500 ng RNA. qPCR was performed as above using primers for LeprE17 (Figure 1) and Socs3 (Table 2). Hypoxanthine phosphoribosyltransferase (Hprt) was selected as a reference gene by geNorm analysis as above. Transcript abundance relative to cDNA from a Leprflox/flox LFD fed control mouse was determined by the Pfaffl method (325).     42 Quantification of hepatic transcripts  Small samples from the left liver lobe were collected immediately from mice euthanized by cervical dislocation while under isoflurane anesthesia, and were placed in RNAlater (Qiagen) overnight at 4˚C. RNAlater was subsequently removed and samples were frozen and stored at -80˚C. RNA was harvested using TRI Reagent (Applied Biosciences, Carlsbad, USA), and subsequently DNase treated (DNase I (RNase-free), New England, Biolabs, Pickering, Canada) according to the manufacturer instructions.RNA samples were stored at -80˚C. cDNA targets were amplified with the appropriate primers (Table 2) in SsoFast EvaGreen Supermix with Low ROX master mix (BioRad, Mississauga, Canada) as described above, with 1:100 diluted cDNA as template, and the following reaction conditions: 95˚C 30 s, and 40 cycles of 95˚C 5 s – 62.5/64 ˚C 20 s, and melt curve 95˚C 15s, 60˚C 60s, +0.3°C/s. Peptidylprolyl Isomerase A (Ppia) was selected as a reference gene by geNorm analysis as above. Transcript abundance relative to cDNA from a non-diabetic control mouse was determined by the Pfaffl method (325).  Table 3. Primer sequences for qPCR. Target Forward Primer Reverse Primer Aqp9 5’-GTGCCTTTGTAGACCAAGTGGTG-3’ 5’-GTGAAGACCTCAAATCCCCATCCT-3’ Glut2 5’-GATGAGTTACGTGAGCATGACTGC-3’ 5’-ACCACCCCAGCGAAGAGGAAG-3’ Hprt1 5’-GCTGACCTGCTGGATTACAT-3’ 5’-TTGGGGCTGTACTGCTTAAC-3’ LeprE17 5’-AAGTTGTTTTGGGACGATGTTCC-3’ 5’-GGGACCATCTCATCTTTATTTTTC-3’ Pepck 5'-GCAGAACACAAGGGCAAGATCATC-3' 5'-GATGTAGCCGATGGGCGTG-3' Pgk1 5’-GCAGATTGTTTGGAATGGTC-3’ 5’-TGCTCACATGGCTGACTTTA-3’ Ppia 5’-AGCTCTGAGCACTGGAGAGA-3’ 5’-GCCAGGACCTGTATGCTTTA-3’ Socs3 5’-AGCTCCAAAAGCGAGTACCAG-3’ 5’-GCTGTCGCGGATAAGAAAGGT-3’ 2.11 HISTOLOGY Tissue processing Mouse pancreata were post-fixed overnight in 4% paraformaldehyde (in PBS, pH 7.2) at 4˚C, rinsed in 70% ethanol, embedded in paraffin and processed for sectioning by Wax-it Histology Services (Vancouver, Canada). Paraffin sections were deparaffinized and hydrated through a series of xylene and ethanol baths, treated by antigen retrieval for 10 minutes at 95˚C, and blocked by serum free protein block (DakoCytomation Inc., Carpinteria, USA).   43 Cre-mediated recombination in mTmG Syncre reporter pancreata To determine whether Syncre recombination occurs in pancreatic islets, EGFP immunopositive area was determined in pancreata from mTmG Syncre and mTmG control mice. As a positive control for EGFP immunopositivity in pancreatic islets, we used a pancreatic section from an mTmG mouse that had been injected with 50 x 1012 viral genomes of adeno-associated virus (AAV) vector carrying a rat insulin promoter driven cre (RIPcre) 26 days prior to pancreas collection. One section per mouse was immunostained for insulin (guinea pig anti-insulin antibody, Cat# I8510, 1:1000, Sigma-Aldrich), glucagon (mouse anti-glucagon antibody, Cat# G2654, 1:1000, Sigma-Aldrich), and EGFP (rabbit anti-GFP antibody, Cat#A-11122, 1:1000, Invitrogen) overnight at 4C. Slides were then incubated with AlexaFluor-conjugated secondary antibodies (Life Technologies, Burlington, Canada) for one hour at room temperature, and mounted using Vectashield Hard Set Mounting Medium for Fluorescence with DAPI (Vector Laboratories, Burlingame, USA). Whole sections were scanned at 20X magnification, using ImageXpress Imaging System (Molecular Devices Corporation, Sunnyvale, USA). Individual images were stitched together to recreate the entire pancreatic section. EGFP positive area was measured relative to total area using Positive Pixel Count Algorithm on MetaXpress software (Molecular Devices) as we have previously described (326). Quantification of β-cell and α-cell area Three sections separated by 200 µm per mouse were immunostained for insulin (guinea pig anti-insulin antibody, Cat# I8510, 1:1000, Sigma-Aldrich) and glucagon (mouse anti-glucagon antibody, Cat# G2654, 1:1000, Sigma-Aldrich) overnight at 4C. Slides were then incubated with AlexaFluor-conjugated secondary antibodies and mounted as above. Whole sections were scanned and stitched as above. Total insulin- or glucagon-positive area relative to total pancreas area using Positive Pixel Count Algorithm as above and averaged across 3 pancreas sections per mouse. Tyrosine hydroxylase (TH) immunoreactivity in BAT  BAT was harvested from mice 19 days post-surgery, post-fixed overnight in 4% paraformaldehyde at 4C, incubated in 25% sucrose in PBS for 72 hours, and subsequently frozen in Tissue Tek OCT Compound (Sakura Finetek, Alphen aan den Rijn, The Netherlands) over an isopentane bath. Frozen BAT blocks were sectioned by Wax-it  44 Histology Services Inc. (Vancouver, Canada). Sections were immunostained for TH (rabbit anti-TH, EMD Millipore, Cat# AB152, 1:1000 dilution) overnight at 4C. Sections were subsequently incubated in AlexaFluor-conjugated secondary antibody and mounted as described above. Whole sections were scanned at 20X magnification with IXMB imaging system, and images were stitched and analyzed by MetaXpress software as previously described (327). Total TH positive area was expressed relative to total cell count (based on DAPI fluorescence), and to total section area. Preparation of brain sections Mice were deeply anesthetized by isoflurane inhalation, and were transcardially perfused with PBS until blood ran clear, followed by perfusion with 4% paraformaldehyde for 12 min. Brains were immediatedly collected and fixed overnight in 4% paraformaldehyde at 4˚C. Brains were subsequently incubated in 25% sucrose in PBS for approximately 72 h at 4˚C until brains had sunk to the bottom of the vial. Brains were subsequently frozen at -39 to -41˚C in an isopentane-dry ice bath for 60 s, and stored at -80˚C. Frozen brains were sectioned (30 μm) on a sliding microtome in a one in six coronal series through the extent of the hypothalamus by Dr. Maria Glavas. Cre-mediated recombination in mTmG Syncre reporter brains Every 6th section containing hypothalamus or NTS was incubated in rabbit anti-GFP antibody (Cat#A-11122, 1:2000 Invitrogen) in 50 mmol/L KPBS with 0.4% Triton X-100) for 2 days at 4˚C. Sections were washed and subsequently incubated for 1 hour in AlexaFluor-conjugated secondary antibodies (Life Technologies) in 50 mmol/L KPBS with 0.4% Triton X-100). Sections were mounted on gelatin-coated glass slides in anatomical order, dried overnight and subsequently dehydrated and cleared through a series of ethanol-xylene washes, and hardmounted with DAPI fluorescence. Whole sections were scanned at 20X magnification, using ImageXpress Imaging System (Molecular Devices Corporation). Individual images were stitched together to recreate the entire brain section. Functional analysis of hypothalamic leptin receptor signalling Brains were collected from mice perfused transcardially with 4% paraformaldehyde (in borate buffer, pH9.5), 45 min after injection of 2 μg/g body weight mouse leptin (Peprotech). Every 6th section containing hypothalamus was incubated in rabbit anti-P-STAT3 (Try705)  45 (cat# 9145, Cell Signalling, 1:250 in 50 mmol/L KPBS with 0.4% Triton X-100) for 2 days at 4˚C. Sections were washed 4x10 min in 50 mmol/L KPBS, followed by 1 hour incubation in secondary antibody (biotinylated donkey-anti rabbit, cat#711-066-152, 1:600 in 50 mmol/L KPBS with 0.4% Triton X-100). Sections were again washed 4x10 min in 50 mmol/L KPBS, and subsequently incubated for 1 hour in A/B solution (Vectastain Elite ABC Kit Standard, PK-6100 diluted in 50 mmol/L KPBS with 0.4% Triton X-100). Sections were washed, and subsequently incubated in 2x10 min 175 mmol/L sodium acetate pH 7.3, followed by Nickel DAB solution (0.5 g nickel (II) sulphate hexahydrate, 200 uL DAB, 16 uL of 3% H2O2 in 20 mL 175 mmol/L sodium acetate). Sections were mounted on gelatin-coated glass slides in anatomical order, dried overnight and subsequently dehydrated and cleared through a series of ethanol-xylene washes, and hardmounted. The number of P-STAT3 positive nuclei was manually counted and totalled over 5 anatomically matched sections per mouse in the ARH, VMH, DMH, LHA and PMV, and averaged for each group.  2.12 DATA ANALYSIS Data and statistics were analyzed using GraphPad Prism (GraphPad Software, La Jolla, USA) with the following exceptions: Chapter 3 Figures 3-4, 6-10, and Chapter 5 Figures 38-39, which were published in Denroche et al. (328) and analyzed using SigmaPlot 12.0 (Systat, San Jose, USA). In Chapter 3 multiple comparisons were performed with student’s t-tests, however analyses made by one-way ANOVA yielded similar statistical results. Also in Chapter 3, tracking data was analyzed by one-way repeated measures ANOVA, but two-way repeated measures ANOVA yielded similar results. In other chapters multiple comparisons were made by one- or two-way ANOVA with either Bonferroni or Tukey post-hoc tests. Data are presented as mean ± standard error of the mean (SEM). The method of statistical analyses is indicated in each figure legend. Statistical significance was set at P < 0.05. The blood glucose effect size between STZ-vehicle controls and STZ-10 or 20 μg/day leptin treated experimental groups  on the C57Bl/6 background was very large (d = 9.1) and hence the power of these studies was calculated to be 0.999 for an n of 3. The effect size between STZ-vehicle controls and STZ-10 or 20 μg/day leptin treated groups in mice on the Leprflox/flox background  was also relatively large (d = 2.2), resulting in a power of 0.448 for an n of 3, and 0.786 for an n of 5.  46 CHAPTER  3: LEPTIN THERAPY REVERSES METABOLIC DISTURBANCES IN STZ-DIABETIC MICE 3.1 INTRODUCTION Since the discovery of insulin almost a century ago (22), insulin therapy has been the predominant treatment for type 1 diabetes. However, recent studies revealed that administration of leptin could reverse hyperglycemia and prevent mortality when administered to rodent models of type 1 diabetes (287-291). Although, leptin has well known glucose lowering effects in leptin deficient Lepob/ob mice, the fact that leptin can restore euglycemia in insulin-deficient rodents is surprising, and the mechanism underlying this effect is unclear. Insulin-deficient diabetes is associated with elevated circulating glucagon, which contributes to hyperglycemia (296,329-331). Yu et al. previously demonstrated that leptin therapy reverses hyperglucagonemia in STZ-diabetic rats and non-obese-diabetic (NOD) mice (288), which may contribute to the restoration of metabolic control in these animals. Insulin resistance is another common characteristic of untreated human and rodent insulin deficiency (286,332-334). Exogenous leptin has been shown to improve insulin sensitivity in STZ-diabetic rats (286,287,335). Therefore the insulin sensitizing effect of leptin may also contribute to lowering blood glucose in type 1 diabetic rodents, yet this pathway has not been fully investigated as many studies have assumed that type 1 diabetic rodents are completely devoid of insulin (288,289).  In this chapter we investigated the metabolic effects of leptin therapy in STZ-induced diabetic mice as a model of type 1 diabetes. Peripheral leptin therapy in the STZ-diabetic mouse model had not been reported in the first studies testing leptin therapy; therefore, we performed a thorough characterization of the effects of leptin therapy on metabolic homeostasis in STZ-diabetic mice, and its effect on insulin sensitivity and counter-regulatory hormones, in order to gain insight onto potential mechanisms of leptin action in type 1 diabetes1.                                                    1 Figures 3,4, and 6-10 were published in the journal Diabetes (328). Figure 5 is unpublished.  47 3.2 RESULTS Leptin therapy ameliorates hyperglycemia in STZ-diabetic mice  We first characterized the effects of leptin therapy on metabolic parameters in STZ-diabetic mice. STZ-diabetic C57Bl/6 mice were treated with 10 μg/day recombinant mouse leptin (STZ-leptin) or saline (STZ-vehicle), delivered by 14 day osmotic pumps implanted on day 0, and were also compared to mice that were injected with acetate buffer instead of STZ (non-diabetic). STZ administration depleted 4-hour fasted plasma leptin levels from 1.2 ± 0.5 ng/mL in non-diabetic controls to below the detection limit (< 0.2 ng/mL) in STZ-vehicle mice (Figure 3A). Administration of 10 μg/day leptin resulted in a 7 fold increase in plasma leptin levels compared to those of non-diabetic controls. Since the level of leptin achieved by leptin administration is within the physiological range for diet-induced obese mice, but is greater than those of non-diabetic lean controls, we defined this as a supraphysiological dose of leptin. Leptin levels on day 14 were not significantly different from values obtained on day 7. By 7 days of treatment, 4-hour fasted blood glucose concentrations in STZ-leptin mice were reduced to non-diabetic levels (6.1 ± 0.7 mmol/L STZ-leptin, 8.0 ± 0.2 mmol/L non-diabetic, 23.2 ± 1.3 mmol/L STZ-vehicle) (Figure 3B), and remained significantly lower than STZ-vehicle controls for the duration of the study. Interestingly, on day 7, fasting blood glucose was significantly lower in STZ-leptin mice compared to non-diabetic controls (P = 0.018). Although blood glucose levels rose in some individual STZ-leptin mice on day 14, average blood glucose was not significantly different compared to non-diabetic controls on day 14 (P = 0.16). We next examined if leptin therapy ameliorated hyperketonemia. β-hydroxybutyrate, a predominant ketone body, was drastically elevated in plasma from 4-hour fasted STZ-vehicle mice compared to non-diabetic controls, an effect which was fully reversed by leptin therapy (Figure 3C). Since high doses of leptin can reduce body weight in some rodent models, we investigated if leptin treatment exacerbated weight loss in STZ-diabetic mice. STZ treatment caused a steady decline in body weight (Figure 3D), however STZ-leptin mice lost a similar amount body weight to STZ-vehicle controls, indicating that leptin did not exacerbate weight loss induced by insulin deficiency.  48  Figure 3. Leptin therapy reverses hyperglycemia and hyperketonemia in STZ-diabetic mice. Four-hour fasted parameters in STZ-leptin treated (red), STZ-vehicle (blue), and non-diabetic (black) mice. Male C57Bl/6 mice were injected with STZ on day -5, and received subcutaneous 14 day osmotic pump implants delivering a 10 μg/day dose of leptin (supplied by NHPP) or saline as vehicle on day 0. Non-diabetic controls received vehicle pumps on day 0. A) Plasma leptin levels  n = 5. Day 7 samples were collected from the saphenous vein, day 14 from cardiac puncture. Leptin was undetectable in plasma samples from STZ-vehicle mice and were assigned the limit of detection (0.2 ng/mL). B) Blood glucose, n = 5. C) Plasma β-hydroxybutyrate , n ≥ 4. D) Body weight, n = 5. A, C) Statistical analyses were performed by Student's t-test. Statistically significant groups were consistent when analysis was performed via one-way ANOVA. B, D) Statistical analyses were performed by one way repeated measures ANOVA with a Holm-Sidak post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle; † P < 0.05 STZ-leptin vs non-diabetic; ‡ P < 0.05 STZ-vehicle vs non-diabetic. Data are expressed as mean ± SEM. Figure adapted from Denroche et al. (328). Hyperglycemia is rapidly restored upon cessation of leptin therapy  We next examined whether the glucose lowering effect of hyperleptinemia in insulin-deficient mice persisted after cessation of leptin therapy. STZ-diabetic C57Bl/6 mice were  49 treated with 5 μg/day leptin or vehicle via 14 day osmotic pumps implanted on day 0 (Figure 4). Blood glucose gradually declined over the treatment period, and hyperglycemia was ameliorated by day 14 of leptin therapy. Following pump explantation, hyperglycemia rapidly returned in the STZ-leptin group. At this point, the STZ-vehicle group had to be euthanized due to deteriorated body condition. When leptin was re-administered to STZ-leptin mice at a dose of 24 μg/day with a second pump implant hyperglycemia resolved within just 3 days of treatment. These data suggest the maintenance of euglycemia in this model requires continuous leptin administration and that the rate of glucose lowering following leptin administration may be dose dependent.  Figure 4. The effect of leptin therapy on STZ-induced hyperglycemia does not persist beyond leptin therapy cessation. Four-hour fasted blood glucose in STZ-leptin (red) and STZ-vehicle (blue) mice, n = 7. Male C57Bl/6 mice received STZ on day -5 and were subsequently treated with 5 μg/day leptin (supplied by NHPP) or vehicle via subcutaneous 14 day osmotic pumps implanted on day 0. Subsequent pump explants and implants are indicated by arrows. STZ-vehicle mice were euthanized on day 19, due to deteriorating body condition. Statistical analyses were performed by one way repeated measures ANOVA with a Holm-Sidak post-hoc test. Data are expressed as mean ± SEM. Figure adapted from Denroche et al. (328).   50  Leptin therapy does not acutely lower blood glucose in STZ-diabetic mice Given that leptin therapy appears to normalize blood glucose over the course of several days, we next examined whether leptin could acutely lower blood glucose in STZ-diabetic mice. To test this we examined the effects 0.5 µg/g leptin (10 µg leptin for a 20 g mouse), delivered by a single i.p. bolus injection in 4-hour fasted STZ-diabetic C57Bl/6 mice that had not received any prior treatment. Plasma leptin levels reached approximately 30 ng/mL, 30 minutes following injection (Figure 5A). Interestingly, there was no effect on blood glucose, measured up to 4 hours post injection (Figure 5B), suggesting that leptin does not acutely lower fasting blood glucose levels. Considered with the finding that hyperglycemia is rapidly restored after leptin therapy is ceased, these data suggest that the anti-diabetic action of leptin in type 1 diabetes requires sustained leptin infusion.   Figure 5. Leptin therapy does not acutely lower blood glucose in STZ-diabetic mice. STZ-diabetic male C57Bl/6 mice were 4-hour fasted and injected with 0.5 µg/g body weight leptin (STZ-leptin, red), or vehicle (STZ-vehicle, blue). Non-diabetic controls were injected with vehicle (non-diabetic, black). A) Plasma leptin levels were measured at 30 min post injection. B) Blood was glucose tracked at the indicated times post injection. Data were analyzed by one-way ANOVA (A) and two way repeated measures ANOVA (B), with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle; ‡ P < 0.05 STZ-leptin vs non-diabetic; † P < 0.05 STZ-vehicle vs non-diabetic. Data are presented as mean ± SEM, n = 3-4.   51 Hyperleptinemia improves post-prandial glucose metabolism in diabetic mice As the above experiments assessed fasting blood glucose levels we next investigated whether leptin therapy improved post-prandial glucose metabolism. Random-fed blood glucose levels were measured 13 days following implantation of osmotic pumps delivering 10 μg/day leptin or vehicle in C57Bl/6 STZ-diabetic male mice and in non-diabetic controls. Non-diabetic mice had random-fed blood glucose levels of 8.1 ± 0.3 mmol/L whereas the majority of STZ-vehicle mice had glucose levels above the limit of detection (33.3 mmol/L), and were assigned a value of 33.3 mmol/L. Random-fed blood glucose levels in STZ-leptin mice were 23.9 ± 3.3 mmol/L, indicating at least a 26% reduction in fed blood glucose compared to STZ-vehicle mice (Figure 6A). In addition, we examined glucose excursion by performing i.p.GTTs on day 9 post pump implantation (Figure 6B) and measuring area under the curve (Figure 6C). STZ-vehicle controls exhibited hyperglycemia throughout the glucose challenge compared to non-diabetic mice, and surprisingly leptin treated mice displayed normal glucose tolerance. These data suggest leptin therapy robustly improves post-prandial glucose metabolism in STZ-diabetic mice.  Figure 6. Leptin improves post-prandial glucose metabolism in STZ-diabetic mice. A) Random-fed blood glucose (11PM) on day 13 post pump implant in STZ-leptin (red), STZ-vehicle (blue) and non-diabetic (black) mice. All but one STZ-vehicle mouse had a blood glucose concentration above the limit of detection and were assigned a value of 33.3 mmol/L, n = 5; statistical analyses were not performed on this group. B) i.p.GTTs were performed on day 9. Mice received an i.p. injection of 1.5 g/kg glucose at time 0, n ≥ 4. C) Area under the curve calculated for the i.p.GTT from time=0 to time=30. Data are expressed as mean ± SEM. Statistical analyses were performed by Student’s t-test, and yielded similar results by one-way ANOVA. Figure adapted from Denroche et al., (328).   52  Leptin therapy reverses STZ-induced dyslipidemia We next sought to determine whether leptin therapy reversed dyslipidemia in STZ-diabetic mice by measuring 4-hour fasted plasma lipids. STZ treatment increased triglyceride and cholesterol levels in STZ-vehicle mice compared to non-diabetic controls, while free fatty acids were unaltered (Figure 7A-C). Leptin administration markedly reduced plasma triglycerides, free fatty acids, and cholesterol in STZ-diabetic mice to levels significantly lower than both STZ-vehicle and non-diabetic controls, suggesting that in addition to improving glucose metabolism, leptin therapy can potently improve lipometabolic disturbances in STZ-diabetic mice.  Figure 7. Leptin reduces plasma lipids in STZ-diabetic mice. Four-hour fasted plasma triglycerides (TG) (A), cholesterol (B), and free fatty acids (FFA) (C), in STZ-leptin (red), STZ-vehicle (blue) and non-diabetic (black) mice on day 14 from cardiac puncture samples, n ≥ 4. Data are expressed as mean + SEM. Statistical analyses were performed by Student’s t-test. Statistical significance was consistent when performed by one-way ANOVA, except in A and B the non-diabetic group was not significantly different from the STZ-leptin group. Figure adapted from Denroche et al., (328). Leptin suppresses counter-regulatory hormone levels and improves insulin sensitivity in STZ-diabetic mice While it is apparent that leptin mimics the effect of insulin on glucose metabolism in STZ-diabetic mice, the mechanism for this is unclear. As previously reported (288), hyperleptinemia may lower blood glucose in STZ-diabetes by alleviating the hyperglucagonemia associated with insulin deficiency. Indeed, STZ-vehicle mice had an approximate 2.5 fold increase in 4-hour fasted plasma glucagon levels compared to non- 53 diabetic controls, while STZ-leptin mice had levels similar to non-diabetic controls (Figure 8A). Four-hour fasted plasma growth hormone was increased approximately 28 fold in STZ-vehicle mice compared to non-diabetic mice; similar to glucagon, growth hormone levels were markedly decreased in response to leptin therapy, although remaining significantly higher than non-diabetic levels (Figure 8B). In contrast, plasma corticosterone was increased in STZ-treated mice compared to non-diabetic controls, but was unaltered by leptin therapy (Figure 8C).  Figure 8. Leptin reduces plasma glucagon and growth hormone levels in STZ-diabetic mice. Four-hour fasted plasma glucagon (A), growth hormone (B), and corticosterone (C), in STZ-leptin (red), STZ-vehicle (blue) and non-diabetic (black) mice on day 14 collected by cardiac puncture, n ≥ 4. Data are expressed as mean + SEM. Statistical analyses were performed by Student’s t-test. Statistical significance between groups was consistent when analysis was performed by one-way ANOVA, except in B where there was no longer a statistical difference between non-diabetic and STZ-leptin groups. Figure adapted from Denroche et al., (328). As expected from previous reports (287-289), leptin therapy did not restore circulating insulin levels in STZ-injected mice; the majority of 4-hour fasted STZ-vehicle and STZ-leptin mice had plasma insulin levels that were below detection limits (< 0.019 ng/ml) even using an ultrasensitive mouse insulin ELISA (Figure 9A). However, using this ultrasensitive assay, insulin was detectable in random-fed STZ-vehicle and STZ-leptin mice, albeit at approximately 9 and 7% that of random-fed non-diabetic controls, respectively. These data highlight that insulin is not completely eliminated by STZ treatment. In light of this, we investigated whether augmented insulin sensitivity may also contribute to the glucose lowering effect of leptin in STZ-diabetic mice by performing ITTs. Both the non-diabetic and STZ-leptin groups exhibited a potent response to exogenous insulin, such that 10 minutes following insulin injection, all but one mouse in both groups had blood glucose levels below  54 4 mmol/L, and had to be rescued by exogenous glucose administration; thus, only one time point post injection for these groups could be examined (Figure 9B). STZ-vehicle controls were significantly insulin resistant compared to non-diabetic mice, as insulin decreased blood glucose by approximately 19% and 56% respectively, 10 minutes post insulin injection. Remarkably, leptin treated mice displayed an approximate 76% drop in blood glucose 10 minutes following insulin injection, and were significantly more insulin sensitive than both STZ-vehicle mice, (P = 0.00016) and non-diabetic controls (P = 0.019). These data indicate that not only does 10 μg/day leptin reverse the insulin resistance in STZ-diabetic mice, it also augments insulin sensitivity beyond that of non-diabetic mice. Therefore, although insulin levels are not altered by leptin therapy, enhanced insulin sensitivity could contribute to leptin-mediated glucose lowering in STZ-diabetic rodents.  Figure 9. Leptin enhances insulin action in STZ-diabetic mice. A) Four-hour fasted plasma insulin (day 7) and random-fed plasma insulin (day 13) were measured in samples collected from the saphenous vein in STZ-leptin (red), STZ-vehicle (blue) and non-diabetic (black) mice. Fasted insulin was undetectable in all but one mouse in both the STZ-leptin and STZ-vehicle groups, n ≥ 4, while random-fed insulin was detectable in most STZ-treated mice tested, n ≥ 3. The limit of detection (0.019 ng/ml) is shown as a broken line. B) An ITT was performed on day 4 post pump implant, n = 5. All but one mouse in both the STZ-leptin and non-diabetic groups had to be rescued with exogenous glucose at 10 min post insulin injection. Statistical analyses were performed by Student’s t-test on 10 minute values. Similar results in A and B were obtained when statistical analyses were performed by one-way ANOVA. * P < 0.05 STZ-leptin vs STZ-vehicle; † P < 0.05 STZ-leptin vs non-diabetic; ‡ P < 0.05 STZ-vehicle vs non-diabetic. Data are expressed as mean ± SEM. Figure adapted from Denroche et al., (328).  To determine whether the improved insulin sensitivity observed in STZ-leptin mice was due to increased glucose uptake, we evaluated tissue specific glucose uptake in vivo by  55 intravenous flash injection of 2-deoxy-D-[14C]glucose as described previously  (321,336). Glucose uptake in the soleus muscle was comparable in STZ-vehicle and STZ-leptin mice (Figure 10). An intravenous insulin bolus significantly increased glucose uptake in the soleus relative to baseline in all groups; however, insulin-stimulated glucose uptake was not significantly increased in response to leptin therapy compared to STZ-vehicle controls. Due to the extreme depletion of WAT in both STZ-vehicle and STZ-leptin mice, WAT glucose uptake could not be determined. These observations suggest that leptin-induced glucose uptake in skeletal muscle is not sufficient to account for the enhanced insulin sensitivity and restoration of euglycemia in STZ-induced diabetes by leptin.  Figure 10. Glucose uptake in the soleus muscle cannot account for the anti-diabetic effect of leptin therapy. Glucose uptake was measured in soleus muscle of STZ-leptin (red), STZ-vehicle (blue) and non-diabetic (black) mice, collected 20 min following intravenous injection of 2-deoxy-D-[14C]glucose with and without insulin, and normalized to tissue weight, n = 3-5. Data are expressed as mean + SEM. Statistical analyses were performed by Student’s t-test. Comparison between non-diabetic, STZ-vehicle and STZ-leptin groups performed by one-way ANOVA yielded similar results except that the difference between non-diabetic + insulin and STZ-vehicle + insulin was P=0.06. Figure adapted from Denroche et al. (328). 3.3 DISCUSSION  In this chapter we examined the effect of leptin therapy on glucose and lipid metabolism in STZ-diabetic mice. We found that a supraphysiological leptin dose normalized fasting blood glucose and ketone levels, and robustly lowered fasting plasma lipids, consistent with previous work in other rodent models of insulin-deficient type 1 diabetes (287,288,337). The glucose lowering effect of leptin was not achieved by a single leptin injection, and was rapidly reversed upon cessation of leptin therapy, revealing that the leptin-mediated glucose lowering is not acute, and requires sustained leptin infusion. In  56 addition, we observed that leptin therapy markedly improved glucose tolerance and post-prandial glucose metabolism. Remarkably, these profound effects of leptin on glucose metabolism were achieved even though insulin levels were unchanged compared to untreated diabetic mice. Confirming the findings of Yu et al. (288), we observed that leptin therapy reversed hyperglucagonemia, and in addition we found that leptin therapy robustly reduced plasma growth hormone levels. Although circulating insulin levels were unaltered, leptin therapy markedly enhanced insulin sensitivity in STZ-diabetic mice. Thus, both reduced counter-regulatory hormones and increased insulin sensitivity could potentially mediate the glucose lowering effect of leptin therapy in type 1 diabetes.   How leptin therapy improves insulin sensitivity in insulin-deficient diabetes remains elusive. It is unlikely that increased insulin sensitivity in diabetic leptin treated mice is secondary to the restoration of euglycemia, since leptin was reported to enhance insulin sensitivity in STZ-diabetic rats to a greater extent than phloridzin, which restores euglycemia by increasing glucose excretion (287). In support of this, the data in this chapter revealed that STZ-leptin C57Bl/6 mice had enhanced insulin sensitivity on day 4 following pump implantation, before blood glucose was fully normalized. Some studies indicate that leptin and insulin signalling cascades intersect at multiple nodes (201,209,258,338,339), and thus leptin signalling could potentially enhance insulin signal transduction through the activation of shared signalling molecules. It is also possible that the enhanced insulin sensitivity of leptin treated mice is due to lower counter-regulatory hormones. However, since glucagon and growth hormone levels were either normalized or partially corrected, this is unlikely to explain the over-correction of insulin sensitivity in leptin treated mice. Alternatively, the lipopenic action of  leptin therapy could enhance insulin sensivity in STZ-diabetic mice, and indeed this is supported by the fact that both plasma triglycerides and free fatty acids were robustly reduced by leptin in this study.  Regardless of the mechanism mediating leptin-induced insulin sensitivity, this chapter highlights that hyperleptinemia may compensate for insulin deficiency in type 1 diabetes by promoting the action of residual insulin. German et al. recently demonstrated that in STZ-diabetic rats, a physiological dose of leptin normalized plasma glucagon and reversed insulin resistance, yet did not restore euglycemia (286). Our observations do not contradict these findings; here STZ-diabetic mice treated with a supraphysiological dose of leptin were more insulin sensitive than non-diabetic controls, whereas German et al. used a physiological dose of leptin resulting in similar insulin sensitivity to non-diabetic controls. This suggests that a certain threshold of insulin-sensitivity may have to be achieved by  57 leptin therapy to compensate for low insulin levels, and that reversal of STZ-associated insulin resistance is not sufficient to reverse hyperglycemia. Although referred to as a state of insulin deficiency, our findings highlight that low levels of circulating insulin are present in the STZ model of type 1 diabetes. Studies claiming the insulin independence of leptin action (including those where leptin was delivered ICV), reported undetectable plasma insulin levels in STZ-treated rodents and NOD mice, using an insulin assay with a limit of detection of 0.1 ng/ml (Ultra-Sensitive Insulin ELISA, Crystalchem) (288,306,309). In the current investigation, using an insulin ELISA with > 5 fold enhanced sensitivity (limit of detection = 0.019 ng/mL), we found that although insulin levels were extremely depleted by STZ (undetectable during fasting), fed insulin levels were detectable. The detection of fed plasma insulin levels suggests that some β-cell mass is retained in STZ-diabetic mice. Indeed, studies quantifying pancreatic β-cells in STZ-treated rodents report a retention of 0.5 to 7% of β-cells compared to non-diabetic controls (288,301). Residual insulin levels can also be detected in mice with autoimmune diabetes, and Wang et al. reported levels of approximately 0.05 ng/ml in vehicle and leptin treated NOD mice (289). Importantly, residual insulin is not specific to rodent models of type 1 diabetes, as more sensitive assays have facilitated the detection of very low circulating C-peptide levels in type 1 diabetic patients. Most patients have a low level of C-peptide production initially following diagnosis (340-347). A recent study found that 59.5% of type 1 diabetic patients have detectable serum C-peptide from 6-10 years following diagnosis, and as much as 39.1% of patients still have detectable C-peptide levels 21-30 years after diagnosis (347). Therefore, while it is clear that leptin does not improve glucose control in type 1 diabetes by raising insulin levels, it is unclear whether the effect of leptin therapy in type 1 diabetes is truly insulin-independent, warranting further investigation.  Despite enhanced whole body insulin sensitivity, glucose uptake in soleus muscle was not increased by leptin therapy, indicating that leptin-induced changes in insulin sensitivity are likely mediated through other target tissues. Chinookoswong et al., reported that under basal conditions, the rate of whole body glucose disappearance in hyperleptinemic STZ-diabetic rats was unaltered compared to diabetic controls, suggesting that the glucose lowering effect of leptin is not due to enhanced whole body glucose uptake (287). Instead, the Chinookoswong study showed that endogenous glucose production was modestly decreased under basal conditions in STZ-diabetic rats. Given the decrease in circulating glucagon levels in STZ-diabetic rodents treated with leptin (288), and the insulin-sensitizing  58 effect that leptin can have on hepatic glucose production in normal rodents (158,159), the glucose lowering effect of leptin may be mediated by enhanced hepatic insulin sensitivity.   Based on observations from the present study and by others, leptin may prove to be a promising therapy for type 1 diabetes. Leptin therapy may have advantages over insulin therapy, particularly in regard to lipid metabolism. However, due to the profound insulin-sensitizing effect of leptin, this study highlights a potential pitfall of leptin administration in conjunction with insulin, namely increased risk of hypoglycemia. In our studies, a single dose of insulin that modestly lowered blood glucose in untreated diabetic animals caused rapid and severe hypoglycemia in leptin treated animals. Given that leptin therapy also robustly lowers circulating glucagon, the potential inability to mount a counter-regulatory response could further heighten the risk of hypoglycemia. Thus while leptin in combination with insulin may dramatically improve treatment for patients with type 1 diabetes, further studies must more rigorously assess the increased risk of hypoglycemia.     59 CHAPTER  4: GENERATION AND CHARACTERIZATION OF MICE WITH DISRUPTED NEURONAL LEPTIN SIGNALLING 4.1 INTRODUCTION Substantial evidence suggests that the metabolic actions of leptin are mediated by the CNS. Lepr-b is abundantly expressed throughout the CNS, including the hypothalamus and brainstem (50,51,68,69). Direct application of leptin to the CNS can mimic the effect of peripherally administered leptin on glucose homeostasis (164). Moreover, deletion of leptin receptors throughout neurons can perturb insulin and glucose levels in rodents (85,87), whereas reconstitution of Lepr-b, or leptin responsive neurons, specifically within the brain, can ameliorate dysglycemia in rodents lacking leptin receptors (86,88,89,107,108,185). In light of these data, we sought to determine whether neuronal leptin signalling is required to mediate the glucose lowering effect of leptin therapy in type 1 diabetes, by generating mice with a neuron-specific deletion of the Lepr-b signalling domain through the Cre-lox method. As the precise anatomical regions that mediate leptin action in type 1 diabetes have not been fully defined, we chose to generate mice with broad neuronal leptin signalling disruption (Leprflox/flox Syncre mice), using a Cre transgene driven by the rat SynapsinI promoter (Syncre) which has broad neuronal expression (348). Prior to testing the efficacy of leptin therapy in Leprflox/flox Syncre mice, we first examined glucose and energy homeostasis in this model.  Limited phenotypic analyses of mice with a Syncre-induced deletion of leptin receptors have been previously reported. While Leprflox/flox Syncre mice specifically lack Lepr-b signalling in neurons, Cohen et al. generated similar mice with a Syncre driven deletion of the entire leptin receptor in neurons (87). Surprisingly, the Cohen study reported that on average, mice with Syncre-mediated deletion of leptin receptors in neurons had no body weight phenotype compared to controls, and only a small subset of mice were obese (4 males and 2 females). These obese mice had the greatest extent of hypothalamic Lepr deletion, and subsequent phenotypic analyses were limited to a comparison between obese experimental mice and all control mice. Thus, the phenotype of the representative population of mice with a Syncre-mediated disruption of leptin receptors is unclear. A second study by Hinoi et al. reported that Leprflox/flox Syncre mice on average have no body weight phenotype but are hyperinsulinemic (182). In this chapter we extended work of the aforementioned studies, by investigating body weight, and glucose metabolism in Leprflox/flox Syncre mice, at several ages and in response to an HFD. By thoroughly examining  60 metabolic and energy homeostasis in the whole population of these mice, it was possible to elucidate the role of leptin receptor signalling in Syncre-expressing neurons2. 4.2 RESULTS Generation of mice with neuronal deletion of leptin receptor signalling domains We employed the Cre-lox approach to generate mice with a neuron-selective loss of leptin receptor signalling (Leprflox/flox Syncre mice). Cre-mediated recombation of the floxed leptin receptor allele (Leprflox) produces the LeprΔ17 allele that encodes a protein with a truncated intracellular signalling domain incapable of initiating JAK2 signalling cascades (310) (shown in Figure 1). The Syncre transgene has been used extensively to induce recombination throughout the CNS, specifically in neurons, with the only reported non-CNS site of expression being the testes (312,348-350). To first examine the suitability of Syncre to mediate widespread recombination in the CNS, we crossed Syncre transgenic mice with mTmG reporter mice that express EGFP permanently in cells that have Cre activity (313). Expectedly, EGFP immunoreactivity was observed throughout the brain, including the hypothalamus (Figure 11A) and nucleus of the solitary tract (NTS) (Figure 11B). Since endogenous SynapsinI expression has been reported in rat pancreatic islets (351), we examined pancreatic islets for Syncre activity in mTmG Syncre mice (Figure 12A-B). As a positive control, we simultaneously performed EGFP immunostaining in the pancreas of an mTmG mouse injected with AAV RIPcre. No EGFP immunoreactivity was observed in the islets of mTmG Syncre mice, relative to mTmG mice without the Syncre transgene, whereas mTmG mice carrying the RIPcre transgene displayed robust EGFP immunoreactivity in pancreatic islets, localized predominantly to insulin positive cells (Figure 12A). Quantification of EGFP positive area, revealed approximately 1.5% positive area in the AAV-RIPcre injected mTmG mouse (Figure 12B). mTmG Syncre mice had a pancreatic EGFP positive area that was <0.04% that of the AAV-RIPcre injected mouse, and was similar to mTmG control mice, indicating the lack of Syncre activity in the pancreas.                                                2 All data in this chapter are unpublished.  61  Figure 11. The Syncre transgene is active throughout the brain. EGFP immunoreactivity in coronal sections from mTmG Syncre and mTmG control mice, demonstrates widespread Syncre-induced recombination in anatomically matched sections containing the hypothalamus (A) and the NTS (B).  Figure 12. The Syncre transgene is not active in pancreatic islets. A) EGFP, insulin and glucagon immunoreactivity were assessed in pancreata from mTmG Syncre and mTmG control mice, n = 3-4 per group. A mouse that had received in injection of AAV-RIPcre was used as a positive control for pancreatic EGFP immunoreacitivity, n = 1. Representative images of islets revealed no detectable EGFP immunoreactivity in mTmG Syncre mice. Scale bar = 100 μm. DAPI fluorescence (white) is shown in the merge. B) EGFP positive area as a percent of whole section area was quantified in 1 section per mouse of each group, n = 3-4. The EGFP positive area from the single   62 section of the AAV-RIPcre is provided as a reference point. Data are presented as mean + SEM, and statistical analysis was performed by Student's t-test. Subsequently, to examine the specificity of Cre-induced Leprflox recombination, PCR analysis was performed in the CNS and peripheral tissues of Leprflox/flox Syncre mice and Leprflox/flox littermate controls. As expected, Leprflox/flox Syncre mice exhibited widespread Leprflox recombination throughout the CNS, and no recombination in the peripheral tissues examined (Figure 13A). To examine whether CNS-wide recombination of Leprflox conferred a reduction in the expression of Lepr-b, we measured the transcript abundance of the wildtype Lepr-b isoform by RT-qPCR in brain regions of Leprflox/flox Syncre mice and Leprflox/flox littermate controls. Indeed, Lepr-b transcript was significantly reduced in several regions of the CNS, particularly in the brainstem, and spinal cord (Figure 13B), both of which contain key leptin responsive neurons implicated in food intake and body weight (69,111). Non-significant reductions in Lepr-b transcript were also observed in the olfactory bulb (P = 0.09) and the hippocampus (P = 0.15). Within the hypothalamus, Lepr-b was reduced by approximately 63%, suggesting that a significant portion of hypothalamic neurons retain Lepr-b expression.  Figure 13. Leprflox/flox Syncre mice have attenuated Lepr-b expression in the CNS. A) Representative images of recombination of Leprflox allele in indicated anatomical brain regions and peripheral tissues, assessed by PCR of genomic DNA from female Leprflox/flox Syncre and Leprflox/flox female littermates, n = 3-4. B) Abundance of wildtype (wt) Lepr-b cDNA in anatomical brain regions of 8 week old Leprflox/flox Syncre and Leprflox/flox male mice, assessed by RT-qPCR with LeprE17 primers (shown in Figure 1) and expressed relative to Leprflox/flox hypothalamus, n = 4-5. Data are presented as mean + SEM. * P < 0.05 between genotypes via Student's t-test. † P < 0.05 between indicated  63 brain region and hypothalamus within the same genotype via two-way ANOVA with a Sidak post-hoc test. To examine the extent of disrupted hypothalamic leptin receptor signalling, we assessed leptin-stimulated phosphorylated-STAT3 (P-STAT3) immunoreactivity in the hypothalamus of Leprflox/flox Syncre and Leprflox/flox mice (Figure 14A), and quantified the number of P-STAT3 positive cells in several hypothalamic nuclei (Figure 14B). Vehicle injected mice did not display robust hypothalamic P-STAT3 immunoreactivity in any hypothalamic nuclei. Leptin injection induced robust P-STAT3 immunoreactivity in the hypothalamus of Leprflox/flox mice, particularly within the ARH, and also within the VMH, DMH, LHA, and PMV (Figure 14A-B). Interestingly, in response to leptin injection, Leprflox/flox Syncre mice displayed a similar number of P-STAT3 positive neurons in the ARH and VMH compared to Leprflox/flox mice (Figure 14A), revealing that leptin signalling is intact in within these nuclei. These data are supported by a recent study showing the Syncre transgene is not active in most POMC and NPY neurons (352). In contrast, Leprflox/flox Syncre mice had a striking reduction of leptin-induced P-STAT3 immunoreactivity in the LHA, and ventral premammillary nucleus (PMV), and significantly attenuated P-STAT3 immunoreactivity in the DMH. As Lepr-b expression was reduced in the spinal cord, brainstem, and cortex, Leprflox/flox Syncre mice also likely have widely disrupted extrahypothalamic signalling, but functional leptin signalling was not analyzed in these regions. Thus, Leprflox/flox Syncre mice are a model of disrupted CNS Lepr-b signalling outside of the ARH and VMH. As much less is known about leptin action in the DMH, LHA and PMV compared to the ARH, these mice provide an interesting tool to dissect the roles of leptin signalling in these hypothalamic nuclei.  64  Figure 14. Functional leptin signalling is disrupted in the DMH, LHA and PMV of Leprflox/flox Syncre mice. Ten week old Leprflox/flox Syncre male mice and Leprflox/flox littermate controls were injected with 2 μg/g leptin or vehicle 45 min prior to tissue harvest. A) Representative images of P-STAT3 staining in hypothalamic nuclei (ARH and PMV top panels, ARH and VMH middle pannels, DMH and LHA lower pannels). B) Total hypothalamic P-STAT3 positive cells. n = 3-4. Data are presented as mean + SEM. * P <0.05 between genotypes, via Student's t-test. Leprflox/flox Syncre mice have normal body weight and fasting glucose levels To assess the impact of disrupted neuronal leptin signalling on glucose and energy homeostasis in Leprflox/flox Syncre mice, we tracked body weight and blood glucose in male and female mice from 4 weeks of age. As expected, on average Leprflox/flox Syncre male (Figure 15A) mice and female (Figure 15B) mice did not have altered body weight compared to Leprflox/flox littermate controls at any age studied. Interestingly, 4-hour fasted blood glucose levels tended to be highly variable in Leprflox/flox Syncre male mice from 4 to 12 weeks of age (Figure 15C). Thus, it appears that male Leprflox/flox Syncre mice have perturbations in glucose homeostasis, but that this does not consistently manifest as overt hyperglycemia. After 12 weeks of age, blood glucose levels stabilized, and resembled those of Leprflox/flox male controls. Female Leprflox/flox Syncre mice had similar blood glucose levels to those of littermate controls at all time points examined (Figure 15D).  65  Figure 15. Male and female Leprflox/flox Syncre mice have normal body weight and fasting blood glucose. Four-hour fasted body weight (A, B) and blood glucose (C, D) were tracked in male (left panels) and female (right panels). Leprflox/flox Syncre mice (red) and Leprflox/flox littermate controls (blue) were tracked from 4 to 58 weeks of age. Data are presented as mean ± SEM, n = 5-8. Statistical analyses were performed by two way repeated measures ANOVA with a Holm-Sidak post-hoc test, *P<0.05.  Cohen et al. (87) demonstrated marked body weight variability in mice with a neuron-selective Lepr deletion at 16 weeks of age, and attributed this to variability in hypothalamic Lepr deletion. Therefore, in the same cohort of male Leprflox/flox Syncre mice that we tracked, we examined body weight variability in male (Figure 16A) and female (Figure 16B) mice. In young adult mice (7 weeks of age) body weight variability was minimal, however as the mice aged, body weight became more variable, reaching a similar spread at 16 weeks of age that was reported in the Cohen study. Leprflox/flox control mice displayed a similar age-related body weight variation to Leprflox/flox Syncre mice, suggesting that the variability in body weight of within this strain is independent of Lepr recombination or the presence of Syncre, and is likely due to the mixed genetic background of this strain. Because Cohen et al. did not report the body weight of individual control mice, it is unknown whether the variability reported in  66 their mice with neuronal Lepr deletion is due to variable Lepr deletion or if it is genotype-independent. To avoid any confounding effects of large variations in body weight, we performed the majority of subsequent metabolic analyses in mice aged 7-8 weeks.  Figure 16. Age-related body weight variability of Leprflox/flox Syncre mice is independent of Syncre genotype. Body weights of individual mice at 7 and 16 weeks (same data as in Figure 15A and B) are plotted for male (A) and female (B) mice. Less variability between individual mice is observed at 7 weeks, but as mice age, variability markedly increases in both Leprflox/flox Syncre mice (red) and Leprflox/flox controls (blue). Mean ± SEM are indicated by black bars.  In addition, we observed that a small subset of Leprflox/flox Syncre mice (approximately 3%) were extremely obese (169 ± 21% increase in body weight compared to littermate controls, P = 0.04, n = 3). The obesity was so robust, we questioned whether these mice may have broader recombination of Leprflox than the rest of Leprflox/flox Syncre mice. Surprisingly, we found that all of the obese mice displayed substantial Leprflox recombination in tail and ear biopsies, indicative of whole body recombination of Leprflox. In these mice hypothalamic Lepr-b transcript abundance was drastically reduced (5.8 ± 1.4% relative to Leprflox/flox controls). We therefore genotyped all experimental mice for this recombination event, and found an additional 3% of mice with partial Leprflox recombination in ear and tail biopsies, and no body weight phenotype. It was previously reported that due to Syncre expression in the testes, approximately 30% of progeny derived from male Syncre mice inherit a recombined gene through germline transmission (312). However, this is distinct from our observations, since germline transmission produces both Syncre positive and Syncre negative progeny with recombined alleles (312), whereas in our study all mice exhibiting recombination were carriers of the Syncre transgene. Furthermore, we specifically  67 maintained the Syncre transgene on female carriers. Therefore, we postulate that in a small minority of mice the Syncre transgene was activated during embryogenesis, resulting in mosaic whole body recombination of Leprflox throughout the brain and periphery. To our knowledge this has not been previously reported for the Syncre transgene, but has been reported for another neuron-specific Cre driver, AgRPcre, which induces early embryonic recombination in a subset of mice (106). All experimental mice were genotyped in order to identify and exclude mice with this recombination event from our data analysis. Although unclear, this begs the question as to whether the small subset of extremely obese Syncre mice in the Cohen study displaying <15% hypothalamic Lepr transcript compared to controls (87) were in fact mice that had undergone embryonic Lepr recombination that was not neuron-specific. Leprflox/flox Syncre mice do not have altered food intake, but have altered lipid oxidation To assess the effect of disrupted neuronal leptin signalling in Leprflox/flox Syncre mice on energy homeostasis, we measured food intake, energy expenditure and RER in Leprflox/flox Syncre and Leprflox/flox males at 10 weeks of age. Food intake (Figure 17A), energy expenditure (Figure 17B), and locomotor activity (Figure 17C) were unaltered in Leprflox/flox Syncre mice, although there were non-significant trends toward increased food intake and decreased energy expenditure and activity compared to littermate controls. These data are consistent with the lack of body weight phenotype, suggesting that neuronal leptin signalling outside of the ARH and VMH Leprflox/flox Syncre is not required for the anorectic effects of leptin. Leprflox/flox Syncre males had significantly increased RER, indicative of reduced lipid oxidation, compared to controls (Figure 18A). Consistent with diminished lipid utilization, Leprflox/flox Syncre mice had a modest increase in fat mass despite a lack of body weight phenotype (Figure 18B). These data suggest that leptin signalling in Syncre-expressing neuronal circuits promotes lipid oxidation, independent of body weight or food intake.  68  Figure 17. Leprflox/flox Syncre mice do not have altered food intake or energy expenditure. Average food intake (A), energy expenditure (B), and locomotor activity (C) in Leprflox/flox Syncre and Leprflox/flox mice during 12-hour light and dark cycles. Energy expenditure values are adjusted to lean body mass, and statistical analyses performed by analysis of covariance. Statistical analyses for food intake and activity were performed by two-way ANOVA with a Sidak post-hoc test. Data are presented as mean + SEM, n = 6-7.  Figure 18. Leprflox/flox Syncre mice have decreased lipid oxidation. A) RER was tracked over two consecutive days (dark cycle indicated in grey, light cycle indicated in white). Statistical analyses were performed on AUC for each light/dark cycle by two-way ANOVA with a Sidak post-hoc test. * P <0.05 between groups. B) Fat and lean mass as a percent of total body weight at 11 weeks of age, following completion of the metabolic run. Data are presented as mean ± SEM, n = 6-7. Leprflox/flox Syncre mice display fasting hyperinsulinemia  After confirming that body weight is not affected in Leprflox/flox Syncre mice, we next examined whether these mice displayed altered 4-hour fasting metabolic profiles at 7 weeks of age. We also examined the metabolic profile in these mice at 47 weeks of age, to  69 determine if any metabolic phenotypes were maintained as mice aged. At 7 weeks of age, Leprflox/flox Syncre male mice displayed hyperinsulinemia (Figure 19A) and hyperleptinemia (Figure 19B). The differences in insulin and leptin levels were age-dependent, and diminished in aged Leprflox/flox Syncre and Leprflox/flox mice. There was a trend toward increased plasma triglycerides (Figure 20A), and free fatty acids (Figure 20B) in 7 week old Leprflox/flox Syncre mice relative to littermate controls, but this did not reach significance. This could reflect the modest increase in fat mass and increased RER in male mice. As mice aged, the difference became more apparent between genotypes. Plasma cholesterol (Figure 20C) was unaffected. Female mice exhibited trends toward hyperinsulinemia and hyperleptinemia but these did not reach statistical significance (Table 3).  Figure 19. Leprflox/flox Syncre male mice are hyperinsulinemic and hyperleptinemic. Four-hour fasted plasma insulin (A), and leptin (B) levels were measured in Leprflox/flox Syncre (red) and Leprflox/flox (blue) males at 7 and 47 weeks of age, n = 5-8. Data are presented as mean + SEM. Statistical analyses were performed by Student's t-test.  Figure 20. Leprflox/flox Syncre male mice do not have significantly altered fasting lipids. Four-hour fasted plasma triglycerides (TG, A), free fatty acids (FFA, B), and cholesterol (C), were measured in Leprflox/flox Syncre (red) and Leprflox/flox (blue) males at 7 and 47 weeks of age, n = 5-8. Data are presented as mean + SEM. Statistical analyses were performed by Student’s t-test.  70 Table 4. Plasma analytes in female Leprflox/flox Syncre mice. Analyte Age (weeks) Leprflox/flox Leprflox/flox Syncre Insulin (ng/mL) 8 0.86 ± 0.13 (5) 1.44 ± 0.52 (5)  47 1.51 ± 0.61 (5) 1.57 ± 0.50 (5) Leptin (ng/mL) 8 4.2 ± 1.6 (4) 5.5 ± 1.9 (5)  47 11.9 ± 3.7 (5) 17.3 ± 6.9 (5) TG (mmol/L) 8 0.79 ± 0.04 (5) 1.07 ± 0.16 (5)  47 0.46 ± 0.04 (5) 0.79 ± 0.16 (5) FFA (mmol/L) 8 0.84 ± 0.15 (5) 0.98 ± 0.12 (5)  47 0.55 ± 0.04 (5) 0.51 ± 0.05 (5) Cholesterol (mg/dL) 47 108 ± 18 (5) 105 ± 22 (4)  Statistical analyses were performed by Student’s t-test. No significant differences were found.  To further examine glucose metabolism in Leprflox/flox Syncre mice, we tested glucose tolerance in young and old male mice. Glucose tolerance was not affected in Leprflox/flox Syncre mice at 4 weeks (Figure 21A) or 55 weeks (Figure 21B) of age. However, Leprflox/flox Syncre mice had elevated glucose-stimulated insulin levels compared to controls at 7 weeks of age (Figure 21C). Just as fasting hyperinsulinemia was no longer observed in aged males, by 53 weeks of age, Leprflox/flox and Leprflox/flox Syncre mice had similar glucose-stimulated insulin levels (Figure 21D).   71  Figure 21. Leprflox/flox Syncre male mice have elevated glucose-stimulated insulin levels. Oral glucose tolerance tests in mice at 4 weeks (A) and 55 weeks (B) of age, n = 5-8. Mice were fasted for 6 hours prior to oral gavage of 1.5 g/kg glucose at time 0. Plasma insulin levels in response to 1.5 g/kg oral glucose gavage were measured at 7 (C) and 53 weeks (D) of age in males, n = 5-8. Statistical analyses were performed by Student's t-test on AUC. AUC is inset in C showing the significant difference between groups. No significant difference was observed in A,B or D. Data are presented as mean ± SEM. To test whether hyperinsulinemia could be attributable to off-target effects of the Syncre transgene, we generated Lepr+/+ Syncre mice on the same genetic background, and examined glucose-stimulated insulin levels compared to Lepr+/+ controls at 8 weeks of age (Figure 22). Lepr+/+ Syncre mice displayed normal fasting and glucose-stimulated insulin levels compared to Lepr+/+ littermate controls, revealing that hyperinsulinemia in Leprflox/flox Syncre mice is directly due to loss of leptin signalling, and not due to the presence of the Syncre transgene. We further investigated whether changes in plasma insulin levels in Leprflox/flox Syncre mice could be due to altered pancreatic β-cell area (Figure 23), however no differences in either were found, suggesting the mechanism involves altered synthesis,  72 secretion, or clearance of insulin. Since Leprflox/flox Syncre mice are hyperinsulinemic but have no difference in body weight or food intake, this suggests a non-redundant, body weight independent role for leptin signalling in neuronal circuits outside of the ARH and VMH in regulating insulin levels.  Figure 22. The Syncre transgene has no effect on basal or glucose-stimulated plasma insulin levels. Eight week old Lepr+/+ Syncre mice (red) and Lepr+/+ controls (blue) were fasted for 6 hours and gavaged with 1.5 g/kg glucose at time 0. Plasma insulin levels were determined at 0, 7, 15 and 60 min post gavage in males (A) and females (B). Data are presented as mean ± SEM, n = 4-6 for females and n = 8-9 for males. Statiscal analyses were performed by Student's t-test on area under the curve, and no significant differences were found.  Figure 23. Pancreatic insulin and glucagon postive area are unaltered in Leprflox/flox Syncre mice. Insulin and glucagon positive area were quantified by immunofluorescence in pancreas sections collected from 8 week old Leprflox/flox Syncre male mice (red) and Leprflox/flox littermate controls (blue), n = 4-5. Data are presented as mean + SEM. Statistical analyses were performed by Student's t-test, and no differences were found.  73  Leprflox/flox Syncre mice have an increased counter-regulatory response  To assess insulin sensitivity we performed ITTs in 8 week old Leprflox/flox Syncre male mice and controls. The overall response, assessed by the area between the curve and baseline, revealed that Leprflox/flox Syncre mice are significantly insulin resistant (Figure 24A). Interestingly, the difference in insulin sensitivity was not exhibited during the initial phase of the ITT where glucose levels are reduced in response to insulin, but rather Leprflox/flox Syncre mice displayed a more rapid return to baseline in the second phase of the ITT, suggesting that Leprflox/flox Syncre mice respond normally to insulin injection, but have an altered counter-regulatory response to insulin-induced hypoglycemia. A similar but non-significant trend was observed in aged males (Figure 24B). To examine whether the more rapid recovery from ITT was due to an increased counter-regulatory response to hypoglycemia, we examined glucagon levels in Leprflox/flox Syncre mice. Overnight fasted plasma glucagon levels were not altered in Leprflox/flox Syncre mice compared to Leprflox/flox controls (Figure 24C). We next injected mice with 2-deoxyglucose, which induces neuroglycopenia, and thus a perceived state of hypoglycemia (Figure 24D). As expected, Leprflox/flox mice displayed increased plasma glucagon levels in response to 2-deoxyglucose injection compared to vehicle injection. Leprflox/flox Syncre mice also showed increased 2-deoxyglucose-stimulated glucagon levels, but the level of stimulation was approximately 2 fold greater than in Leprflox/flox controls. These data suggest that Leprflox/flox Syncre mice have enhanced glucagon secretion in response to hypoglycemia, which may account for the enhanced recovery from insulin-induced hypoglycemia during ITTs in Leprflox/flox Syncre mice.   74  Figure 24. Leprflox/flox Syncre male mice have an increased counter-regulatory response. ITTs were performed at 8 weeks (A) and 57 weeks (B) of age, n = 5-8. Mice received an i.p. injection of insulin (0.8 U/kg) at time 0. Net AUC relative to baseline is inset. Plasma glucagon levels were assessed after an overnight fast in 8 week old males (C), n = 5. Plasma glucagon levels in response to neuroglycopenia were measured in 8 week old mice (D), n = 4-5. Mice were fasted overnight, and received an i.p. injection of 250 mg/kg 2-deoxyglucose (2-DG) or vehicle (V) at time 0, and plasma glucagon was measured 15 min following injection. Data are presented as mean ± SEM. Statistical analyses were performed by Student's t-test (A-C) and two-way ANOVA (D) with a Sidak post-hoc test. Leprflox/flox Syncre  mice are protected from HFD-induced weight gain and glucose intolerance Having assessed energy and glucose homeostasis in Leprflox/flox Syncre mice on a regular diet, we next sought to examine whether disrupted neuronal leptin signalling outside of the ARH and VMH may result in worsened glucose metabolism during a metabolic stress such as high fat feeding. At 8 weeks of age, Leprflox/flox Syncre mice and Leprflox/flox mice were placed on either a 45% fat diet (HFD), or a 10% fat control diet (LFD). Leprflox/flox mice placed  75 on HFD rapidly gained weight relative to LFD fed Leprflox/flox mice (Figure 25A). Surprisingly however, Leprflox/flox Syncre mice displayed a dramatic protection from HFD-induced weight gain compared to HFD fed Leprflox/flox controls. Both HFD fed groups trended towards higher fasting blood glucose levels than LFD controls, but were not different between genotypes over the course of the study (Figure 25B).  Figure 25. Leprflox/flox Syncre mice are protected from weight gain on an HFD. Four-hour fasted body weight (A) and blood glucose (B) were tracked over the course of diet manipulation. Data are presented as mean ± SEM. n = 5-7 for HFD, and 4-6 for LFD groups. Statistical analyses were performed by two-way repeated measures ANOVA with a Tukey post-hoc test. ‡ P < 0.05 Leprflox/flox mice HFD vs LFD; † P < 0.05 Leprflox/flox Syncre mice HFD vs LFD; * P < 0.05 Leprflox/flox Syncre vs Leprflox/flox fed HFD. After 5 weeks of high fat feeding, we examined glucose tolerance by OGTTs. Consistent with the protection against weight gain, Leprflox/flox Syncre mice on an HFD had similar glucose tolerance to LFD controls, whereas Leprflox/flox HFD controls already displayed glucose intolerance (Figure 26A, analyzed by AUC in Figure 26C). An OGTT after 14 weeks of diet manipulation revealed that both HFD fed groups had similarly impaired glucose tolerance (Figure 26B, analyzed by AUC in Figure 26C), suggesting that in Leprflox/flox Syncre mice, HFD-induced glucose intolerance is delayed relative to Leprflox/flox mice but not completely prevented. We also performed an insulin tolerance test after 15 weeks of HFD feeding, and similarly saw that Leprflox/flox Syncre mice had retained normal insulin sensitivity, whereas Leprflox/flox mice had a non-significant trend toward insulin resistance on a HFD (Figure 26D).  76  Figure 26. Leprflox/flox Syncre mice are protected from HFD-induced glucose intolerance. A) OGTT performed 5 weeks post diet manipulation. B) OGTT performed 14 weeks post diet manipulation. C) AUC analysis from both OGTTs. D) ITT performed 15 weeks post diet manipulation, area between baseline and curve is inset. Data are presented as mean ± SEM. n = 5-7 for HFD, and 4-6 for LFD groups. Statistical analyses were performed by two-way ANOVA. At the end of the study, body composition was assessed. Leprflox/flox mice had significantly more lean mass on HFD than Leprflox/flox Syncre mice (P = 0.0004) and also had a non-significant trend toward increased fat mass (Figure 27A). However, Leprflox/flox and Leprflox/flox Syncre mice had similar fat and lean mass when considered relative to body weight (Figure 27B). These data suggest that Leprflox/flox Syncre mice have decreased linear growth on an HFD, but that the relative ratios of fat and lean mass remain similar between genotypes.  77  Figure 27. Leprflox/flox Syncre mice gain similar relative fat and lean mass to Leprflox/flox mice on HFD. Body composition at 24 weeks expressed as absolute values (A) and as percent of body weight (B). Data are presented as mean + SEM, n = 5-7 for HFD, and 4-6 for LFD groups. Statistical analyses were performed by two-way ANOVA with a Sidak post-hoc test. † P < 0.05 Leprflox/flox mice HFD vs LFD; ‡ P < 0.05 Leprflox/flox Syncre mice HFD vs LFD; * P < 0.05 Leprflox/flox Syncre vs Leprflox/flox fed HFD.  We repeated the study in a second cohort of mice, and confirmed that Leprflox/flox Syncre mice are protected from HFD-induced weight (Figure 28A). Overall blood glucose levels were not different between Leprflox/flox and Leprflox/flox Syncre mice as we had observed in the first cohort (Figure 28B). To determine if protection from HFD-induced weight gain was due to differences in energy balance, we examined food intake (Figure 29A-B), energy expenditure (Figure 29C), and locomotor activity (Figure 29D) after 10 weeks of high fat feeding. No significant differences between any parameters were observed between genotypes on an HFD, however there was a trend toward decreased food intake, and increased energy expenditure and activity in Leprflox/flox Syncre mice. It is plausible that these non-significant changes in energy balance are biologically meaningful, and could protect against diet-induced weight gain in Leprflox/flox Syncre mice over the course of the study. To assess this, we estimated the net caloric balance in HFD-fed Leprflox/flox Syncre and Leprflox/flox mice from average daily food intake, and energy expenditure values. Indeed, Leprflox/flox mice were estimated to have an energy surplus of approximately 1.2 kcal/day, whereas Leprflox/flox Syncre have an estimated energy balance of approximately -0.5 kcal/day. Therefore, the subtle differences in food intake and energy expenditure between Leprflox/flox Syncre and Leprflox/flox mice on an HFD could result in significantly different weight gain over time.   78  Figure 28. Leprflox/flox Syncre mice are protected from HFD-induced weight gain (cohort 2). Four-hour fasted body weight (A) and blood glucose (B) tracked over the course of diet manipulation. Data are presented as mean ± SEM, n = 3-6 for HFD, and 4-5 for LFD groups. Statistical analyses were performed by two-way repeated measures ANOVA with a Tukey post-hoc test. ‡ P < 0.05 Leprflox/flox mice HFD vs LFD; † P < 0.05 Leprflox/flox Syncre mice HFD vs LFD; * P < 0.05 Leprflox/flox Syncre vs Leprflox/flox fed HFD.   79  Figure 29. Leprflox/flox Syncre mice do not have significantly altered energy homeostasis on HFD. Average daily food intake (A), cumulative food intake (B), energy expenditure (C), and locomotor activity (D) were measured in HFD-fed mice following 10 weeks of diet manipulation. Data are presented as mean ± SEM, n = 3-6. Energy expenditure values in (C) were adjusted to lean body mass and analyzed by analysis of covariance. Statistical analyses were performed by Student's t-test. To assess whether protection from HFD-induced weight gain in Leprflox/flox Syncre mice could be due to changes in leptin signal transduction component, we measured Lepr-b and Socs3 transcript abundance in the hypothalamus of HFD fed mice. The levels of Lepr-b transcript were non-significantly increased in HFD-fed Leprflox/flox controls compared to HFD-fed Leprflox/flox Syncre mice (1.8 ± 0.5 vs 0.99 ± 0.12 respectively relative to Leprflox/flox-LFD mice, P = 0.09). Socs3 transcript was significantly increased in Leprflox/flox Syncre mice on  80 HFD relative to Leprflox/flox mice on HFD (1.6 ± 0.2 vs 0.79 ± 0.19 respectively relative to Leprflox/flox-LFD mice, P = 0.02). Although increased Socs3 transcript levels could be indicative of hypothalamic leptin resistance in Leprflox/flox Syncre mice, this would be expected to worsen diet-induced obesity and thus cannot account for the protection against HFD.   We next examined plasma insulin and leptin levels over the duration of the study. Interestingly, while Leprflox/flox mice displayed marked diet-induced hyperinsulinemia by 4 weeks of age and increasing thereafter, Leprflox/flox Syncre mice did not display significant diet-induced hyperinsulinemia relative to LFD controls for the duration of the study, until a slight increase was observed after 18 weeks (Figure 30A). Despite having a lower fat mass, and decreased weight gain, HFD fed Leprflox/flox Syncre mice had similar diet-induced hyperleptinemia to HFD fed Leprflox/flox controls (Figure 30B). We confirmed the lack of diet-induced hyperinsulinemia in the second cohort of HFD fed Leprflox/flox mice (Figure 31). These data suggest that lack of hyperinsulinemia on a HFD could play a causal role in the metabolic and body weight protection observed in Leprflox/flox Syncre mice. Hyperinsulinemia has been shown to play a causal role in the development of diet-induced obesity (353). Moreover, we have found that hyperinsulinemia in mice with a deletion of the leptin receptor signalling domain in β-cells drives insulin resistance, as correction of hyperinsulinemia with diazoxide can reverse insulin resistance in these mice (354).  Figure 30. Leprflox/flox Syncre mice do not develop HFD-induced hyperinsulinemia. Four-hour fasted plasma insulin (A) and leptin (B) were tracked from 0 to 18 weeks of diet manipulation. Data are presented as mean ± SEM, n = 5-7 for HFD, and 4-6 for LFD groups. Statistical analyses were performed by two way repeated measures ANOVA with a Tukey post-hoc test. ‡ P < 0.05 Leprflox/flox mice HFD vs LFD; † P < 0.05 Leprflox/flox Syncre mice HFD vs LFD; * P < 0.05 Leprflox/flox Syncre vs Leprflox/flox fed HFD.  81  Figure 31. Leprflox/flox Syncre mice do not develop HFD-induced hyperinsulinemia (cohort 2). Four-hour fasted plasma insulin was tracked from 0 to 30 weeks of diet manipulation. Data are presented as mean ± SEM, n = 3-6 for HFD, and 4-5 for LFD groups. Statistical analyses were performed by two way repeated measures ANOVA with a Tukey post-hoc test. ‡ P < 0.05 Leprflox/flox mice HFD vs LFD; † P < 0.05 Leprflox/flox Syncre mice HFD vs LFD; * P < 0.05 Leprflox/flox Syncre vs Leprflox/flox fed HFD. 4.3 DISCUSSION In this chapter we examined the metabolic consequences of widespread neuron-specific deletion of the Lepr-b signalling domain in mice. Similar mice with a Syncre-mediated deletion of all leptin receptor isoforms in neurons were generated by Cohen et al., who reported that while on average these mice had no body weight phenotype, a small subset of mice were obese and had neuroendocrine abnormalities at 22 weeks of age (87). The work in this chapter extends the phenotypic analysis of mice with a Syncre-mediated loss of leptin signalling, by performing a rigorous investigation of metabolic and energy homeostasis in the whole population of mice. Specifically, we found that Syncre induced the robust depletion of Lepr-b expression throughout the CNS, and partly in the hypothalamus. Partial reduction of hypothalamic Lepr-b expression resulted in diminished leptin signalling in the LHA, PMV and DMH, while ARH and VMH leptin signalling remained intact. More extensive deletion of Lepr-b was observed in the brainstem and spinal cord, but the precise anatomical regions affected were not defined.  Disruption of leptin signalling outside of the ARH and VMH had no effect on body weight, food intake or energy expenditure, but mice had modestly increased RER and fat mass, indicative of decreased lipid oxidation. Despite having normal body weight, disruption of neuronal leptin receptor signalling in male mice resulted in hyperleptinemia, transient  82 fasting and glucose-stimulated hyperinsulinemia, and increased counter-regulatory glucagon secretion, without significant alterations in blood glucose or glucose tolerance. The differences in insulin and leptin levels were age-dependent, and were no longer observed in aged male mice. Interestingly, male mice also showed faster recovery from insulin-induced hypoglycemia, consistent with an increased counter-regulatory response. Although female mice with attenuated neuronal leptin receptor signalling displayed trends towards hyperinsulinemia and hyperleptinemia, these did not reach statistical significance. This may relate to the role of sex hormones in modulating glucose metabolism, and also the higher degree of body weight variability in females manifesting in a more variable metabolic profile. Unexpectedly, when fed an HFD, male mice lacking neuronal leptin signalling outside of the ARH and VMH were protected from diet-induced weight gain, and diet-induced glucose intolerance. Although no significant differences in energy balance were observed between HFD-fed Leprflox/flox and Leprflox/flox Syncre mice, it is possible that over several weeks, the trends toward lower food intake and increased activity in Leprflox/flox Syncre mice could contribute to the protection from diet-induced weight gain. Interestingly, Leprflox/flox Syncre mice showed a marked suppression of diet-induced hyperinsulinemia relative to HFD fed controls. Collectively these data reveal a non-redundant role for neuronal leptin signalling outside of the ARH and VMH, in regulating insulin and glucagon levels but not the regulation of food intake. In young mice on a chow diet, these neurons contribute to the suppression of insulin and glucagon levels, and in response to HFD, these neurons contribute to the development of diet-induced weight gain and hyperinsulinemia. The results of this study reveal an important link between leptin signalling in neurons outside of the ARH and VMH, and the regulation of pancreatic endocrine hormone levels. Interestingly, the effect of disrupted neuronal leptin signalling outside of the ARH and VMH appears to have opposite effects on circulating insulin levels in young mice on chow diet, and in response to several weeks on an HFD. While young Leprflox/flox Syncre male chow-fed mice did not have altered pancreatic β-cell area, this remains to be measured in mice after HFD feeding, and warrants further investigation. Although leptin can inhibit the secretion of insulin and glucagon through direct action on β-cells (146,152,188-191,193-195) and α-cells (197,198), neuronal leptin signalling outside of the ARH and VMH may contribute to the fine tuning of this suppressive effect by integrating cues of nutritional status, such as high fat feeding and hypoglycemia. Leptin action in hypothalamic neurons, particularly within the ARH is well known to inhibit food intake, and increase energy expenditure promoting weight loss. The  83 preservation of functional leptin signalling in these nuclei likely explains the lack of body weight phenotype, despite widespread disruption of neuronal leptin signalling domains in chow-fed Leprflox/flox Syncre mice. Interestingly however, our data suggest that neuronal leptin signalling outside of the ARH and VMH plays a critical role in body weight gain in response to HFD, and that leptin action in these sites contributes to, rather than inhibits, weight gain. Although this initially seems surprising, this is consistent from the view point of selective leptin resistance. In diet-induced obese rodents, leptin resistance occurs specifically within the ARH, while leptin signalling is maintained in other hypothalamic nuclei (123,124). Thus, the contribution of leptin resistance to weight gain is likely primarily due to defects in ARH anorectic circuits. In contrast, the maintenance of leptin signalling in the DMH during diet-induced obesity has been suggested to play a causal role in many deleterious actions of leptin, including over activation of the sympathetic nervous system and hypertension (124,125,355). Therefore, we postulate that in Leprflox/flox Syncre mice, diminished leptin signalling in specific neuronal circuits that would normally exert the deleterious effects of leptin action, results in protection from diet-induced obesity. Due to the broad Lepr-b depletion in Leprflox/flox Syncre mice, the precise identity of these neurons is unclear, but the DMH is a potential candidate. While leptin action in the ARH has been rigorously assessed in the literature, the specific roles of leptin action in other hypothalamic nuclei, including the LHA, DMH and PMV are ill-defined. Results from studies where leptin is affected in the whole hypothalamus cannot dissect the distinct roles of these extra-arcuate circuits. Thus, the Leprflox/flox Syncre model has provided a unique opportunity to assess the role of neuronal leptin signalling outside of the ARH. Collectively, the data in this chapter reveal a novel role for leptin action neurons located outside of the ARH and VMH, in the regulation of pancreatic endocrine hormone levels, and provocatively, in the exacerbation of diet-induced obesity and its associated metabolic disturbances.     84 CHAPTER  5: THE ROLE OF NEURONAL AND HEPATIC LEPTIN SIGNALLING IN LEPTIN-MEDIATED GLUCOSE LOWERING IN DIABETES 5.1 INTRODUCTION We and others have demonstrated the profound ability of leptin therapy to reverse hyperglycemia in rodent models of type 1 diabetes, without raising circulating insulin levels (287-291,327,328). However, the precise target tissues that mediate the effect of leptin in type 1 diabetes are unknown. Substantial evidence suggests that anti-diabetic action of leptin in type 1 diabetes can be mediated through CNS. Central administration of leptin and central leptin gene therapy have both been shown to reverse hyperglycemia in insulin-deficient diabetic rodents in a similar manner to peripheral administration (304-307,356), and euglycemia can be achieved with lower doses of ICV leptin than are necessary with peripheral leptin administration (305). Leptin signals initiated within the CNS can be relayed to peripheral tissues by the autonomic nervous system to influence glucose metabolism. Central administration of leptin stimulates sympathetic outflow to tissues including BAT, and skeletal muscle (357,358), and can induce glucose uptake in these tissues in a manner dependent upon sympathetic innervation (176,178,187). In addition, the parasympathetic nervous system can mediate central leptin action on hepatic glucose metabolism by increasing hepatic insulin sensitivity and modulating enzymes controlling glycogen and glucose production. Restoration of leptin signalling to the hypothalamus of leptin receptor deficient rats results in improved hepatic insulin sensitivity through vagal innervation (185). This body of evidence suggests that leptin signalling in the CNS and subsequent relays through the autonomic nervous system play a key role in the metabolic actions of leptin in type 1 diabetes. Recent reports have found that leptin injection in the VMH is sufficient to achieve euglycemia in STZ-diabetic rats, but that VMH leptin receptors are not required for this leptin-mediated effect in STZ-diabetic mice (308), and that deletion of leptin receptors in POMC neurons only modestly blunts ICV leptin action in diabetic mice (309). Therefore, while direct application of leptin to the CNS is sufficient to ameliorate diabetes, it remains unclear whether CNS leptin receptors are necessary for this effect. Thus, utilizing the Leprflox/flox Syncre mice (generated and characterized in Chapter 4) as a model of widespread CNS attenuation of leptin receptor signalling outside the ARH and VMH, we investigated whether leptin receptors in the CNS are required to mediate the anti-diabetic action of leptin therapy in type 1 diabetes. Although Leprflox/flox Syncre mice did not display  85 the expected body weight phenotype resultant from neuronal disruption of leptin receptors, presumably due to intact leptin responsivity in the ARH and VMH, our analysis in Chapter 4 revealed that these mice have disrupted leptin signalling in the DMH, LHA and PMV. Thus, this model offered the unique opportunity to test whether these hypothalamic sites are required for leptin’s anti-diabetic effects, while leaving the ARH and VMH intact, and complementing previously published studies that tested the requirement for leptin signalling in POMC and VMH neurons specifically (308,309). To further investigate the CNS-mediated effects of leptin therapy, we examined whether autonomic efferent pathways are necessary to mediate leptin action in type 1 diabetes. To this end, we assessed whether subdiaphragmatic vagotomy or chemical sympathectomy could attenuate leptin action in mice with STZ-induced diabetes. Alternative to a neuronally mediated mechanism, it is possible that the anti-diabetic action of leptin in type 1 diabetes could be mediated by direct leptin action in peripheral tissues. The liver is a key Lepr-b expressing organ (70) that controls glucose flux in response to many metabolic cues, and disturbed hepatic nutrient metabolism is a major contributor to hyperglycemia, dyslipidemia and hyperketonemia in insulin-deficient diabetes. Attenuated insulin action on the liver alone contributes to perturbations in glucose homeostasis (359). In Chapter 3, we found that despite enhanced whole body insulin-sensitivity, glucose uptake was not increased in the soleus muscle. This led us to postulate that reduced hepatic glucose production may be involved in the anti-diabetic action of leptin. Intriguingly, studies conducted by our laboratory and others have revealed that direct action of leptin on hepatocytes can modulate hepatic insulin action (147,199,201,206). Based on these data, we postulated that direct leptin activation of hepatic leptin receptor signalling could mediate leptin-induced glucose lowering in type 1 diabetes. Therefore, in addition to assessing whether neuronal leptin signalling is required for the anti-diabetic action of leptin, we examined whether hepatic leptin signalling is required for the anti-diabetic action of leptin. We utilized mice previously generated and characterized in our laboratory (Leprflox/flox Albcre mice), which have a liver specific attenuation of hepatic leptin receptor signalling, achieved through an Albumin promoter driven Cre (Albcre) (206)3.                                                   3 Figures 38-40 in this chapter were published in Diabetes (328). All other data are unpublished.  86 5.2 RESULTS Mice with disrupted neuronal leptin receptor signalling outside of the ARH and VMH do not have a blunted response to leptin therapy  To determine whether leptin receptor signalling in neurons outside of the ARH and VMH are required for the action of leptin in type 1 diabetes, we examined whether peripheral leptin therapy could ameliorate STZ-induced hyperglycemia in Leprflox/flox Syncre mice as effectively as in littermate controls. Mice aged 12-14 weeks were treated with STZ; both Leprflox/flox Syncre mice and Leprflox/flox controls became similarly hyperglycemic following STZ administration (Figure 32A), and subsequently received osmotic pump implants delivering 10 µg /day leptin (Leprflox/flox-leptin; Leprflox/flox Syncre-leptin) or vehicle (Leprflox/flox-vehicle; Leprflox/flox Syncre-vehicle) on day 0. Since we determined in Chapter 4 that the phenotype of Leprflox/flox Syncre mice was not due to the presence of the Syncre transgene itself, we did not include Lepr+/+ Syncre controls in this study. Following pump implantation, glycemia continued to rise in Leprflox/flox Syncre-vehicle and Leprflox/flox-vehicle mice (Figure 32A), whereas diabetes was expectedly ameliorated in Leprflox/flox-leptin mice. Despite widespread deletion of neuronal Lepr-b in Leprflox/flox Syncre mice, diabetes was ameliorated as effectively by leptin therapy as it was in Leprflox/flox mice. On day 10, osmotic pumps were replaced to deliver 20 μg/day leptin or vehicle. With this higher dose of leptin, still no difference between Leprflox/flox-leptin and Leprflox/flox Syncre-leptin groups were observed. STZ injection initiated a continuous decline in body weight in all groups (Figure 32B). There was a trend toward increased weight loss in the leptin treated groups compared to their vehicle treated counterparts, but this did not reach significance (Figure 32B). Endogenous leptin levels were expectedly decreased by STZ injection, as observed in Leprflox/flox Syncre-vehicle and Leprflox/flox-vehicle groups compared to pre-STZ values (P = 0.035 and 0.040 respectively), and leptin therapy restored plasma leptin to pre-STZ levels in both Leprflox/flox Syncre mice and Leprflox/flox mice (Figure 32C). We noted that, independent of genotype, plasma leptin levels prior to STZ-administration were relatively high in some mice of the Leprflox/flox Syncre strain. Leptin levels corresponded to body weight (Figure 33A), which were more variable on this mixed genetic background compared to what we observed in C57Bl/6 mice. The variable leptin levels inherent in this strain allowed us to examine whether the responsivity to leptin therapy correlated to endogenous leptin levels prior to STZ. Interestingly, there was a nearly significant negative correlation between endogenous pre-STZ leptin levels, and the glucose lowering response to leptin therapy (Figure 33B),  87 indicating that high endogenous leptin levels prior to diabetes may decrease sensitivity to leptin therapy. Measurement of plasma insulin prior to STZ and on day 13 post-pump implant confirmed that STZ diminished circulating insulin levels similarly in both Leprflox/flox Syncre and Leprflox/flox mice (Figure 32D), and that amelioration of hyperglycemia by leptin was not accompanied by an increase in circulating insulin. Collectively, these data reveal that widespread deletion of the Lepr-b signalling domain in neurons outside the ARH and VMH does not blunt the therapeutic action of leptin in STZ-diabetic mice.  Figure 32. Attenuated neuronal leptin receptor signalling does not block therapeutic leptin action in STZ-diabetic mice. Leprflox/flox Syncre and Leprflox/flox male mice aged 12 to 14 weeks were injected with STZ, 7 days prior to implantation of osmotic pumps delivering 10 μg/day leptin or vehicle on day 0. On day 10 post-pump implant, osmotic pumps were replaced delivering 20 μg/day leptin or  88 vehicle. Four-hour fasted blood glucose (A) and body weight (B) were tracked for the study duration. Groups are as follows: Leprflox/flox-vehicle treated mice (open blue circles, n = 3); Leprflox/flox-leptin treated mice (filled blue circles, n = 5); Leprflox/flox Syncre-vehicle treated mice (open red squares, broken line, n = 5); Leprflox/flox Syncre-leptin treated mice (filled red squares, broken line, n = 5). Four-hour fasted plasma leptin (C) and insulin (D) were measured prior to STZ injection on day -9 (pre-STZ), and on day 13 post pump in vehicle (STZ-vehicle) and leptin (STZ-leptin) treated groups, n ≥ 3 (Leprflox/flox mice in blue bars, Leprflox/flox Syncre mice in red bars). Data are presented as mean ± SEM. Statistical analyses were performed by repeated measures two-way ANOVA (A,B) or two-way ANOVA (C,D) with a Tukey post-hoc test. † P < 0.05 leptin vs vehicle treated Leprflox/flox mice; ‡ P < 0.05 leptin vs vehicle treated Leprflox/flox Syncre mice. No significant differences were found between Leprflox/flox Syncre vs Leprflox/flox mice receiving the same type of treatment.   Figure 33. Leptin responsivity in STZ-diabetic mice negatively correlates with pre-STZ leptin levels. A) Correlation between endogenous leptin levels measured on day -9 prior to STZ-administration and body weight on the same day, in all Leprflox/flox (blue) and Leprflox/flox Syncre (red) mice (regardless of subsequent vehicle or leptin treatment) used in this study. B) The change in blood glucose on day 13 compared to baseline blood glucose at day -1 is plotted on the Y axis, and correlated to pre-STZ leptin levels on day -9 on the X axis, for Leprflox/flox -leptin (blue) and Leprflox/flox Syncre -leptin (red) mice. Linear fit (solid black line) and 95% confidence band (broken black line) are shown. Significant correlation was determined by Pearson's correlation test. Subdiaphragmatic vagotomy does not block leptin action in STZ-diabetes  While robust and widespread attenuation of the Lepr-b signalling domain in neurons outside of the ARH and VMH of Leprflox/flox Syncre mice had no impact on the ability of leptin to ameliorate diabetes, we next assessed whether parasympathetic efferents are necessary to mediate glucose lowering by leptin therapy. To test this, subdiaphragmatic vagotomies were performed in C57Bl/6 male mice at 6 weeks of age. Control mice received a sham operation. Sham and vagotomized mice were injected with STZ at 14 weeks of age. Notably, vagotomized mice were very sensitive to STZ effects, and 10 of 14 mice reached humane endpoint within several days of STZ injection, whereas all sham mice survived. We  89 suspect that this heightened sensitivity is due to the combined gastroparesis effect of vagotomy and STZ. The 4 surviving vagotomized mice received pump implants delivering 10 μg/day leptin on day 0 (vagotomy-leptin), and sham controls received implants on the same day delivering either vehicle (sham-vehicle) or 10 μg/day leptin (sham-leptin). Sham and vagotomized mice became similarly hyperglycemic following STZ, and as expected, sham-vehicle controls remained severely diabetic for the duration of the study, whereas 10 μg/day leptin treatment robustly lowered blood glucose levels in sham-leptin mice (Figure 34A). Interestingly, leptin therapy potently reversed hyperglycemia in vagotomy-leptin mice, and in fact blood glucose levels were significantly lower in vagotomy-leptin mice compared to sham-leptin mice at most time points during leptin therapy. Vagotomy-leptin mice tended to lose more weight than sham groups, but this only reached significance at one time point (Figure 34B). Plasma leptin was significantly increased in sham-leptin and vagotomy-leptin groups compared to pre-STZ values (P≤0.0003) (Figure 34C). Insulin levels (Figure 34D) were significantly decreased by STZ in all groups (P≤0.008) and remained low for the duration of the study. To confirm that the reduction in glycemia of vagotomized mice was due to leptin therapy rather than surgical complications in more sensitive mice, leptin therapy was discontinued after 19 days, at which point hyperglycemia rapidly returned to pre-treatment levels in both vagotomized and sham-operated mice, concomitant with a drop in plasma leptin to sham-vehicle levels (Figure 3C).  90  Figure 34. Subdiaphragmatic vagotomy does not attenuate leptin action in STZ-diabetic mice. C57Bl/6 male mice that received vagotomy or sham operations were treated with STZ to induce diabetes at 14 weeks of age, 7 days prior to pump implantation. On day 0, osmotic pumps were implanted delivering either 10 μg/day leptin or vehicle for a calculated delivery lasting 17.7 days. Groups are as follows: sham operated mice receiving vehicle (sham-vehicle, open blue circles/bars, n = 6); sham operated mice receiving leptin (sham-leptin, filled blue circles/bars, n = 7); vagotomized mice receiving leptin (vagotomy-leptin, filled red squares/bars, n = 4). Four-hour fasted blood glucose (A) and body weight (B) were tracked throughout the study. Four-hour fasted plasma leptin (C) and insulin (D) were measured 3 weeks prior to STZ administration, and on day 13 (during leptin therapy), and day 29 (after leptin therapy had ceased). Limits of detection are indicated by broken horizontal line. Statistical analyses were performed by repeated measures two-way ANOVA (A,B) or two-way ANOVA (C,D) with a Tukey post-hoc test. Data are presented as mean ± SEM. * P < 0.05 vagotomy-leptin vs sham-leptin mice; † P < 0.05 sham-leptin vs sham-vehicle mice; ‡ P < 0.05 vagotomy-leptin vs sham-vehicle mice. Given the well-known effect of the vagus to inhibit gastric distension (316,360), we validated the loss of vagal efferents by measuring stomach weight in mice at the end of the study. All of the vagotomy-leptin mice in the study had substantially enlarged stomachs compared to sham-vehicle and sham-leptin groups, consistent with vagal denervation  91 (Figure 35A). In addition, we examined vagally mediated CCK-induced satiety 1 week prior to STZ-injection (316,360). While sham-operated control mice displayed the expected decrease in food intake in response to CCK injection, this response was blunted in mice that underwent vagotomy (Figure 35B). Together, these data reveal that subdiaphragmatic vagal efferents are not required to mediate the action of leptin on blood glucose in STZ-induced diabetes.  Figure 35. Validation of subdiaphragmatic vagotomy. Empty stomach weight was measured in STZ-injected sham and vagotomized mice following sacrifice on day 32 (A) and analyzed by one-way ANOVA with a Tukey post-hoc test., n ≥ 4. Food intake 1 hour following cholecystokinin (CCK) or vehicle (V) injection was measured on day -15 prior to pump implantation (8 days prior to STZ injection), n ≥ 6 per group (sham in blue bars, vagotomy in red bars), and analyzed by two-way ANOVA with a Bonferroni post-hoc test. Data are presented as mean + SEM. * P < 0.05 vagotomy-leptin vs sham-leptin mice; † P < 0.05 sham-leptin vs sham-vehicle mice; ‡ P < 0.05 vagotomy-leptin vs sham-vehicle mice. Partial chemical sympathectomy does not block leptin action in STZ-diabetes Next, we examined whether sympathetic efferents are a requisite for leptin-mediated glucose lowering in type 1 diabetes. To do this, we chemically sympathectomized 12 week old C57Bl/6 male mice by i.p. injection of 6OHDA, which when given peripherally, causes selective degeneration of noradrenergic neurons (314,361). Control (sham) mice received injections of buffer only instead of 6OHDA. Administration of 6OHDA produced symptoms in mice consistent with decreased sympathetic function, including ptosis and piloerection (362,363).  Sham controls and 6OHDA treated mice were injected with STZ at 15 weeks of age, which induced similar degrees of hyperglycemia (Figure 36A) and similar reductions in body  92 weight (Figure 36B), although 6OHDA mice exhibited a trend toward greater hyperglycemia. Diabetic sham mice were subsequently treated with 20 μg/day leptin (sham-leptin) or vehicle (sham-vehicle), and diabetic 6OHDA mice were treated 20 μg/day leptin (6OHDA-leptin) by implantation of osmotic pumps on day 0. Notably, 20 μg/day leptin administration resulted in complete reversal of hyperglycemia in both sham-leptin and 6OHDA-leptin mice, whereas sham-vehicle mice remained hyperglycemic throughout the study. As expected, leptin therapy had no impact on body weight throughout the study (Figure 36B). Leptin therapy was subsequently ceased after day 9, at which point hyperglycemia rapidly returned to pre-treatment levels in 6OHDA-leptin and sham-leptin groups, as leptin levels decreased. Treatment of 6OHDA and sham mice with 20 μg/day leptin resulted in supraphysiological elevations in plasma leptin levels compared to pre-STZ values (P < 0.0001 for both groups), whereas plasma leptin levels were significantly reduced in sham-vehicle mice by STZ injection (P = 0.04; (Figure 36C). Following the cessation of leptin therapy, plasma leptin levels were similarly diminished in 6OHDA and sham mice. Expectedly, following STZ administration, plasma insulin dropped substantially in all groups (< 3 mice per group had detectable insulin levels after STZ, so statistical analysis could not be performed), and remained low for the duration of the study (Figure 36D).  93  Figure 36. Injection of 6OHDA to chemically sympathectomize mice does not attenuate therapeutic leptin action in STZ-diabetes. Sympathectomy was induced in male C57Bl/6 mice by injection of 6OHDA at 12 weeks of age. Sham controls received buffer only injections instead of 6OHDA. Mice were injected with STZ to induce diabetes 14 days prior to implantation of osmotic pumps delivering 20 μg/day leptin or vehicle on day 0, for a calculated delivery period of 9.2 days. Groups are as follows: sham-vehicle treated mice (sham-vehicle, open blue circles/bars, n = 5); sham-leptin treated mice (sham-leptin, filled blue circles/bars, n = 5); 6OHDA injected, leptin treated mice (6OHDA-leptin, filled red squares/bars, n = 5). Four-hour fasted blood glucose (A) and body weight (B) were tracked over the course of the study. Four-hour fasted plasma leptin (C) and insulin (D) levels were measured on indicated days relative to pump implantation. Limit of detection for insulin assay is shown by the broken horizontal line, and numbers above indicate the number of samples which yielded detectable insulin levels out of total mice in the group. Plasma leptin was detectable in all mice at all time points. Data are presented as mean ± SEM. Statistical analyses were performed by repeated measures two-way ANOVA (A,B) or two-way ANOVA (C) with a Tukey post-hoc test. Statistical analyses in D were performed by one-way ANOVA with a Tukey post-hoc test on day -8 values only. † P < 0.05 sham-leptin vs sham-vehicle mice; ‡ P < 0.05 6OHDA-leptin vs sham-vehicle mice. No significant differences were observed between 6OHDA-leptin and sham-leptin mice. To examine the extent of 6OHDA-mediated sympathectomy, we quantified TH immunoreactivity, a sympathetic neuron marker, in BAT – a tissue densely innervated by sympathetic neurons. Mice injected with 6OHDA displayed an obvious decrease in TH  94 immunoreactivity within BAT (Figure 37A). TH positive area relative to whole section area (Figure 37B), and TH positive area relative to total cell number (Figure 37C), revealed an approximate 50% decrease in TH immunoreactivity in 6OHDA injected mice compared to sham controls. Thus, we conclude collectively from these results that partial ablation of sympathetic neurons is insufficient to blunt leptin’s therapeutic actions in mice. With only a partial sympathectomy, it remains possible that leptin’s therapeutic action in this model could still be mediated by the sympathetic neurons that survived after 6OHDA injection. Interestingly however, the fact that these mice display physical symptoms of decreased sympathetic function and yet the efficacy of leptin therapy is not even modestly attenuated indicates that the mechanism of leptin action is likely independent of sympathetic efferents.  Figure 37. Verification of reduced sympathetic neurons in 6OHDA injected mice. BAT tissue was harvested on day 15 at the end of the study, and immunostained for TH, n=5 per group. Representative images for sham (left) and 6OHDA (right) mice are shown in (A), scale bar is 50 μm. Quantification of TH immunopositive area was expressed as a percent of total section area (B) and relative to total cell number (C). Data are presented as mean + SEM. Statistical analyses were performed by Student's t-test.  95 Hepatic leptin signalling is not required for the therapeutic effect of leptin in STZ-diabetes In addition to examining whether leptin therapy was dependent upon neuronal leptin receptor signalling, we assessed whether leptin receptor signalling in hepatocytes was required to mediate the effect of leptin therapy in STZ-induced diabetes. To accomplish this, we utilized mice Leprflox/flox Albcre mice, which have a liver specific attenuation of the Lepr-b signalling domain due to Albcre-induced hepatocyte specific recombination of the Leprflox allele (206,364). RT-PCR analysis of Lepr-b transcript from liver cDNA generated an amplicon similar in size to the 343 bp product expected from wildtype Lepr-b transcript in Leprflox/flox controls, whereas liver cDNA from Leprflox/flox Albcre mice produced an amplicon consistent with the expected 267 bp size of the Lepr Δ17 transcript (Figure 38). Thus in the livers of Leprflox/flox Albcre mice the majority of Lepr-b transcripts are truncated (refer to Figure 1). We previously characterized Leprflox/flox Albcre mice and found that these mice are protected from diet-induced glucose intolerance, have increased hepatic insulin sensitivity, but do not have altered blood glucose levels during fasting (206).  Figure 38. The long form of the leptin receptor is truncated in livers of Leprflox/flox Albcre mice. RNA was extracted from livers of Leprflox/flox Albcre mice and Leprflox/flox controls for use as template for generating cDNA (RT+). Reactions lacking reverse transcriptase (RT-) were run in parallel. The cDNA generated was PCR amplified using primers G and 60 (indicated in Figure 1). These primers generate a 343 bp amplicon for the Leprflox transcript and a 267 bp amplicon for the LeprΔ17 transcript. Arrows on the left denote the migration of molecular weight markers. Data generated by Dr. S. Covey.    To determine if leptin therapy reverses STZ-induced diabetes through direct action on the liver, age-matched male Leprflox/flox Albcre mice and Leprflox/flox littermate controls were first treated with STZ to induce a state of insulin deficiency. It was previously determined that the presence of the Albcre transgene itself did not impact glucose homeostasis in Leprflox/flox Albcre mice, therefore Lepr+/+ Albcre transgenic controls were not used in this  96 experiment (206). Following development of hyperglycemia, both Leprflox/flox controls and Leprflox/flox Albcre mice were divided into two groups, one group treated with 10 μg/day leptin, the other with saline, delivered via 14 day subcutaneous osmotic pump implants. Non-diabetic Leprflox/floxAlbcre and Leprflox/flox mice had similar body weight, blood glucose, and plasma leptin and insulin levels (Figures 39 and 40), as previously described (206). STZ injection produced an approximate10 fold reduction in plasma leptin in both Leprflox/flox Albcre and Leprflox/flox mice respectively (Figure 39A), and treatment with 10 μg/day leptin increased plasma leptin levels by >19 fold in both types of mice.  97  Figure 39. Leptin reverses STZ-induced hyperglycemia in Leprflox/flox Albcre mice. Four-hour fasted parameters in Leprflox/flox Albcre and Leprflox/flox mice that were STZ injected on day -4, and received subcutaneous 14 day osmotic pumps delivering 10 μg/day leptin (supplied by NHPP) or saline as vehicle on day 0. A) Plasma leptin, n ≥ 4. B) Blood glucose, n ≥ 4. C) Plasma β-hydroxybutyrate, n ≥ 4. D) Body weight, n ≥ 4. A, C) Leprflox/flox mice (blue solid bars), Leprflox/flox Albcre (red solid bars). B, D) Leprflox/flox Albcre mice treated with: STZ-leptin (red filled triangles) or STZ-vehicle (red open triangles). Leprflox/flox controls treated with: STZ-leptin (blue filled circles) or STZ-vehicle (blue open circles). A, C) Statistical analyses were performed by Student's t-test. B, D) Statistical analyses were performed by one-way ANOVA with a Holm-Sidak post-hoc test on values. * P < 0.0001 Leprflox/flox Albcre STZ-leptin vs STZ-saline; # P < 0.0001 Leprflox/flox STZ-leptin vs STZ-saline. Data are expressed as mean ± SEM.  98  We then examined whether hyperleptinemia in Leprflox/flox Albcre mice could lower blood glucose to the same extent as in Leprflox/flox controls. Following STZ administration, all groups displayed a similar extent of hyperglycemia (Figure 39B). STZ-vehicle treated Leprflox/flox Albcre mice and STZ-vehicle treated littermate controls were severely hyperglycemic for the duration of the study, whereas Leprflox/flox Albcre and Leprflox/flox mice treated with leptin showed a marked reduction in fasting blood glucose; interestingly, leptin treatment was equally effective at alleviating hyperglycemia in mice with disrupted hepatic leptin signalling as in their wildtype littermates (Figure 39B). However, unlike our studies in C57Bl/6 mice, a 10 µg/day dose of leptin was not able to fully restore fasting blood glucose levels to pre-STZ levels in Leprflox/flox mice. The partial amelioration of hyperglycemia was similar to what we observed in the Leprflox/flox Syncre strain (Figure 32A). This may be explained by differences in genetic background, which can alter leptin-related phenotypes (311,365). Nevertheless, comparing Leprflox/flox Albcre mice to littermates of identical genetic background reveals that liver leptin signalling is not required for the glucose lowering action of leptin in STZ-diabetes.  In addition to assessing the impact of attenuated hepatic leptin signalling on the glucose lowering action of leptin, we also examined the impact on the ketone lowering action of leptin. Pre-STZ plasma β-hydroxybutyrate levels were significantly elevated by approximately 10 and 5 fold following STZ administration in Leprflox/flox Albcre and Leprflox/flox mice respectively (Figure 39C). Similar to C57Bl/6 mice, leptin therapy reduced plasma β-hydroxybutyrate levels in Leprflox/flox controls to pre-STZ values. This effect was even more pronounced in Leprflox/flox Albcre mice, which despite inactivated hepatic leptin signalling, exhibited an approximate 96% reduction in plasma β-hydroxybutyrate levels in response to leptin therapy. In fact leptin therapy reduced plasma β-hydroxybutyrate to levels that were significantly lower than pre-STZ values in the Leprflox/flox Albcre mice. Thus leptin action on hyperketonemia does not depend on hepatic leptin signalling.  Leprflox/flox Albcre mice and Leprflox/flox controls injected with STZ displayed a gradual decrease in body weight, and all groups had similar weight loss over the duration of the study (Figure 39D). STZ administration decreased 4-hour fasted insulin levels to approximately 13% and 10% of pre-STZ levels in Leprflox/flox controls and Leprflox/flox Albcre mice respectively, and insulin levels were not restored by leptin therapy (Figure 40A). Thus, similar to C57Bl/6 mice, the effects of leptin on glucose and ketone levels are not attributable to changes in insulin levels or body weight.  99  Figure 40. Leptin therapy enhances insulin sensitivity in Leprflox/flox Albcre mice. A) Four-hour fasted plasma insulin, n ≥ 4. Only 2 STZ-leptin treated Leprflox/flox Albcre mice had detectable insulin, and statistical analyses could not be performed on this group. B) An ITT was performed on day 11, after a 4 hour fast, n ≥ 3. Mice received 0.65 U/kg insulin i.p. at time 0. AUC values are inset. Statistical analyses were performed by Student's t-test. Data are expressed as mean ± SEM. Since hyperleptinemia could alleviate the symptoms of insulin-deficient diabetes in mice with disrupted hepatic leptin signalling, we predicted that enhanced insulin sensitivity would still be observed in these mice in response to leptin therapy. To test this we performed ITTs. Both STZ-diabetic Leprflox/flox Albcre and Leprflox/flox mice displayed significantly enhanced insulin sensitivity in response to leptin treatment compared to their vehicle treated counterparts (Figure 40B). Therefore, hepatic leptin signalling is not required for the augmentation of insulin action by leptin therapy.  5.3 DISCUSSION In this chapter, we examined potential direct target tissues that mediate the glucose lowering effect of leptin in STZ-induced diabetes. The CNS has long been viewed as the primary mediator of the metabolic actions of leptin. Here we examined whether systemic leptin therapy requires leptin receptors in the CNS to exert its anti-diabetic effect. To this end, we tested leptin therapy in Leprflox/flox Syncre mice, which our analysis in Chapter 4 revealed have a partial hypothalamic reduction of Lepr-b, and widespread reduction of Lepr-b throughout extra-hypothalamic sites in the CNS. Importantly, within the hypothalamus, functional leptin signalling is maintained in the ARH and VMH, but attenuated specifically in the DMH, LHA and PMV of these mice. We found that disruption of neuronal leptin signalling  100 in these regions had no impact on the ability of peripheral leptin administration to reverse STZ-induced hyperglycemia. Furthermore, this study revealed that attenuation of autonomic efferent communication from the CNS to peripheral tissues, either by subdiaphragmatic vagotomy or partial sympathectomy did not blunt the glucose lowering action of leptin in STZ-diabetic mice. While this study demonstrates that widespread deletion of the Lepr-b signalling domain in neurons has no impact on the ability of leptin to reverse diabetes, the preserved leptin signalling in the ARH and VMH warrants careful consideration. Leptin action in POMC neurons and VMH neurons is sufficient to mediate leptin’s effects in STZ-diabetic rodents (308,309), and therefore, Leprflox/flox Syncre mice may respond normally to leptin therapy due to these intact hypothalamic neuronal circuits. Given the robust ablation of leptin signalling in the PMV and LHA in Leprflox/flox Syncre mice, our data strongly suggest that PMV and LHA hypothalamic neurons are not required to mediate the anti-diabetic action of leptin. Moreover, the strong depletion of Lepr-b transcript in the brainstem and spinal cord indicates that brainstem neurons are likely not necessary to mediate this effect of leptin. Although leptin signalling in POMC and VMH neurons is sufficient to mediate the therapeutic effect of leptin in STZ-diabetes, these neuronal populations appear to have redundant roles. Meek et al. demonstrated that while direct application of leptin to the VMH could reverse STZ-induced hyperglycemia in rats, leptin receptor deletion within the VMH was unable to attenuate the effect of central leptin therapy in STZ-diabetic mice (308). In addition, Fujikawa et al., found that deletion of leptin receptors in POMC neurons only mildly attenuated the effect of ICV leptin therapy in STZ-diabetic mice (309). Interestingly, considered collectively with the aforementioned studies, the data in this chapter suggest that there may not be a non-redundant neuronal population that mediates the anti-diabetic effect of leptin. Instead, it is likely that either redundant neuronal circuits, or a redundant peripheral signalling pathway can mediate the effect of leptin therapy on blood glucose. However, in order to definitively test whether neuronal leptin signalling is essential for the anti-diabetic action of leptin in type 1 diabetes, a mouse model with disrupted leptin signalling throughout the CNS, including the ARH and VMH neurons must be employed. The finding in this chapter that the attenuation of autonomic efferent pathways did not blunt glucose lowering by leptin is consistent with the possibility that leptin action in the CNS is not required to reverse hyperglycemia. Although 6OHDA produced a partial sympathectomy, several recent studies support that the anti-diabetic action of leptin is not mediated through increased sympathetic tone. Administration of β-adrenergic receptor  101 agonists is unable to lower blood glucose in STZ-diabetic rodents (366), and ICV leptin was found to reverse hyperglycemia in STZ-diabetic mice lacking β-adrenergic receptors (309). Similarly guanethidine mediated reduction in sympathetic nerve terminals did not blunt leptin action in STZ-diabetic rats (367). Regarding the role of the parasympathetic nervous system, we previously found that subdiaphragmatic vagotomy had no impact on the ability of leptin administration to lower blood glucose in Lepob/ob mice (316). These findings, in combination with those of the current study indicate that activation of the autonomic efferents is not critical for the anti-diabetic action of leptin. Since CNS leptin action can also alter neuroendocrine function, future studies investigating the role of hypothalamic-pituitary axes in the anti-diabetic action of leptin are warranted.  Several lines of evidence point to the liver as a primary tissue mediating the anti-diabetic effects of leptin. Leptin treated STZ-diabetic rats exhibit reduced glucose appearance rates, and ICV leptin administration decreases expression/activation of hepatic gluconeogenic factors (287,288,304). Although leptin can modulate liver glucose metabolism through direct hepatic leptin signalling (147,185), the current study reveals that this pathway is not required for leptin therapy to ameliorate STZ-induced hyperglycemia, hyperketonemia or to enhance insulin sensitivity. While it is possible that some hepatocytes retain expression of functional leptin receptors that compensate for attenuated hepatic leptin signalling, RT-PCR analysis indicated that wildtype Lepr-b expression is robustly reduced in the liver of Leprflox/flox Albcre mice. Furthermore, Leprflox/floxAlbcre mice trended toward a more potent response to hyperleptinemia in regard to plasma β-hydroxybutyrate and blood glucose levels than wildtype littermates, as opposed to an attenuated response.  Although direct action on the liver is not required for the glucose lowering action of leptin therapy, leptin-induced effects on liver metabolism through indirect mechanisms may be critical. Reconstitution of leptin receptors in the hypothalamus of leptin receptor deficient rats enhances the inhibitory action of insulin on hepatic glucose production (185). Furthermore, central leptin therapy can inhibit hepatic glucose production in STZ-diabetic rats (305). However, considered with the findings from vagotomized and sympathectomized mice in this chapter, it is unlikely that the glucose lowering action of leptin would be mediated through autonomic innervation in the liver. Alternatively, leptin’s effects on circulating factors that influence hepatic insulin sensitivity or hepatic glucose production may mediate to the therapeutic action of leptin. By decreasing free fatty acid levels as we observed in Chapter 3, possibly through increased lipid oxidation in adipose tissue (368), hyperleptinemia may enhance hepatic insulin sensitivity (369). Furthermore, the suppression  102 of glucagon and growth hormone likely reduces hepatic glucose output in STZ-diabetes. Therefore, direct and/or indirect leptin action on other tissues including adipocytes, and pancreatic α-cells may also be important for the therapeutic action of leptin (164,197,213). To investigate the role of direct leptin signalling in α-cells, our laboratory recently tested leptin therapy in STZ-diabetic Leprflox/flox mice in which disruption of leptin signalling specifically in α-cells was induced by a glucagon-cre transgene (240). The ability of leptin to lower STZ-induced hyperglycemia and hyperglucagonemia was not blunted in these mice, but the interpretation of these results is confounded by the fact that glucagon-cre activity was only present in half of the α-cells. Thus, a model with more complete ablation of leptin receptors in α-cells is necessary to definitively assess the role of α-cell leptin signalling in the reversal of hyperglycemia. Although the β-cell is a key target of leptin (195), it is unlikely to mediate the therapeutic action of leptin after STZ mediated β-cell depletion. The inhibitory effect of leptin on insulin secretion (190,195) would not benefit insulin-deficient rodents, and hyperleptinemia does not appreciably alter insulin levels or β-cell mass in rodents with type 1 diabetes (288).  While the mechanism of leptin action remains to be determined, this study suggests that neither the CNS nor the liver is the sole requisite target that conducts leptin’s glucose lowering effects in diabetic rodents. This leads us to postulate that leptin therapy may act either through a non-neuronal mechanism to lower blood glucose, or that it may act through redundant CNS-dependent and CNS-independent pathways. Since peripheral administration will expose widespread areas to leptin, the potential role of leptin signalling in peripheral tissues other than hepatocytes should be considered. Identification of the primary target tissues that are necessary for leptin action in type 1 diabetes will lead to a better understanding of the mechanism by which leptin therapy lowers blood glucose. Furthermore, identification and characterization of these leptin-induced pathways that lower blood glucose in type 1 diabetes may unveil new strategies to improve glycemic control in this disease.    103 CHAPTER  6: LEPTIN DIMINISHES SUBSTRATES FOR HEPATIC GLUCOSE PRODUCTION IN DIABETIC MICE 6.1 INTRODUCTION Upon leptin administration, multitudinous changes occur in organs that influence glucose levels. In Chapter 3, we confirmed that leptin administration reverses hyperglucagonemia (288), and found that leptin lowers growth hormone and enhances insulin sensitivity (328). Additional insight into the mechanism of leptin action has been gained from studies applying leptin directly to the CNS of type 1 diabetic rodents, which have shown a dramatic increase in BAT glucose uptake, and decreased glucose production by the liver (305,309). Furthermore, metabolomic analysis of livers from NOD mice treated with peripheral leptin showed remarkably similar results to those of mice treated with insulin (289). In contrast, we and others have found that glucose uptake in skeletal muscle does not appear robust enough to account for the glucose lowering effect of leptin (305,328), whereas others have shown robust skeletal muscle glucose uptake (309). With the plethora of metabolic changes that occur along with leptin-mediated glucose lowering, it is difficult to determine which pathways play a causal role. Three major substrates for hepatic gluconeogenenesis are lactate, alanine and glycerol. Circulating alanine and lactate are derived from protein catabolism and anaerobic glycolysis respectively, and can be subsequently taken up by hepatocytes for conversion into pyruvate for gluconeogenesis. Increased gluconeogenesis from both alanine and lactate has been reported in subjects with type 2 diabetes (370,371). The importance of alanine as a gluconeogenic substrate is also evident in Kruppel-like factor 15 knockout mice, which have defects in the conversion of pyruvate from alanine, and become severely hypoglycemic during fasting (372). In respect to the role of lactate in gluconeogenesis, it is interesting to note that the hypoglycemic action of metformin in diabetic rodents has been attributed in part to the inhibition of hepatic gluconeogenesis from lactate (373). Glycerol is a critical molecule in lipid and glucose homeostasis, as it serves as both a substrate for gluconeogenesis during fasting, and as the backbone for triglyceride synthesis during feeding. However, the role of glycerol in diabetes is poorly defined. A recent clinical study found that glycerol levels are increased in men with increased fasting glycemia, impaired glucose tolerance and recent onset type 2 diabetes, and that elevated fasting glycerol levels predict type 2 diabetes incidence (374). Gluconeogenesis from glycerol (glycerol gluconeogenesis) accounts for approximately 10% of hepatic glucose production in  104 type 2 diabetic patients (375), which results from both increased lipolysis and altered hepatic glycerol handling (376). Glycerol infusion at physiological concentrations (0.5 mmol/L), stimulates a > 2 fold increase in hepatic glucose output from perfused murine livers, and cultured primary hepatocytes (377). Further evidence for a critical role of glycerol gluconeogenesis in diabetes is derived from mouse models in which glycerol channels have been deleted. At physiological concentrations, glycerol permeates cells through aquaglyceroporins (AQPs). Genetic deletion of AQP9, the major channel facilitating glycerol entry into the liver for gluconeogenesis, reduces fasting glucose levels in Leprdb/db mice (378). Deletion of AQP7, believed to be the major channel through which glycerol is released from adipocytes during lipolysis, reduces circulating glycerol and results in fasting hypoglycemia (379). Both AQP9 and AQP7 are upregulated in obese and type 2 diabetic subjects, and are down-regulated by insulin and leptin application in vitro (380). These data suggest that glycerol levels may play a key role in determining circulating glucose concentrations, and that therapeutic strategies that reduce glycerol levels could exhibit anti-diabetic actions. Indeed, pharmacological inhibition of lipolysis, which reduces glycerol and free fatty acid levels, improves glycemia in rats with STZ-induced diabetes (381). To elucidate the mechanism of leptin-mediated glucose lowering in type 1 diabetes, we performed a rigorous analysis of glucose metabolism in leptin treated STZ-diabetic mice. This chapter reveals a surprising finding that leptin therapy markedly depletes several key energy substrates, and presents evidence that leptin therapy reduces hepatic glucose production, not through inhibition of hepatic gluconeogenic pathways, but through depletion of circulating glycerol levels4. 6.2 RESULTS Leptin therapy attenuates the metabolic and energy defects of STZ-induced diabetes in mice The studies in this chapter were conducted in the CDM, with leptin supplied from Peprotech. Under these conditions, preliminary evidence revealed that the dose we had previously used (10 μg/day leptin from NHPP at D.H. Copp) only resulted in partial amelioration of hyperglycemia. Thus for these studies we used a dose of 20 μg/day leptin. We first confirmed the extent of glucose lowering in STZ-diabetic mice with 20 μg/day leptin, and examined the levels of leptin and insulin in response to this treatment. Mice were                                                4 All data in this chapter are unpublished.  105 injected with STZ, and 5 days later following the onset of hyperglycemia, implanted with subcutaneous osmotic pumps delivering 20 μg/day leptin or vehicle. Non-diabetic controls received buffer injection instead of STZ, and sham surgery instead of pump implants. As we have previously shown in Chapter 3 (328), leptin therapy lowered 4-hour fasted blood over the course of several days, whereas vehicle treated controls remained hyperglycemic; blood glucose reached 10 mmol/L within 5 days of leptin treatment, and reached levels similar to non-diabetic controls within 7 days (Figure 41A). Similar to our findings in Chapter 3, STZ-injection elicited a steady decline in body weight, which was not altered by leptin therapy (Figure 41B). Plasma leptin levels were measured on day 8, and were expectedly lowered in STZ-vehicle mice compared to non-diabetic controls (Figure 41C), whereas 20 μg/day leptin treatment raised circulating leptin to supraphysiological levels. Plasma insulin levels were reduced by STZ-treatment, and were not restored by leptin therapy (Figure 41D), confirming that leptin can lower blood glucose without restoring circulating insulin levels. Corresponding to the similar body weight in STZ-vehicle and STZ-leptin treated mice, both groups had similar fat and lean mass relative to body weight (Figure 41E).    106 Figure 41. Leptin therapy reverses hyperglycemia and the symptoms of insulin-deficient diabetes. On day 0, STZ-diabetic mice received 20 µg/day leptin (STZ-leptin, red) or vehicle (STZ-vehicle, blue) on day 0. Non-diabetic mice (black) were injected with STZ buffer only, and received sham surgery on day 0. A) Leptin therapy lowered 4-hour fasted blood glucose levels during 8 days of treatment. B) Leptin therapy did not significantly lower 4-hour fasted body weight compared to STZ-vehicle controls. Plasma leptin levels (C) and insulin levels (D) were measured on day 8 post pump. E) Both lean and fat mass, measured by DEXA on day 6 post pump were reduced by STZ, and not further impacted by leptin therapy. Data are presented as mean ± SEM, n = 7-8 for panels A-D and n = 5-6 for panel E. Statistical analyses were performed by repeated measures two-way ANOVA (A,B), and one-way ANOVA (C-E) with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle, † P < 0.05 STZ-vehicle vs non-diabetic, and ‡ P < 0.05 STZ-leptin vs non-diabetic. We next examined the effect of leptin therapy on energy balance. Both polyuria (Figure 42A) and polydipsia (Figure 42B) were diminished by leptin therapy, consistent with the previously reported reduction in glucose excretion following leptin therapy (289). As expected (285), leptin therapy reversed diabetic hyperphagia in STZ-injected mice (Figure 42C). Despite the normalization of food intake, pair-feeding studies have already demonstrated that the metabolic action of leptin therapy is not mimicked by caloric restriction (287-289). Analysis of energy expenditure revealed no significant alterations between any groups, although there was a trend toward increased energy expenditure in STZ-vehicle mice (Figure 42D). In contrast a marked decrease in locomotor activity was observed in STZ-vehicle mice, which was fully rescued by leptin therapy (Figure 42E). Collectively, these results show that leptin therapy robustly normalizes the disturbances in energy balance and the major symptoms of uncontrolled diabetes.  107  Figure 42. Leptin therapy reverses the symptoms and energy disturbances of STZ-induced diabetes. STZ-diabetic mice received pump implants delierving either 20 µg/day leptin (STZ-leptin, red) or vehicle (STZ-vehicle, blue) on day 0. Non-diabetic mice (black) were injected with STZ buffer only on day -5, and received sham surgery on day 0. A) The appearance of bedding in cages from singly housed representative mice from each treatment group (from day 3-6), demonstrate reversal of polyuria in STZ-leptin mice. Cumulative water intake (B), and food intake (C), average energy expenditure adjusted for lean mass by analysis of covariance (D), and average locomotor activity (E) were measured from day 3-6 in metabolic cages. Because STZ-leptin mice had difficulty training with new food hoppers, food pellets were placed on the cage bottoms and difference in food weight before and after was added to cumulative feeding from the hoppers. Data are presented as mean ± SEM, n = 5-6. Statistical analyses were performed by one-way ANOVA with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle, † P < 0.05 STZ-vehicle vs non-diabetic, and ‡ P < 0.05 STZ-leptin vs non-diabetic. Leptin treatment induces hypoglycemia during prolonged fasting in STZ-diabetic mice  We had previously found a more pronounced effect of leptin on lowering glucose in the fasted state compared to the fed state, indicating that leptin may specifically target hepatic glucose production to reverse hyperglycemia (328). Consequently, as an indirect measure of hepatic glucose production we assessed whether leptin treated mice could maintain  108 steady blood glucose levels during a prolonged fast. We tested this in 2 cohorts of mice using different doses of leptin therapy: 10 μg/day leptin which partially attenuates 4-hour fasting hyperglycemia, and 20 μg/day leptin which fully normalizes 4-hour fasting blood glucose levels. Prior to food removal, 10 μg/day leptin treated mice had significantly lower blood glucose levels compared to STZ-vehicle controls, albeit they were still severely hyperglycemic (Figure 43A). Following food removal, blood glucose levels rapidly declined in the STZ-leptin group, whereas blood glucose levels were maintained in the other groups. One STZ-leptin mouse was removed from the experiment at 14 hours due to dangerous hypoglycemia (< 2.0 mmol/L), and the experiment was ceased at 15 hours when 2 additional STZ-leptin mice became hypoglycemic. The same phenomenon was observed when mice were treated with 20 μg/day leptin (Figure 43B). In this case, the initial free feeding blood glucose of STZ-leptin mice was only slightly above non-diabetic controls, and severe hypoglycemia occurred after only 5 hours of fasting. The inability of leptin treated mice to maintain fasting blood glucose levels is indicative of decrased endogenous glucose production.  109  Figure 43. Leptin treated STZ-diabetic mice are sensitive to prolonged fasting. A) STZ-diabetic mice were treated with 10 µg/day leptin (red) or vehicle (blue), and compared to non-diabetic controls. On day 11 of leptin therapy, mice were fasted at 9AM, and blood glucose was prior to food removal, and periodically throughout the fast until 2 STZ-leptin mice became dangerously hypoglycemic, n = 10. When blood glucose levels were > 33.3 mmol/L, blood was diluted with blood from a non-diabetic mouse and measured for glucose. One STZ-leptin treated mice was pulled from the experiment at 14 hours due to hypoglycemia. B-D) STZ-diabetic mice were treated with 20 µg/day leptin (red) or vehicle (blue), and compared to non-diabetic controls. On day 7 of leptin therapy, the same experiment as in A was performed, and continued until most STZ-leptin mice reached dangerous hypoglycemia, n = 5. Blood glucose (B), plasma β-hydroxybutyrate (C) and hepatic glycogen (D). Data are represented as mean ± SEM. Statistical analyses were performed by two way repeated measures ANOVA (A,B) and two-way ANOVA (C, D) with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle; † P < 0.05 STZ-vehicle vs non-diabetic; and ‡ P < 0.05 STZ-leptin vs non-diabetic. As ketone bodies serve as an alternative fuel source during hypoglycemia, we examined whether these were altered in leptin treated mice. Prior to fasting, leptin treated mice had β-hydroxybutyrate levels similar to non-diabetic controls, whereas ketones were markedly elevated in STZ-vehicle mice (Figure 43C). However, while non-diabetic mice  110 showed an approximate 6 fold elevation of circulating β-hydroxybutyrate upon fasting, levels remained markedly low in STZ-leptin mice, despite severe hypoglycemia.  As the first source of endogenous glucose production by the liver is hepatic glycogen, we next assessed whether the severe hypoglycemia in STZ-leptin mice was due to an inability to mobilize liver glycogen. Interestingly, prior to fasting glycogen stores were already approximately 60% lower in STZ-leptin mice compared to STZ-vehicle (Figure 43D). However, in response to fasting, hepatic glycogen was substantially depleted in all groups, including STZ-leptin mice, suggesting that glycogenolysis is not impaired by leptin therapy. Given that hepatic glycogen stores in STZ-leptin and non-diabetic mice are reduced to the same level during 9 hours of fasting, the severe hypoglycemia in STZ-leptin treated mice suggests an impairment in gluconeogenesis after glycogen stores are exhausted, such that STZ-leptin mice cannot maintain safe blood glucose levels. Leptin therapy does not inhibit gluconeogenesis, but depletes hepatic energy stores in STZ-diabetic mice To examine gluconeogenic pathways we first measured hepatic Pepck transcript levels in leptin treated mice. STZ administration produced the expected rise in Pepck transcript compared to non-diabetic levels, but surprisingly this was not lowered by leptin (Figure 44A). Interestingly, leptin therapy reduced transcript levels of the bidirectional glucose transporter Glut2 (Figure 44A). To examine whether gluconeogenesis was inhibited at any point along the gluconeogenic pathway we measured gluconeogenesis by injecting 7-hour fasted mice with pyruvate, and measuring blood glucose levels for 2 hours. Interestingly, leptin treated mice exhibited a normal response to pyruvate, and this acutely rescued the hypoglycemic effect of prolonged fasting. Thus surprisingly, in spite of the inability of mice to maintain fasting blood glucose levels, gluconeogenesis from pyruvate was not defective in leptin treated mice (Figure 44B).  111  Figure 44. Leptin therapy depletes hepatic energy substrates, but does not inhibit gluconeogenesis. Mice were treated 20 µg/day leptin (STZ-leptin, red), vehicle (STZ-vehicle, blue) and non-diabetic mice (black) for 8 days. Livers were harvested following a 4 hour fast on day 8 (A, C-J) and a pyruvate tolerance test was performed on day 5 (B). A) Abundance of hepatic Pepck and Glut2 transcripts was measured by RT-qPCR, and is expressed relative to a non-diabetic calibrator sample, n = 4-6. B) Leptin treated mice have normal pyruvate gluconeogenesis. Mice were fasted for 7 h, prior to injection of pyruvate (2 g/kg bodyweight, solid lines) or vehicle (broken lines) at time 0. Blood glucose was measured at indicated time points post-injection, n = 4-5. # P < 0.05 pyruvate vs vehicle injection in the treatment group with the corresponding color. Leptin treatment depleted hepatic glucose (C), glycogen (D), triglycerol (E), but had no effect on hepatic cholesterol (F). Leptin corrected hepatic acetyl CoA levels, without affecting free CoA levels (G), resulting in a non-significant reduction in acetyl CoA: free CoA ratio (H), n = 6-8. Leptin therapy reduced hepatic HMG-CoA (I) and had no effect on succinyl CoA (J). Data are represented as mean ± SEM. Statistical analyses were performed by one-way ANOVA with a Tukey post-hoc test, except in B, in which statistical analyses were performed by two way repeated measures ANOVA with a Sidak post-hoc  112 test. * P < 0.05 STZ-leptin vs STZ-vehicle, † P < 0.05 STZ-vehicle vs non-diabetic, and ‡ P < 0.05 STZ-leptin vs non-diabetic. To search for other potential mechanisms that could explain diminished gluconeogenesis during fasting, we measured hepatic energy sources in 4-hour fasted mice. Hepatic glycogen levels were robustly depleted in STZ-leptin mice during a 4-hour fast (C). Interestingly, there was an extreme deprivation of intrahepatic glucose (D), and a robust depletion of hepatic triglycerides (E), all of which were significantly lower compared to non-diabetic and STZ-vehicle mice. Hepatic cholesterol levels were unaltered by uncontrolled diabetes or leptin therapy (F). To assess whether the depletion of hepatic triglycerides and glucose could be due to altered lipid or glucose oxidation, we examined hepatic levels of short CoA esters. Acetyl CoA levels were reduced beyond that of non-diabetic mice, indicative of decreased overall oxidation (G). While the ratios of acetyl CoA: free CoA were not significantly different between groups, STZ-vehicle mice had a trend towards higher ratios, whereas STZ-leptin was more similar to non-diabetic levels, indicative of a restoration of glucose oxidation relative to lipid oxidation by leptin therapy (H). Similarly, the levels of HMG CoA were significantly reduced in STZ-leptin mice (I); HMG CoA is an intermediate in ketogenesis, and thereby further supports that lipid oxidation is reduced in leptin treated mice. Moreover, the constant succinyl CoA levels between all 3 groups (J), suggests that despite the depletion of these energy sources the TCA cycle is functioning normally in the liver. Leptin treated mice have diminished energy substrates during prolonged fasting  We next examined whether the energy deprived state of the liver may reflect an overall deprivation of gluconeogenic and lipid substrates in the body. We measured whether plasma gluconeogenic substrates were diminished during the prolonged fasting experiment in STZ-leptin mice. Plasma measurements were taken prior to food removal at time 0, and after 9 hours of fasting. Lactate (Figure 45A) and alanine (Figure 45B), 2 major gluconeogenic substrates, were significantly reduced in all treatment groups in response to fasting, indicating the expected utilization of these substrates for gluconeogenesis. Leptin treatment had no effect on fed lactate or alanine levels, and leptin treated mice had similar lactate and alanine levels to non-diabetic controls during fasting. In contrast, plasma glycerol levels appeared substantially reduced in STZ-leptin mice in the fed and prolonged fasted state (46% and 28% those of STZ-vehicle mice respectively), although this did not reach statistical significance (P = 0.11 and 0.10 respectively compared to STZ-vehicle controls)  113 (Figure 45C). We also measured plasma free fatty acids (Figure 45D). Expectedly, there was a trend toward increased free fatty acids in non-diabetic mice in response to fasting. Free fatty acid levels were increased in STZ-vehicle mice, but not further enhanced by fasting. In stark contrast, whereas STZ-leptin treated mice had fed plasma free fatty acid levels similar to non-diabetic mice, in response to fasting, plasma free fatty acids were robustly reduced, indicating either an increase in utilization or a decrease in supply. The extremely low β-hydroxybutyrate levels after 9 hours of fasting (Figure 43C), and reduced hepatic acetyl CoA: free CoA ratio in leptin treated mice (Figure 44H), suggest that the depletion of free fatty acids is not due to enhanced lipid-oxidation, but instead could be due to impaired adipose tissue lipolysis. Consistent with a decrease in lipid substrates, circulating triglycerides were also robustly depleted by leptin therapy in both the fed and fasted state (Figure 45E). There was also a trend toward reduced plasma cholesterol, but this was not as robust as the depletion in triglycerides or free fatty acids (Figure 45F). Collectively, these data suggest that the robust lipopenic action of leptin therapy in STZ-diabetic mice may limit the amount of glycerol available for gluconeogenesis during fasting.   114  Figure 45. Gluconeogenic substrate availability is altered during prolonged fasting in leptin treated mice. STZ-diabetic mice were treated with 20 µg/day leptin (red) or vehicle (blue), and compared to non-diabetic controls (black). On day 7 of leptin therapy, 5 mice/group were sacrificed at time 0 prior to food removal (9AM), and 5 mice/group underwent a prolonged fast (same mice tracked in Figure 3B), and were sacrificed at 9 hours of fasting. Lactate (A), alanine (B), glycerol (C), free fatty acids (FFA, D), triglycerides (TG, E), and cholesterol (F) were measured in the plasma of 0- and 9 hour-fasted mice. Data are presented as mean + SEM, n = 5. Statistical analyses were performed by two-way ANOVA with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle, † P < 0.05 STZ-vehicle vs non-diabetic, and ‡ P < 0.05 STZ-leptin vs non-diabetic. # P < 0.05 for same group 0- hour vs 9-hour fasted.  115 Leptin therapy depletes circulating energy substrates concomitant with glucose lowering We treated STZ-diabetic C57Bl/6 male mice with 20 μg/day leptin, and measured fasting blood glucose and energy substrates daily to examine the kinetics of leptin-mediated glucose lowering and energy deprivation. Substantiating our data above that leptin does not acutely lower blood glucose in diabetic mice, we found that initially there was a significant but only modest decrease in blood glucose levels, and subsequently after 2 days, blood glucose rapidly began to drop, reaching non-diabetic levels by day 4 (Figure 46A). This was not due to a slow rise in plasma leptin via pump infusion, as maximal leptin levels were achieved by the first day of leptin therapy (Figure 46B). Interestingly, there was an immediate depletion of hepatic glycogen levels by day 1 (Figure 46C), however, this alone is not sufficient to reverse hyperglycemia, as blood glucose levels remained high until day 2. At the same time that blood glucose levels began to drop to normal levels, there was a drop in several energy substrate concentrations in the plasma. Of the 3 major gluconeogenic substrates (lactate, alanine and glycerol, Figure 46D-F) there was a depletion of circulating glycerol, which matched the kinetics of glucose lowering (Figure 46F). Circulating free fatty acids, β-hydroxybutyrate, and triglycerides followed the same kinetics (Figure 47A-C), dramatically falling by day 3 of leptin therapy. In fact, by the end of the study free fatty acids and triglycerides were depleted in STZ-leptin mice to approximately 39% and 27% the levels of STZ-vehicle mice. Cholesterol levels also were significantly reduced on days 4 and 5 of leptin therapy (Figure 47D). Collectively, these data show that glucose lowering by leptin corresponds to a marked depletion in energy substrates, namely free fatty acids, triglycerides, and glycerol, and indicate that the fall in these energy substrates may play a causal role in the glucose lowering action of leptin. The decreased free fatty acid and glycerol levels likely reflect diminished lipolysis in leptin treated mice.  116  Figure 46. Diminished plasma glycerol and hepatic glycogen coincide with amelioration of hyperglycemia by leptin. STZ-diabetic mice were implanted with osmotic pumps delivering 20 µg/day leptin (STZ-leptin, red) or vehicle (STZ-vehicle, blue) on day 0. Non-diabetic controls received sham surgery on day 0. Four-hour fasted plasma analytes were tracked on day -1, and from day 1-5 of treatment. A) Blood glucose. B) Plasma leptin. C) Hepatic glycogen content was dramatically reduced by leptin therapy on day 1. Leptin therapy did not alter plasma lactate (D), or alanine (E), but diminished plasma glycerol (F) over the course of treatment. Data are represented as mean ± SEM, n = 5-10. Statistical analyses were performed by two way repeated measures ANOVA (A,B,D-F) or one-way ANOVA (C) with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle; † P < 0.05 STZ-vehicle vs non-diabetic; and ‡ P < 0.05 STZ-leptin vs non-diabetic.   117  Figure 47. Leptin therapy reduces fasting plasma lipids with time. STZ-diabetic mice were implanted with osmotic pumps delivering 20 µg/day leptin (STZ-leptin, red) or vehicle (STZ-vehicle, blue) on day 0. Non-diabetic controls received sham surgery on day 0. Four-hour fasted plasma analytes were tracked on day -1, and from day 1-5 of treatment. Leptin reduced plasma free fatty acids (FFA, A), β-hydroxybutyrate (B), and triglycerides (TG, C) on a similar time frame to glucose lowering. Plasma cholesterol was also reduced by day 5 in leptin treated mice (D). Data are represented as mean ± SEM, n = 5-10. Statistical analyses were performed by two way repeated measures ANOVA with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle; † P < 0.05 STZ-vehicle vs non-diabetic; and ‡ P < 0.05 STZ-leptin vs non-diabetic. Acute glycerol infusion prevents fasting hypoglycemia in leptin treated mice Glycerol is a major substrate for gluconeogenesis, and it has previously been shown that mice with impaired glycerol mobilization from adipocytes exhibit fasting hypoglycemia (379)  – reminiscent of our observation in leptin treated diabetic mice. Thus, we subsequently examined whether the leptin-induced depletion of circulating glycerol could  118 contribute to the glucose lowering effect of leptin. First, we examined whether hepatic glycerol levels reflected the depletion of circulating glycerol in leptin treated mice. Indeed, 4-hour fasted STZ-leptin mice showed a substantial depletion of hepatic glycerol content after 7 days of leptin therapy (Figure 48A). Interestingly, transcript abundance of Aqp9, the major transporter for glycerol in the liver, was also decreased by leptin therapy (Figure 48B). To test whether glycerol could play a causal role in glucose lowering by leptin, we examined whether acute injection of glycerol could increase blood glucose levels in leptin treated mice, and prevent fasting induced hypoglycemia. Mice were fasted for 7 hours, and subsequently received vehicle or glycerol injection at time 0. Vehicle injection had no impact on blood glucose in STZ-leptin mice, and glucose levels continued to drop reaching dangerous hypoglycemic levels over the course of 2 hours (Figure 48C). In contrast, injection of glycerol, acutely increased blood glucose levels in STZ-leptin treated mice, and temporarily prevented leptin-induced hypoglycemia. By 2 hours, blood glucose had decreased again trending toward hypoglycemia. Collectively, these data indicate that glycerol can acutely raise blood glucose in leptin treated mice and reverse fasting-induced hypoglycemia, and thus, when coupled with the depletion of hepatic glycogen, glycerol depletion may be a key mechanism of leptin-mediated glucose lowering in diabetic mice.  Figure 48. Glycerol injection can raise blood glucose in STZ-leptin treated mice. Hepatic glycerol content (A) and transcript abundance of aquaporin9 (aqp9) in livers harvested from 4-hour fasted STZ-leptin (red), STZ-vehicle (blue), and non-diabetic (black) mice, on day 8 post pump implant, n = 6-7. Aqp9 transcript abundance is expressed relative to a non-diabetic calibrator sample. C) STZ-diabetic mice were implanted with osmotic pumps delivering 20 µg/day leptin (red) or vehicle (blue), and compared to non-diabetic controls (black) receiving sham surgery. On day 5 post pump implant, mice were fasted for 7 h, and subsequently injected with either 2 g/kg bodyweight glycerol (solid lines) or vehicle (broken lines). Blood glucose was tracked at the indicated time points post injection. Data are represented as mean ± SEM, n = 4-8. Statistical analyses were performed by one- 119 way ANOVA with a Tukey post-hoc test (A,B) or two way repeated measures ANOVA with a Sidak post-hoc test (C). * P < 0.05 STZ-leptin vs STZ-vehicle, † P < 0.05 STZ-vehicle vs non-diabetic, and ‡ P < 0.05 STZ-leptin vs non-diabetic. # P < 0.05 pyruvate vs vehicle injection in the treatment group with the corresponding colour. Leptin-mediated glucose lowering and gluconeogenic substrate depletion is consistent with the activation of an energy sink  Collectively, the fact that leptin reversed STZ-induced hyperglycemia, contributed to profound fasting-hypoglycemia, and the depletion of hepatic gluconeogenic substrates, led us to hypothesize that leptin therapy may achieve these effects through the activation of an energy substrate sink. A physiological process that acts as a potent energy sink is thermogenesis in BAT. Thus we next examined whether leptin-induced BAT thermogenesis. To first test this, we implanted temperature transponders at on day 0 in STZ-leptin, STZ-vehicle and non-diabetic mice, to measure interscapular temperature (Figure 49A). Interscapular temperature was decreased by STZ administration, indicative of decreased BAT thermogenesis. Remarkably, leptin therapy increased interscapular temperature, normalizing it to non-diabetic levels by day 2 post leptin therapy, indicating that leptin therapy enhanced BAT thermogenesis. In these same mice, we measured RER from days 3-6 to determine whether glucose oxidation was enhanced, which could be caused by enhanced thermogenesis (Figure 49B). Indeed, STZ-leptin mice showed a marked increase in RER, indicative of increased glucose oxidation, compared to STZ-vehicle mice, although this did not reach non-diabetic levels. Moreover, interscapular BAT weight on day 6, was markedly decreased by leptin therapy, indicative of triglyceride lipolysis and lipid oxidation (Figure 49C).  Figure 49. The effects of leptin in STZ-diabetic mice are consistent with the activation of an energy sink. STZ-diabetic mice were implanted with osmotic pumps on day 0 delivering 20 µg/day  120 leptin (STZ-leptin, red), or vehicle (STZ-vehicle, blue). Non-diabetic controls were not treated with STZ, and received sham surgery on day 0 (non-diabetic, black). A) RER was tracked for 48 hours from day 4 to 5. Statistical analyses were performed by two-way ANOVA on area under the curve for each light and dark cycle with a Tukey post-hoc test. B) Intrascapular temperature was tracked over course of therapy by implanted temperature transponders. Statistical analyses were performed by two-way repeated measures ANOVA. C) BAT mass harvested 6 days post therapy. Data are presented as mean ± SEM, n = 4-6. Statistical analyses performed by one-way ANOVA with a Tukey post-hoc test. * P < 0.05 STZ-leptin vs STZ-vehicle. † P < 0.05 STZ-vehicle vs non-diabetic. ‡ P < 0.05 STZ-leptin vs non-diabetic. 6.3 DISCUSSION In the current study we found that leptin therapy lowers blood glucose concomitantly with a dramatic depletion of key energy substrates, namely glycerol, free fatty acids, and triglycerides. In addition, while leptin therapy normalized 4-hour fasting blood glucose levels in STZ-diabetic mice, leptin treated mice were unable to cope with prolonged fasting, displaying severe hypoglycemia, hypoketonemia, and impaired free fatty acid mobilization. Our data support that the glucose lowering action of leptin therapy is mediated by reduced hepatic glucose production, however this is not achieved through inhibition of gluconeogenic pathways, but rather depletion of substrates for gluconeogenesis and glycogenolysis. Moreover, acute administration of glycerol was able to increase blood glucose levels in leptin treated mice, and delay leptin-mediated hypoglycemia in fasted mice. Several interesting concepts can be drawn from the fact that STZ-leptin mice cannot maintain sufficient blood glucose levels during prolonged fasting. Firstly, the dose of leptin necessary to reverse hyperglycemia also induces severe hypoglycemia during fasting. This highlights a currently underappreciated effect of leptin therapy in type 1 diabetes that warrants caution in using leptin as an anti-diabetic therapy for type 1 diabetes. We have reported similar hypoglycemic effects in leptin treated STZ-diabetic mice during insulin tolerance tests (328) and glucose tolerance tests (328). While other studies claim that leptin does not induce hypoglycemia (309), it should be noted that these studies were conducted in 2-hour fasted mice. Secondly, the degree by which leptin lowers glycemia is highly-dependent upon fasting time, and this should be considered between different studies. Presumably, given the substantially lower fed blood glucose levels achieved with 20 μg/day compared to 10 μg/day leptin, if a high enough dose of leptin is used, fed blood glucose levels could be normalized. Normal fed blood glucose levels were reported in diabetic NOD mice treated with 20 μg/hour leptin (289). The mechanism driving leptin-mediated hypoglycemia during fasting is likely linked to the same mechanism driving leptin-mediated reversal of STZ-induced hyperglcycemia.   121 The inability of leptin treated mice to cope during fasting suggests that there are insufficient energy stores to maintain blood glucose levels for survival, and thus energy substrates including glucose are feeding-derived, allowing leptin treated mice to maintain sufficient circulating energy substrates only in the fed state. The extreme energy deprivation induced by leptin during fasting, is also seen within the liver, with a dramatic depletion of glycogen, triglycerides, and glucose. While it could be postulated that decreased hepatic and circulating lipids are due to increased oxidation, the lack of ketogenesis and decreased hepatic acetyl CoA: free CoA levels indicate the absence of lipid oxidation. Interestingly, while circulating glycerol is depleted, other gluconeogenic substrates, namely circulating pyruvate, alanine and lactate are unaffected; thus, these substrates alone are insufficient to support glucose production in the presence of depleted hepatic glycogen stores and diminished glycerol gluconeogenesis. This indicates that the fall in plasma glycerol levels could be the key mediator of glucose lowering by leptin. The fasting phenotype of leptin treated STZ-diabetic mice is remarkably similar to lipoatrophic A/ZIP mice, which have a complete lack of white adipose tissue (42), and display a marked reduction in glucose, free fatty acid, and ketone levels during prolonged fasting. This suggests that WAT plays a critical role in maintaining blood glucose levels during fasting. Consistent with a role for lipolysis in influencing glucose metabolism, inhibition or haploinsufficiency of hormone sensitive lipase (HSL), which is involved in lipolysis of WAT triglyceride stores, results in improved insulin sensitivity and glucose metabolism in HFD-fed mice, without altering fat mass (382). Furthermore, pharmacological inhibition of HSL has been shown to lower free fatty acid, glycerol, and glucose levels in STZ-diabetic rats (381). Taken together, these findings indicate that the leptin-induced depletion of glycerol and glucose in STZ-diabetic mice could be mediated by alterations in WAT lipid stores or lipolysis. Interestingly, the postulated mechanism of leptin-mediated glucose lowering through glycerol depletion aligns with the already well-known lipopenic actions of leptin. The remarkable property of leptin is that while stimulating lipolysis, it does not raise free fatty acid levels, as occurs in canonical lipolysis stimulating pathways (166). This property of leptin has been attributed to simultaneously increased lipid oxidation and lipolysis within adipocytes (167), but does explain why glycerol levels would be reduced by chronic leptin therapy in STZ-diabetic mice. One possibility is that the intracellular triglyceride stores in WAT become depleted by leptin therapy, diminishing triglyceride as a source of lipolysis-induced glycerol and free fatty acid release. The robustly depleted circulating triglycerides in  122 STZ-leptin treated mice are consistent with this possibility. One study found that while acute leptin administration stimulated lipolysis, prolonged high-dose leptin application to white adipocytes resulted in decreased glycerol release, decreased lipolysis, and decreased glyceroneogenesis and fatty acid re-esterification within white adipocytes (383). Therefore, decreased glyceroneogenesis in WAT could also result in diminished glycerol release. Alternatively, leptin application was found to inhibit AQP7 expression in white adipocytes (380); as this is the major channel through which glycerol is released during lipolysis, this could also mediate the depletion of circulating glycerol. Interestingly, AQP7 knockout mice also exhibit lower circulating glycerol levels and hypoglycemia during fasting (379). The findings in this chapter present a novel aspect of leptin therapy, namely the depletion of energy substrates, in particular glycerol, which could mediate leptin-mediated glucose lowering in STZ-diabetic mice. Although glycerol is a major gluconeogenic substrate, and glycerol levels are elevated in uncontrolled diabetes, and predict type 2 diabetes incidence (374), the role of glycerol in hyperglycemia is largely understudied. Thus, the current study has revealed an unexpected link between lipolysis-generated substrates and glucose homeostasis in type 1 diabetes. While glycerol presents an obvious mechanism for leptin-mediated glucose lowering, the depletion of free fatty acids could also be critical in mediating leptin action, given that free fatty acids can stimulate gluconeogenesis (384), and that depleted free fatty acids could alleviate inhibition of glucose oxidation in tissues through the glucose-fatty acid cycle (385). This study highlights that caution is warranted in the use of leptin therapy for type 1 diabetes, as the depletion of energy stores induced by leptin results in life threatening hypoglycemia when access to food is restricted. Further investigation of the role of white adipose tissue lipolysis in leptin-mediated glucose lowering will help to elucidate the anti-diabetic action of leptin in type 1 diabetes, and could lead to novel therapeutic targets, namely inhibitors of lipolysis, that could be used to treat type 1 diabetes.     123 CHAPTER  7: THE THERAPEUTIC POTENTIAL OF LEPTIN CO-THERAPY AS AN ADJUNCT TO ISLET TRANSPLANTATION 7.1 INTRODUCTION The current state of the art for achieving long-term glycemic control in type 1 diabetic patients is transplantation of cadaveric donor islets. Whereas frequent episodes of hyper and hypoglycemia occur with insulin therapy, islet transplantation can effectively eliminate these excursions and maintain glycemia within a target range of 3.3 to 7.8 mmol/L (386). Unfortunately, islet transplantation is not widely available due to limited donor islet supply. Most transplant recipients require islets from at least 2 cadaveric donors to achieve target glycemia (386,387), and the decline of graft function within 5 years of transplant necessitates that most patients resume insulin therapy (387). Thus, a strategy to reduce the number of islets needed to achieve insulin independence is essential for wide-spread application of islet transplantation from cadaveric donor islets.  The preceding chapters, and previous studies, have demonstrated that high-dose leptin administration reverses hyperglycemia and dyslipidemia in type 1 diabetic rodent models (287-290,328). At present, it is unclear whether leptin monotherapy could replace insulin as a therapy for type 1 diabetes. Alternatively, glycemic control and insulin requirements for type 1 diabetic patients may be improved by leptin and insulin co-therapy. Indeed in diabetic mice, leptin administration reduced the insulin dose needed to ameliorate hyperglycemia (388), and combined leptin and insulin administration achieved better glycemic control than insulin alone (289).  Since islet transplantation provides superior metabolic control over insulin injections, we investigated whether leptin as an adjunct to islet transplantation could provide tighter glycemic control with fewer transplanted islets. Such an effect could increase the availability and efficacy of islet transplantation as a treatment. To test this, in this chapter we examined whether leptin administration could reduce the number of transplanted islets needed to reverse STZ-induced diabetes in mice. As high-dose leptin alone can restore normoglycemia in STZ-diabetic rodents (287-290,328), which can thereby enhance islet graft function (389,390), we first performed a dose response study in STZ-diabetic mice to identify a leptin dose that was insufficient to reverse hyperglycemia. Subsequently, we administered this dose of leptin to diabetic mice transplanted with 50 or 125 syngeneic islets (17% and 42% of an optimal dose of 300 islets respectively), to determine whether leptin co- 124 therapy could enhance the ability of these suboptimal islet doses to achieve metabolic control5. 7.2 RESULTS Leptin administration dose-dependently reverses STZ-induced hyperglycemia  To examine whether leptin administration can enhance islet transplant efficacy, we first determined a dose of leptin that alone was insufficient to achieve normoglycemia. We performed a dose response experiment, in which mice were injected with STZ on day -6 to induce diabetes, and subsequently implanted with osmotic pumps infusing 1, 3, 5 or 10 µg/day leptin continuously for 4 weeks. One group of diabetic mice received pump implants delivering vehicle only (STZ-vehicle), and mice that were not STZ-injected (non-diabetic) served as controls. Blood glucose was measured for 4 weeks post-surgery. As we previously reported (328), 10 µg/day leptin administration normalized blood glucose levels within 5 days (Figure 50), as did 5 µg/day leptin. Treatment with 3 µg/day leptin decreased blood glucose moderately within this time frame, eventually normalizing blood glucose levels by day 15. Mice receiving 1 µg/day leptin had a trend toward decreased blood glucose, but values never significantly decreased compared to STZ-vehicle controls. Unexpectedly, the ability of 10 µg/day leptin to normalize blood glucose waned after 12 days of treatment, and hyperglycemia returned (24.7 ± 6.4 mmol/L on day 29). Blood glucose rose similarly in 5 µg/day treated mice, but to a lesser extent (14.1 ± 2.5 mmol/L on day 29). Regardless, AUC analysis of blood glucose tracking from days 5-12 revealed that blood glucose levels were dose-proportionally lowered by leptin, and it was clear that 1 μg/day leptin was a suitable dose to test the effect of leptin on islet transplantation as it did not significantly lower blood glucose levels compared to STZ-vehicle controls.                                                 5 All data in this chapter were published in Diabetes (327).  125  Figure 50. Leptin reverses STZ-diabetes in a dose-dependent manner. Mice were treated with STZ, and 6 days later received implantation of osmotic pumps (day 0, vertical broken line) delivering doses of either leptin ranging from 1 to 10 µg/day (STZ-1/3/5/10 µg/day), or vehicle only (STZ-vehicle). Non-diabetic mice served as controls. A) Fasting blood glucose and (B) change in body weight relative to day -1. AUC analysis of blood glucose levels from day 5 to day 12 is presented as part of panel A. AUC analysis of body weight change from day 5 to 12 revealed no significant differences and is not shown. C) Four-hour fasted plasma insulin levels on day 25, limit of detection shown by broken horizontal line. D) Four-hour fasted plasma leptin levels on day 12 and 25. Four of 5 STZ-vehicle mice had leptin levels below the detection limit (indicated by broken line) on both days, and this group was not included in statistical analyses. Statistical analyses were performed by two-way ANOVA with a Tukey post-hoc test. E) Four-hour fasted plasma IGFBP2 levels on day 12. Data are presented as mean ± SEM, n = 4-5. * P < 0.05; † P < 0.05 vs. non-diabetic controls; ‡ P < 0.05 vs. STZ-vehicle controls. We also examined the effect of leptin on body weight in STZ-induced diabetic mice (Figure 50B). Prior to pump implantation, body weight was significantly reduced from 24.1 ± 0.3 g in non-diabetic controls to an average of 23.0 ± 0.2 g in STZ-injected mice (P = 0.04, t-test). Body weight change was not significantly altered between STZ-injected groups in response to leptin or vehicle administration. However, mice treated with 5 or 10 μg/day leptin initially tended to lose weight, and subsequently gained weight after day 12. This correlates with the diminished long-term blood glucose response to the highest leptin doses.  126 In agreement with our findings in the previous chapters, and work by others (287-289,304-306,328), plasma insulin was significantly reduced by STZ injection, and leptin did not increase insulin levels compared to STZ-vehicle mice (Figure 50C).  We next measured the concentration of plasma leptin 12 and 25 days post-implant (Figure 50D). Leptin levels on day 12 were reduced below the limit of detection in STZ-vehicle mice, consistent with the expected fall in leptin following STZ-administration (276). Plasma leptin levels were restored to non-diabetic levels by 1 µg/day treatment, and increased dose-proportionally up to 5 µg/day treatment. Notably, the lowest dose of leptin that achieved normoglycemia at this time-point (5 µg/day) produced circulating leptin levels significantly higher than those of non-diabetic mice (P = 0.0001), revealing that supraphysiological leptin levels are needed to achieve normoglycemia in STZ-diabetic mice. Treatment with 10 µg/day produced a lower plasma leptin concentration on day 12 than 5 µg/day leptin. Given that both of these doses initially yielded similar glucose lowering activity, and that at day 12 the efficacy of 10 µg/day leptin began to wane, we postulate that plasma leptin levels were initially higher in the 10 µg/day group but had fallen by the time of measurement. Supporting this, plasma leptin fell from day 12 to 25 in the 5 µg/day treated group (P = 0.0095), corresponding to the loss of efficacy of this dose at day 21. In contrast, the 1 and 3 µg/day leptin doses, which maintained constant efficacy for the duration of the study, did not show a reduction of plasma leptin over time. To determine whether plasma leptin concentrations corresponded to biologically active leptin, we measured plasma concentrations of IGFBP2 on day 12 (Figure 50E). Circulating levels of IGFPB2 are dose-dependently elevated by leptin (149,316). STZ-administration caused a 1.9 fold rise in IGFBP2 levels compared to non-diabetic mice, supporting previous reports that STZ increases hepatic Igfbp2 expression in rats (286). While 1 µg/day leptin did not further change IGFBP2 levels, 3 and 5 µg/day leptin resulted in dose-dependent rises in plasma IGFPB2. Mice receiving 10 µg/day leptin had a non-significant drop in plasma IGFBP2 compared to 5 µg/day treated mice. Thus plasma IGFBP2 levels corresponded with plasma leptin concentrations on day 12, supporting that plasma leptin levels correlated with biologically active leptin. We postulate that the return to hyperglycemia in mice treated with supraphysiological doses of leptin was due to a decrease in circulating biologically active leptin levels.   127 Leptin treated mice have improved glucose tolerance and hypoglycemia during an OGTT  We next assessed whether leptin could dose-dependently lower glucose excursions in STZ-induced diabetic mice by performing OGTTs on day 19 post-pump implantation (Figure 51A). Overall glucose excursions were assessed by AUC (Figure 51B). All 5 STZ-vehicle and 4 of 5 STZ-1 µg/day treated mice had blood glucose levels above the limit of detection (33.3 mmol/L) at one or more time-point post-gavage. Values of 33.3 mmol/L were assigned for these time-points, and these groups were not included in statistical analyses. In agreement with their rising fasted blood glucose levels prior to the day of OGTT (Figure 50A), 10 µg/day leptin treated mice showed a trend toward glucose intolerance compared to non-diabetic controls and mice treated with 3 or 5 µg/day leptin. Treatment with 3 and 5 µg/day leptin nearly normalized glucose tolerance, but trended toward hyperglycemia during the glucose peak, and hypoglycemia during the recovery phase of the test. In fact, 2 of 5 mice receiving 5 µg/day leptin and 1 of 5 mice receiving 3 µg/day leptin had blood glucose <2.9 mmol/L at 90 min, and had to be excluded from the remainder of the test.  Figure 51. Leptin improves glucose tolerance in STZ-diabetic mice in a dose dependent manner. Oral glucose tolerance tests were performed in non-diabetic controls, and STZ-induced diabetic mice receiving different doses of leptin (STZ-1/3/5/10 µg/day) or vehicle only (STZ-vehicle), following a 6 hour fast on day 19 post-surgery. Mice were gavaged with 2 g/kg glucose at time 0. A) Time course of blood glucose tracking and (B) AUC from 0-90 min. Four of 5 mice treated with 1 µg/day and all STZ-vehicle treated mice had blood glucose above the detection limit (33.3 mmol/L) at one or more time points, in which case values of 33.3 mmol/L were assigned. These groups were not included in statistical analyses. Two STZ - 5 µg/day mice and 1 STZ - 3 µg/day mouse had blood glucose < 2.9 mmol/L at 90 min and were rescued with exogenous glucose. Data from these mice are omitted from the 120 min time point. n = 5 for 0-90 min, and 3-5 for 120 min. Data are presented as mean ± SEM. Statistical analyses were performed on AUC values by one-way ANOVA with a Tukey post-hoc test.  128 Collectively, our leptin dose study revealed two key points: a) 1 µg/day leptin does not reverse hyperglycemia or glucose intolerance, making it an appropriate dose to test whether leptin can improve islet transplant performance; b) long-term leptin efficacy and risk of hypoglycemia may be limitations associated with high-dose leptin monotherapy for diabetes, thus strengthening the argument to combine low-dose leptin therapy with a regulated, cell-based source of insulin for achievement of well-controlled blood glucose levels. Low-dose leptin administration enhances islet transplant efficacy We subsequently examined whether leptin administration could improve the ability of islet transplantation to ameliorate STZ-induced diabetes. Six days following STZ administration (day 0), all mice underwent two surgeries simultaneously: transplantation of syngeneic islets under the left kidney capsule (or sham surgery) and subcutaneous implantation of 6 week osmotic pumps infusing 1 µg/day leptin (or vehicle). Previous reports (389), as well as data from our group, indicate that transplantation of less than 200 islets under the kidney capsule is insufficient to reverse hyperglycemia in STZ-diabetic mice. Therefore, we transplanted diabetic mice with suboptimal doses of either 50 or 125 islets, along with infusion of either leptin (50/125 islets-leptin), or vehicle (50/125 islets-vehicle). These groups were compared alongside diabetic mice given an optimal islet transplant dose of 300 islets (300 islets-vehicle), mice receiving leptin only (sham-leptin), untreated diabetic mice (sham-vehicle), and healthy non-diabetic controls that received sham surgery and vehicle infusion. We first examined the effect of leptin and islet co-therapy on 4-hour fasted blood glucose levels (Figure 52A). Untreated diabetic mice maintained blood glucose levels above 21 mmol/L for the study duration. For statistical analyses, AUC was calculated using fasting blood glucose data from day 5 to 43 post-surgery (Figure 52B). Transplantation of 300 islets rapidly restored normoglycemia, revealing that the donor islets were functional and viable. As expected, neither 50 islets nor 125 islets alone significantly lowered blood glucose compared to sham-vehicle mice, nor did leptin treatment alone. However, the combination of leptin with either 50 or 125 islets led to a robust and significant lowering of blood glucose levels compared to sham-vehicle mice (P = 0.026 and 0.0019, respectively). In fact, mice treated with 125 islets in the presence of leptin had mean fasting blood glucose levels nearly as low as mice receiving 300 islets (11.5 ± 0.4 mmol/L vs 7.6 ± 1.2 mmol/L).  129  Figure 52. Leptin administration enhances the efficacy of islet transplantation for treatment of STZ-diabetes. Mice were treated with STZ on day -6, and subsequently received transplants of 50, 125 or 300 islets or sham surgery (day 0), and simultaneous osmotic pump implants delivering 1 µg/day leptin or vehicle for 6 weeks. A) Four-hour fasted blood glucose levels. B) Blood glucose data from day 5 to 43 analyzed by AUC. C) Four-hour fasted HbA1c levels in whole blood presented as percent and mmol/mol equivalents in parentheses. D) Four-hour fasted body weight gain normalized to day -1 and (E) net AUC from day 5 to 43. F) Four-hour fasted plasma leptin measured from cardiac puncture samples collected 6 weeks post-surgery. Data are presented as mean ± SEM, n = 3-5. Statistical analyses were performed by one-way ANOVA with a Tukey post-hoc test. * P < 0.05;  † P < 0.05 vs. non-diabetic controls;  ‡ P < 0.05 vs. sham-vehicle controls. HbA1c levels in mice 6 weeks post-surgery (Figure 52C) revealed similar effects on long-term glycemic control. As expected there was a dramatic rise in HbA1c in sham-vehicle mice compared to non-diabetic controls (P < 0.0001), and transplantation of 300 islets normalized HbA1c levels to non-diabetic levels. Mice treated with leptin alone, and 50 or 125 islets alone showed non-significant trends toward reduced HbA1c levels compared to sham-vehicle mice. In contrast, combination of leptin with either 50 or 125 islets led to a  130 substantial reduction in HbA1c compared to sham-vehicle mice (P = 0.006 and 0.002, respectively). We also tracked the effect of islet transplantation with leptin co-therapy on body weight (Figure 52D). Prior to implantation, body weight was significantly reduced to 24.3 ± 0.4 g in STZ-injected mice compared to 26.4 ± 0.4 g in non-diabetic controls (P = 0.025, t-test). Cumulative weight gain over the 6 weeks post-surgery was determined by AUC analysis (Figure 52E). With the exception of the 300 islets-vehicle group, cumulative weight gain was lower in STZ-injected groups compared to non-diabetic controls. Neither suboptimal islet transplant, nor leptin, nor combination therapy significantly altered net weight gain compared to sham-vehicle controls, although we noted that all transplant recipient groups exhibited a positive cumulative weight gain, whereas sham-vehicle and sham-leptin treated mice had no weight gain. Plasma leptin levels were measured at 6 weeks post-surgery (Figure 52F). Leptin levels were significantly reduced in sham-vehicle mice compared to non-diabetic controls and administration of 1 µg/day leptin restored plasma leptin concentrations to non-diabetic levels. Mice transplanted with islets had a trend toward increased leptin concentrations compared to sham-vehicle mice that was proportional to the amount of islets transplanted. Transplantation of 300 islets yielded leptin levels similar to non-diabetic controls (1.6 ± 0.3 ng/mL), consistent with stimulation of endogenous leptin production by graft-derived insulin. Mice treated with islet and leptin co-therapy had similar plasma leptin concentrations to sham-leptin and non-diabetic mice. Therefore, while unable to reverse hyperglycemia alone, a replacement-dose of leptin can enhance the efficacy of suboptimal islet transplants to lower blood glucose in diabetic mice. Leptin enhances islet transplant efficacy without increasing circulating insulin or endogenous β-cell recovery We next determined whether the improvement of islet transplant efficacy in the presence of leptin co-therapy was due to an increase in circulating insulin levels (Figure 53A). Leptin alone had no effect on plasma insulin levels, whereas transplantation of islets alone significantly increased plasma insulin levels above those of sham-vehicle controls. Interestingly, despite the effect of leptin co-therapy to enhance islet transplant efficacy, leptin did not further increase plasma insulin concentrations compared to mice receiving islet transplants without leptin. In fact, there was a non-significant decrease in recipients of 125 islets co-treated with leptin compared to recipients of 125 islets alone. Measurement of  131 pancreatic β-cell area revealed an approximate 85% decrease in β-cell area in sham-vehicle mice compared to non-diabetic controls (Figure 53B,C). Transplantation of 125 islets alone or with leptin, did not increase β-cell area compared to sham-vehicle controls, supporting that the increase in plasma insulin following islet transplantation was from graft-derived insulin. Representative pancreatic sections from non-diabetic and STZ-injected mice demonstrate the extreme loss of β-cells (Figure 53C). In contrast, pancreatic α-cell area did not differ between non-diabetic controls and sham-vehicle mice (Figure 53B,C). Increased α-cell area as a proportion of islet area has been interpreted as α-cell expansion following STZ-administration (391). However, when α-cell area is normalized to total pancreatic area, there is clearly no expansion of α-cells in STZ-injected mice compared to non-diabetic mice. Islet transplantation with or without leptin had no effect on α-cell area.  132   133 Figure 53. Leptin co-therapy does not alter circulating insulin levels or β-cell recovery. A) Four-hour fasted plasma insulin levels were measured 6 weeks post-transplant in all groups by ultrasensitive insulin ELISA, limit of detection (0.019 ng/mL) shown as horizontal broken line, n = 3-5. B) Immunofluorescent quantification of β-cell and α-cell area were performed in non-diabetic controls and STZ-induced diabetic mice treated with either 125 islets (125 islets-vehicle), 125 islets and leptin (125 islets-leptin) or left untreated (sham-vehicle) n = 3-4. Data are presented as mean + SEM. † P < 0.05 vs. non-diabetic controls. Statistical analyses were performed by one-way ANOVA with a Tukey post-hoc test. ‡ P < 0.05 vs. sham-vehicle controls. C) Representative images of pancreata from non-diabetic and sham-vehicle groups, costained for insulin (red), glucagon (green) and DAPI (white), n = 3-4. Scale bars on left set of images represent 1000 µm, and on right set of images represent 100 µm. Leptin and islet co-therapy improve glucose tolerance in STZ-diabetic mice To assess whether leptin and islet co-therapy also improved glucose excursions, we performed OGTTs on day 20 post-surgery (Figure 54A). As expected, sham-vehicle mice had severely impaired glucose tolerance compared to non-diabetic controls, and transplantation of 300 islets nearly normalized glucose excursion. At the glycemic peak, some mice had blood glucose concentrations greater than the 33.3 mmol/L limit of detection; thus we collected blood from all mice at 10 and 20 min post-gavage and measured plasma glucose by the glucose-oxidase method (Figure 54B). Plasma glucose concentrations in mice treated with suboptimal islet doses or leptin alone were similar to sham-vehicle controls at 10 and 20 min. In contrast, the combination of leptin and 125 islets significantly lowered plasma glucose at 10 min, whereas there was a non-significant trend toward lower plasma glucose in mice treated with leptin and 50 islets. During this test, we also measured glucose-stimulated plasma insulin levels (Figure 54C). Only non-diabetic controls and mice transplanted with 300 islets had detectable glucose-stimulated insulin levels, further supporting that leptin and islet co-therapy achieves metabolic control similar to recipients of an optimal islet transplant, without restoring plasma insulin levels.  134  Figure 54. Leptin and islet co-therapy improves glucose tolerance. A) OGTT on day 20, following a 6 hour fast in mice transplanted with islets with or without leptin, and untreated diabetic (sham-vehicle), leptin treated diabetic (sham-leptin), and non-diabetic controls. B) As some mice had blood glucose levels over the limit of detection post-gavage, blood was collected at 10 and 20 min for plasma glucose measurement. C) Plasma insulin levels post-gavage. The limit of detection (0.188 ng/mL) is indicated by a broken horizontal line. Only non-diabetic and 300 islets-vehicle groups had detectable plasma insulin levels at any given time. The remainder of mice were assigned values of 0.188 ng/mL. Data are presented as mean ± SEM, n = 3-5. Statistical analyses were performed by one-way ANOVA with a Tukey post-hoc test. * P < 0.05. † P < 0.05 vs. non-diabetic controls. ‡ P < 0.05 vs. sham-vehicle controls. Low-dose leptin administration reverses dyslipidemia in STZ-diabetic mice Since leptin clearly enhanced the ability of islet transplantation to improve glycemic control, we subsequently examined whether leptin and islet co-therapy also improved lipid homeostasis. We measured plasma triglycerides (Figure 55A), free fatty acids (Figure 55B)  135 and β-hydroxybutyrate levels (Figure 55C) on day 12 following surgery. As expected, STZ-administration caused elevations in plasma triglycerides, free fatty acids and β-hydroxybutyrate levels from non-diabetic values. Transplantation of 300 islets normalized all three metabolites in diabetic mice, whereas transplantation of 50 or 125 islets without leptin led to intermediate values between those of non-diabetic and sham-vehicle controls. Interestingly, despite having a minimal impact on glucose metabolism, 1 µg/day leptin alone completely normalized all 3 of these metabolites. This is consistent with the profound lipopenic actions of leptin, which occur in part through stimulating lipid oxidation in multiple tissues, and through inhibiting hepatic triglyceride synthesis and secretion (166,207,289,368,392,393). Consequently, combination of leptin with 50 or 125 islets led to plasma triglyceride, free fatty acid, and β-hydroxybutyrate concentrations similar to those of healthy non-diabetic controls. We also measured fasting plasma glycerol levels (Figure 55D), as we found in Chapter 6 that supraphysiological doses of leptin depleted circulating glycerol levels in STZ-diabetic mice. Similar to the effects on lipids, low-dose leptin alone, or in combination with suboptimal islet doses, was associated with lower plasma glycerol levels compared to STZ-vehicle controls, although this did not reach significance in the 50 islets with leptin group. These data support that leptin potently lowers glycerol levels in the STZ-diabetic mouse model, however it is important to note that this was insufficient to normalize blood glucose levels in low-dose leptin treated mice. This could be due to the fact that glycerol levels were normalized but not depleted in the 1 µg/day leptin treated group relative to non-diabetic controls. Regardless, it is possible that the normalization of circulating glycerol, along with the normalization of free fatty acids and triglycerides, could contribute to the enhanced efficacy of islet transplantation when combined low-dose leptin administration.  136  Figure 55. Low dose leptin administration normalizes lipid levels in STZ-diabetic mice. Four-hour fasted plasma triglycerides (A), free fatty acids (B), β-hydroxybutyrate (C), and glycerol (D) were measured in mice transplanted with islets with or without leptin, and in untreated diabetic (sham-vehicle), leptin treated diabetic (sham-leptin), and non-diabetic controls on day 12 post-surgery. Data are presented as mean + SEM, n = 3-5. Statistical analyses were performed by one-way ANOVA with a Tukey post-hoc test. * P < 0.05; † P < 0.05 vs. non-diabetic controls; ‡ P < 0.05 vs. STZ-vehicle controls. 7.3 DISCUSSION We and others have found that high-dose leptin administration reverses hyperglycemia in rodent models of type 1 diabetes (287-290,328). However, the dose response profile of glucose lowering by leptin has not been previously investigated. This chapter reveals that leptin lowers blood glucose in STZ-diabetic mice in a dose-dependent manner, and that a supraphysiological level of leptin was necessary to achieve normoglycemia. This supports a previous report that a physiological leptin dose in STZ-diabetic rats only modestly reduced blood glucose levels (286).  There is some rationale to support the use of leptin as a monotherapy. Firstly, type 1 diabetes at least in rodents, results in relative leptin deficiency (276), a state in which leptin  137 therapy is most effective. Secondly, leptin has beneficial effects on lipid metabolism, which opposes the potentially deleterious, lipogenic action of insulin (289). Furthermore, unlike insulin, there is no weight gain associated with leptin-induced normalization of glucose levels in type 1 diabetic rodents (288,289,328). However, our study revealed key aspects of high-dose leptin therapy that may make it inappropriate for use as a monotherapy. With the doses of leptin sufficient to achieve rapid normoglycemia (5 and 10 µg/day), the efficacy of leptin was transient. We postulate that the transient nature of high-dose leptin could be due to a loss of leptin bioactivity within long-term implanted pumps, or to a physiological response in mice to counteract high-dose leptin treatment. Additionally, the lowest leptin doses that achieved fasting normoglycemia, produced severe hypoglycemia during an OGTT in approximately 30% of the mice. Previously we observed that injection of an insulin dose that only modestly lowered glucose levels in diabetic mice, resulted in severe hypoglycemia when combined with high-dose leptin therapy (328). Consequently, the addition of leptin to unregulated high insulin doses may confer increased hypoglycemic risk; thus we propose that combination of leptin with either low-dose insulin or a regulated source of insulin such as islets, could be a safer and more effective method to lower blood glucose. In the current study, administration of low-dose leptin, while insufficient to ameliorate diabetes alone, robustly augmented the ability of a suboptimal dose of syngeneic islets to improve fasting glycemia and glucose tolerance in STZ-diabetic mice. Provocatively, when combined with leptin, 125 islets functioned almost as well as a transplant of 300 islets in regard to achieving glycemic control, whereas alone 125 islets failed to ameliorate diabetes. Furthermore, although suboptimal islet transplantation alone did not reverse dyslipidemia, low-dose leptin alone normalized lipid metabolism and ketone levels in diabetic mice. A potential mechanism by which leptin could improve the ability of islet transplantation to lower blood glucose is through increased insulin secretion from transplanted or endogenous β-cells. However, leptin and islet co-therapy did not affect pancreatic β-cell area in our study. Moreover, although leptin can protect islets against lipotoxicity, and β-cell apoptosis (394,395), we found that leptin co-therapy did not increase circulating insulin levels in islet transplant recipients. Rather, in mice transplanted with 125 islets, administration of leptin produced a non-significant decrease in circulating insulin. This supports multiple studies showing that leptin has an inhibitory action on insulin secretion (146,151,152,190,191,290). The finding that low-dose leptin alone had no glucose lowering ability, indicates that the mechanism of leptin co-therapy is dependent on the islet graft providing a source of insulin. Alternatively, leptin therapy has been shown to enhance insulin  138 sensitivity in diabetic rodents (286,287,328,335). In our study, low-dose leptin alone was effective in reversing the dyslipidemia, and in lowering plasma glycerol levels, which could contribute to enhanced insulin sensitivity in peripheral tissues or decreased hepatic glucose production, and could collectively provide a more lipopenic, insulin-sensitive, environment for islet grafts in a transplant recipient, allowing graft-derived insulin to have more profound metabolic effects. Our data reveal that as few as half the number of islets normally required to restore euglycemia in mice, can achieve glycemic control when combined with low-dose leptin administration. This suggests that the number of diabetic patients treated with cadaveric islet transplantation could be virtually doubled by simply adding leptin to their therapeutic regimen. Furthermore, the profound lipopenic action of low-dose leptin may provide a particular advantage by protecting against cardiovascular complications associated with dyslipidemia (396,397). However, in our opinion, the potentially deleterious effects of leptin administration on the underlying autoimmune aspect of type 1 diabetes should be assessed with caution. High-dose leptin administration in non-obese diabetic (NOD) mice has been shown to accelerate autoimmune β-cell destruction (398), whereas mutations in the leptin receptor are protective in NOD mice (399,400). However, Kruger and colleagues found that pre-treatment of BB rats with leptin prevented immune destruction of both endogenous and transplanted β-cells (290). Likewise, leptin treatment confers clinical benefit to patients with autoimmune lipodystrophy and type 1 diabetes without exacerbation of the underlying autoimmune disorder (401). Thus, while the impact of leptin on autoimmune destruction of β-cells should be carefully assessed, the potential therapeutic benefit of leptin co-administration with islet transplantation for type 1 diabetes warrants consideration.     139 CHAPTER  8: CONCLUSIONS AND FUTURE DIRECTIONS 8.1 CONCLUSIONS Initial studies of leptin generated excitement that this fat-derived hormone could be the cure for obesity. Hopes were rapidly diminished when it was realized that the vast majority of obese people have high circulating leptin, and must therefore be resistant to the anorectic actions of leptin (40,46). However, as the number of studies demonstrating a primary role for leptin in glucose metabolism, independent of body weight regulation have accumulated, the concept that leptin could be a powerful anti-diabetic agent (for both type 1 and type 2 diabetes) is gaining momentum, and renewing hopes of the therapeutic potential of this hormone. The substantial metabolic improvements achieved by leptin administration in states of leptin deficiency are remarkable. Given that for almost a century, insulin has been assumed to be the only hormone that could lower blood glucose and reverse the relentless catabolic state of type 1 diabetes, the profound metabolic action of leptin is underscored by its ability to normalize blood glucose levels in this disease. Rapidly accumulating studies have demonstrated that leptin therapy, either through peptide administration or genetic overexpression, reverses hyperglycemia, prolongs life, and diminishes long term complications in rodent models of type 1 diabetes (287-289,291). Evidence has already shown that these effects occur without restoring circulating insulin levels or reducing body weight, and are independent of the inhibitory effect of leptin on food intake (287-289). What this thesis now shows is that leptin therapy also reverses hyperglycemia, hyperketonemia, and dyslipidemia in STZ-diabetic mice. In addition, leptin therapy reverses the hyperphagia, and the diminished activity levels, associated with insulin-deficient diabetes in mice. Others have shown that leptin reverses glycosuria (288,289), and this thesis now reveals that leptin therapy also diminishes polyuria and polydipsia in a mouse model of type 1 diabetes. Therefore, with the exception of weight loss, leptin therapy reverses the predominant symptoms and metabolic disturbances of insulin-deficient type 1 diabetes in mice, without restoring circulating insulin levels. Furthermore, this thesis reveals information regarding the kinetics and dose dependence of leptin therapy. Leptin lowers blood glucose in STZ-diabetic mice in a dose dependent manner, and supraphysiological leptin levels are necessary to fully normalize blood glucose. Furthermore, the glucose lowering action of leptin is not acute, and is rapidly reversed upon the cessation of leptin therapy.   140 The overarching goal of this thesis was to perform preclinical tests of leptin therapy in type 1 diabetic rodents to elucidate the mechanism by which leptin reverses hyperglycemia, both in respect to the primary leptin target tissues that mediate this action and in regard to the downstream metabolic pathways that result in glucose lowering. Substantial evidence in the literature suggests that central neuronal circuits mediate the anti-diabetic action of leptin in rodent models of type 1 diabetes, since central delivery of leptin or central leptin gene therapy can mimic the anti-diabetic effect of peripheral leptin therapy (304-307,309,356), and reconstitution of leptin signalling in GABAergic neurons has been shown to be sufficient to reverse STZ-induced diabetes in leptin receptor deficient mice (309). Investigation of candidate leptin responsive neurons have focused on the ARH and VMH; direct injection of leptin into the VMH can reverse hyperglycemia (308), and reconstitution of leptin signalling in POMC and GABAergic neurons has been shown to be sufficient to reverse STZ-induced diabetes in leptin receptor deficient animals (309). However, leptin receptors are expressed throughout the CNS (68), and in peripheral tissues (70), and neither leptin signalling in POMC neurons nor SF1 neurons are required for leptin-mediated reversal of insulin-deficient diabetes in rodents (308,309). This thesis now contributes to our understanding of required leptin responsive targets, and our findings along with those mentioned above have now ruled out several key target sites (summarized in Figure 56). We found that neuronal leptin signalling in hypothalamic nuclei outside of the ARH and VMH are not necessary for the glucose lowering action of leptin in a mouse model of type 1 diabetes. In addition, this thesis suggested a novel potential role for LHA, PMV and DMH neurons in non-insulin deficient states; leptin action in neurons may mediate some of the deleterious outcomes of diet-induced obesity. In light of the aforementioned studies demonstrating that leptin signalling in POMC neurons and SF1 neurons are not required to mediate the actions of central leptin therapy, this thesis work suggests that leptin action in type 1 diabetes may be mediated through redundant neuronal circuits or redundant peripheral leptin-induced pathways. Moreover, this thesis revealed that surgical ablation of subdiaphragmatic parasympathetic efferents, or partial ablation of sympathetic efferents, does not blunt the anti-diabetic action of leptin in a mouse model of type 1 diabetes. This supports a recent study showing that central leptin therapy can reverse diabetes in mice lacking all β-adrenergic receptors (309). The evidence that central leptin action is sufficient, but that no precise leptin responsive hypothalamic nucleus is required for leptin-mediated reversal of diabetes, suggests the involvement of either redundant neuronal or peripheral targets. In regard to peripheral pathways, this thesis also reveals that direct leptin action in hepatocytes  141 is not a requisite for leptin-mediated glucose lowering in mice with STZ-induced diabetes. In light of our findings, continued investigation into the precise target tissues required for leptin action in type 1 diabetes warrants future investigation.   Figure 56. Putative targets and neural circuits that mediate leptin-induced glucose lowering in rodent models of type 1 diabetes. Candidate leptin targets that could mediate glucose lowering are leptin responsive neurons, and peripheral tissues including the pancreatic α-cells, liver, skeletal muscle, and white adipose tissie (WAT) and brown adipose tissue (BAT). Investigations performed in this thesis are indicated by green symbols, while investigations performed outside of this thesis are indicated by orange symbols. Leptin receptors in POMC neurons, GABAergic neurons, and injection of leptin into the ventromedial hypothalamic nucleus (VMH) are each sufficient to reverse type 1 diabetes in rodents (indicated by an orange S). However, leptin signalling in POMC, and SF1 neurons is not necessary for the anti-diabetic action of leptin (indicated by an orange X). This thesis has ruled out several additional candidate targets, showing that leptin signalling in the lateral hypothalamic area (LHA), ventral premammillary hypothalamic nucleus (PMV), or the liver is not required for this action of leptin (green X). Our data also suggest that extra-hypothalamic neuronal leptin signalling, and leptin signalling in the dorsomedial hypothalamic nucleus (DMH) is not required, but Lepr-b deletion  142 was partial in these regions (broken green X). Moreover, this thesis reveals that subdiaphragmatic vagal efferents are not necessary, nor are sympathetic efferents (note that vagal efferents to tissues above the diaphragm were not tested). Although the sympathectomy was partial in our study, this conclusion is supported by a study showing that β-adrenergic receptors are not required for the anti-diabetic action of leptin. Additional studies are needed to clarify the role of other leptin responsive neurons and peripheral tissues in the anti-diabetic action of leptin therapy in type 1 diabetes. In regard to the downstream leptin-activated pathways that mediate glucose lowering, investigation of the metabolic and neuroendocrine effects of leptin therapy has revealed multiple possibilities. Several studies indicate that leptin may lower glucose through either an insulin-mimetic or insulin-sensitizing effect, or through the reduction of counter-regulatory hormones. Yu et al. (288) proposed that leptin lowers blood glucose through the normalization of circulating glucagon levels. Other reported effects of leptin therapy that could mediate an insulin-like effect on glucose metabolism, are the enhanced levels and signal transduction of IGF1 in peripheral tissues (288), and the increase in IGFBP2 (149), which could modulate IGF1-receptor interactions, although recent data suggests that IGFBP2 is not required for leptin’s metabolic actions (295). This thesis now reveals that leptin could modulate an insulin-like effect on glucose metabolism, both by leptin-induced insulin sensitivity and by gluconeogenic substrate depletion. Although the action of leptin in rodent models of type 1 diabetes has been assumed in several studies to be insulin-independent due to the lack of effect on extremely depleted insulin levels (288,305,306,309), here we found that leptin therapy dramatically enhances insulin sensitivity beyond the level in non-diabetic animals, which therefore could compensate for the extremely low circulating insulin. Our study showed that glucose uptake in the soleus muscle cannot account for increased insulin sensitivity in leptin treated STZ-diabetic mice. Studies conducting ICV leptin administration in type 1 diabetic rodent models have shown that glucose uptake in some muscle types but not others is induced (305,309). Alternatively, if enhanced insulin sensitivity mediates glucose lowering by leptin, evidence suggests that this may be through the liver. Leptin therapy has been shown to have striking similarities to insulin therapy on hepatic glucose metabolism in type 1 diabetic mice (289). Moreover, a reduction in hepatic glucose production in leptin treated STZ-diabetic rats has been shown (287,305). In this thesis, we present novel evidence that leptin robustly inhibits endogenous glucose production by the liver, as leptin treated mice are profoundly sensitive to prolonged fasting and cannot maintain glucose levels for extended periods without food. Thus, this thesis implicates hepatic glucose production as a major contributor to the mechanism of leptin action in a mouse model of type 1 diabetes. However, while reduced hepatic glucose  143 production could be achieved by enhanced insulin sensitivity, many of the leptin-mediated effects on hepatic metabolism uncovered in this thesis are inconsistent with increased hepatic insulin sensitivity.  With evidence in this thesis strongly underlining hepatic glucose production in the mechanism of leptin action, a predominant component of this work focused on elucidating the changes in hepatic glucose metabolism that occur in the STZ mouse model of type 1 diabetes treated with leptin. Inconsistent with enhanced hepatic insulin sensitivity, we found that leptin therapy robustly depleted fasting and free-feeding hepatic glycogen content, and that the mobilization of hepatic glycogen was not inhibited during fasting. Furthermore, although endogenous glucose production was clearly diminished, leptin therapy did not inhibit gluconeogenic flux or transcript levels of Pepck. The findings in this thesis suggest that rather than lowering hepatic glucose production through inhibition of gluconeogenesis per se, leptin may achieve this effect through the depletion of glucose-producing substrates, namely glycerol and glycogen (summarized in Figure 57). After rapidly depleting hepatic glycogen stores, leptin therapy gradually diminishes circulating lipids, including free fatty acids, and triglycerides. While insulin can reduce circulating lipids by enhancing intracellular storage of lipids in adipose (402), leptin depletes adipose lipid stores by stimulating lipolysis. Central administration of leptin has been shown to acutely stimulate the expression of hormone sensitive lipase (HSL) in WAT (403), and activation of the central melanocortin pathway stimulates lipolysis through the expression of both HSL and adipose triglyceride lipase (ATGL) (404). However, since HSL and ATGL expression are upregulated during insulin deficiency (405,406), it is unclear whether leptin therapy would stimulate lipolysis beyond the level found in untreated STZ-diabetic controls. Interestingly, leptin-induced lipolysis is distinct from other physiological conditions that enhance lipolysis, because it results in a depletion of adipose tissue without increasing the release of free fatty acids (166), which has been attributed to the simultaneous induction of lipolysis and lipid oxidation within adipocytes (167). As glycerol is normally released during lipolysis, the free fatty acid and glycerol depleting action of leptin therapy revealed in this thesis is consistent with the severe depletion of intracellular triglycerides in WAT, diminishing the body’s source of free fatty acids and glycerol during fasting. Indeed, in vivo leptin administration has been shown to activate AMPK, inhibit acetyl CoA carboxylase, and decrease expression of fatty acid synthase in WAT (167), which would collectively limit lipogenesis and promote lipid oxidation internally within adipocytes. Moreover, the fact that leptin takes several days to lower circulating glycerol in the STZ-diabetic model, is consistent with the timeline for the reported  144 depletion of intracellular lipid droplets and conversion of WAT to a lipid oxidizing phenotype by leptin treatment in normal rats (167). Subsequently, the depletion of glycerol, concomitant with reduced hepatic glycogen, may lead to the robust reduction in hepatic glucose production achieved by leptin therapy. The leptin-induced depletion of glycerol as a gluconeogenic substrate reconciles why hepatic glucose production is diminished but hepatic gluconeogenic flux from pyruvate is unaltered compared to uncontrolled diabetes. Therefore, the contribution of glycerol gluconeogenesis to blood glucose levels is diminished by the depletion of glycerol by leptin. Indeed, the depletion in circulating free fatty acids and glycerol corresponds tightly with the reversal of hyperglycemia by leptin therapy, and furthermore acute administration of glycerol can delay the dangerous hypoglycemia in response to prolonged fasting in leptin treated mice.    145 Figure 57. Leptin therapy lowers glycemia in a mouse model of type 1 diabetes by depleting glycerol and glycogen for hepatic glucose production. Glycerol and free fatty acids (FFA) are severely depleted in the plasma of leptin treated mice, resulting in diminished glycerol gluconeogenesis. The depletion of hepatic glycogen stores may increase the dependence on gluconeogenesis, thereby exacerbating the depletion of glcyerol. Hepatic glucose production could be futher diminished by decreased counter-regulatory hormone levels, increased insulin sensitivity, and reduced hepatic Glut2 expression. Likewise, the depletion of circulating FFA drives a decrease in β-oxidation, resulting in reduced ketone levels, and reduced substrates for lipid synthesis and storage in the liver as triglyceride. The leptin-induced depletion of circulating FFA and glycerol, could be driven by either a severe depletion of glycerol and FFA release from WAT, (due to a reduction in triglyceride (TG) storage), or by the activation of BAT thermogenesis acting as an energy sink. Pathways including increased glucose excretion or muscle glucose uptake are less likely to mediate leptin-induced glucose lowering. Data in support of this model were generated in type 1 diabetic rodents by: this thesis work (green circles), findings of other labs (orange circles), and findings of other labs that were confirmed by this thesis work (purple circles). Portions of this model that have not been validated experimentally in type 1 diabetic rodents are shown in black circles. Arrow thickness corresponds to the relative strengths of these pathways in uncontrolled type 1 diabetes. Despite the fact that leptin is known to promote lipid oxidation, the extreme deprivation of circulating free fatty acids likely limits the use of lipids as a source for oxidative phosphorylation. This is supported by the following evidence in this thesis: the normalization of ketone bodies by leptin therapy, which are a side-product of lipid oxidation; the reduction of hepatic acetyl CoA, and trend towards reduced acetyl CoA: free CoA levels which supports a shift towards glucose oxidation; and the increased RER observed in leptin treated mice, indicative of decreased lipid oxidation. These data collectively form a model whereby the well-known lipopenic actions of leptin play a critical role in the improvements in glycemia achieved by leptin therapy in rodent models of type 1 diabetes. Moreover, the glucose and lipid lowering actions of leptin could be initiated by a 2 pronged mechanism that centres on adipocytes: the depletion of energy reserves, namely triglycerides as a source of free fatty acids and glycerol within WAT, and/or the activation of an energy sink that rapidly utilizes fuel reserves, and potentially glucose. Notably, a major energy sink known to be activated by leptin is BAT thermogenesis (124,215,216,305,309), and our finding that leptin treated mice have increased interscapular temperature, increased RER, and decreased BAT mass are indicative of activated BAT thermogenesis. Further investigation of the mechanism of energy depletion and whether it plays a causal role in the therapeutic effects of leptin therapy is warranted. The impact of hyperleptinemia on metabolism in rodent models of type 1 diabetes appears to be consistent with the role of leptin in limiting lipid accumulation and promoting negative energy balance. Since leptin normally circulates in proportion to adipose stores (40,46), leptin treated type 1 diabetes mimics a physiological state in which insulin levels are extremely low, but adipocyte stores and energy reserves are plentiful. Our data suggest that  146 this promotes the utilization of energy through thermogenesis, lipolysis, the oxidation of lipids, and increased insulin sensitivity which would promote glucose utilization. Due to supraphysiological leptin levels starvation is not perceived by the brain, resulting in decreased food intake, and increased locomotor activity, which we confirmed in our studies. Leptin therapy has also been shown to increase thyroid hormone levels in insulin-deficient diabetic rats, which would further enhance energy expenditure (366). Given the known effects of leptin therapy in leptin deficient states on the restoration of fertility and immune function, reviewed by Mantzoros et al. (130), we postulate that high leptin levels in type 1 diabetic rodents are likely to restore these energy expensive processes, furthering the utilization of energy stores. However, since in reality lipids as an energy source are not plentiful due to insulin deficiency, energy stores become rapidly depleted by leptin therapy, and fasting becomes extremely dangerous due to hypoglycemia, and the depletion of gluconeogenic substrates. Further exacerbating hypoglycemia is the inhibition of circulating glucagon by leptin (288).  The profound effects of leptin therapy on glucose and lipid metabolism in type 1 diabetes also raise questions as to whether the fall in circulating leptin that occurs in insulin-deficiency contributes to the metabolic disturbances of type 1 diabetes, and what the physiological role of hypoleptinemia in this condition is. Based on the overview above, hypoleptinemia likely plays a critical role in mediating the metabolic adaptation to insulin deficiency, much like it has been shown to mediate adaptation to starvation (49). Previous studies have shown that hyperphagia, insulin resistance and hyperglucagonemia in insulin-deficient diabetes are directly caused by hypoleptinemia (281,284-286). Based on these studies and the findings of this thesis, we postulate that hypoleptinemia induced by insulin deficiency acts as a signal to conserve energy, mobilize adipose tissue lipid stores as a fuel source, and raise counter-regulator hormones, in order to prevent life threatening hypoglycemia during fasting. Hypoleptinemia would alleviate the effects of leptin on intracellular lipid oxidation with adipocytes (167), which would preserve lipids as a fuel for other tissues to support essential cellular processes during insulin deficiency. Thus hypoleptinemia may directly contribute to the elevated circulating lipid levels that accompany insulin deficiency. Supporting this, in Chapter 7 we observed that a leptin replacement dose reversed elevations in free fatty acids, triglycerides, and β-hydroxybutyrate. Interestingly, this occurred despite the maintenance of hyperglycemia, suggesting that lipid metabolism is more sensitive to changes in leptin than glucose metabolism. Notably, the profound sensitivity of leptin treated mice to fasting-induced hypoglycemia, is remarkably similar to  147 that of mice lacking WAT (42), supporting that lipid stores are critical for the maintenance of blood glucose levels during fasting. Also, by both inducing insulin resistance in peripheral tissues, and alleviating suppression on glucagon levels, hypoleptinemia would help to minimize peripheral glucose utilization, preserving glucose for the brain. The results from this thesis revealed that several caveats exist with leptin therapy. Firstly, leptin therapy may increase the risk of hypoglycemia, which we found occurs both during prolonged fasting and during the recovery phase of an OGTT in leptin treated mice. Thus although the anti-diabetic actions of leptin are therapeutically promising, the dangers of hypoglycemia warrant caution, and the limitations of aggressive leptin and insulin therapy are likely similar. Furthermore, the proposed therapeutic combination of leptin and insulin therapy must be carefully investigated, as we observed that leptin treated mice became dangerously hypoglycemic during an ITT. A second limitation of leptin therapy brought to light by this thesis is the potential for leptin resistance. Intriguingly, in our leptin dose response study, we observed that the highest doses of leptin that initially achieved normoglycemia were immediately followed by a decrease in plasma leptin levels and the restoration of hyperglycemia. This could either be due to a biological response that decreases circulating leptin (leptin clearance or a leptin binding protein) or due to impaired release of leptin from the pump. Importantly, some data in the literature suggest that this effect may not be specific to leptin peptide administration via pump, as a waning of leptin efficacy was also reported 12 days following the adenoviral induction of hyperleptinemia in diabetic rats, and corresponded to a decrease in hyperleptinemia (288). Notably, most studies testing leptin therapy are limited to a duration of approximately 2 weeks (288,289,305,306). In contrast, the protective effect of hyperleptinemia in insulinopenic Akita mice carrying a Leptin transgene is prolonged, and leptin levels do not appear to wane (291). Thus, the feasibility of long-term leptin therapy requires further investigation, however our data suggest that leptin may be best utilized for type 1 diabetes at low doses, to limit the risk of hypoglycemia and leptin resistance.  The clinical trial for leptin in type 1 diabetes is testing the efficacy of leptin in combination with insulin injection therapy to determine whether this can achieve better glycemic control, and reduce insulin dosing (www.ClinicalTrials.gov identifier: NCT01268644). This was based on evidence that occasional leptin injections in NOD mice receiving continuous insulin infusion improved glycemia, compared to insulin alone, and at a lower insulin dose (58). Therefore, the addition of leptin to insulin therapy regimens for patients with type 1 diabetes may allow for tighter glycemic control, with less frequent insulin  148 dosing. This thesis now reveals that in a similar manner leptin can be combined with islet transplantation to improve the efficacy of suboptimal islet doses and to reduce the number of islets required to achieve normoglycemia. Given that islet transplantation has been the most effective long term curative treatment for type 1 diabetes to date but is limited by insufficient donor tissue, low dose leptin therapy as an adjunct to islet transplantation could greatly improve the feasibility of islet transplantation as widely available treatment option. Expectedly, low-dose leptin co-therapy does not enhance islet transplant efficacy by increasing insulin levels, but through its metabolic actions is more likely to alleviate glucolipotoxicity and metabolic demand on β-cells, which are known to impair islet graft function (169,407). Although leptin replacement doses do not reverse hyperglycemia, this thesis work revealed that leptin replacement can fully reverse hyperlipidemia. Thus the marked lipopenic action of leptin could be advantageous when combined with insulin therapy or islet transplantation, to prevent complications and comorbidities associated with dyslipidemia and the lipogenic effects of insulin therapy. Thus therapies that utilize low-dose leptin as an adjunct to insulin replacement strategies may greatly improve metabolic control in type 1 diabetes, while minimizing risks associated with high doses of either hormone. 8.2 FUTURE DIRECTIONS Since the work in this thesis has shed light on the mechanism of leptin therapy in glucose homeostasis, it has raised several avenues of investigation that are worth pursuing to uncover the pathways that play a causal role in leptin-mediated metabolic improvements in type 1 diabetes. Given that central leptin action is sufficient to mediate the anti-diabetic effect of leptin therapy, but no particular requisite neuronal circuit has yet been identified, future studies could benefit from the use of multiple Cre drivers to maximize disruption of leptin receptors throughout the CNS, similar to what has been done for leptin receptor reconstitution in the brains of Leprdb/db mice (86). One strategy would be to combine the power of Syncre to induce recombination broadly throughout the CNS, with a Cre transgene that has improved hypothalamic expression, such as CamKII-cre (85). Another strategy would be to deliver Cre under a constitutively active promoter directly to the CNS through microinjection or ICV infusion of viral vectors. Furthermore, the role of peripheral leptin receptors has not been adequately assessed to rule out the involvement of direct leptin signalling in peripheral tissues in leptin-mediated glucose lowering. Strategies to test the relative contribution of central and peripheral leptin receptor signalling could involve comparing the effect of peripheral versus centrally administered tamoxifen in mice with an  149 inducible, ubiquitously expressed Cre transgene and a floxed leptin receptor gene. Another question is whether leptin therapy acts on glucose metabolism in rodent models of type 1 diabetes through a mechanism that is Lepr-b dependent. We attempted to assess this using Leprdb/db mice, which specifically lack the Lepr-b isoform. Unfortunately, we found these mice are remarkably resistant to STZ-mediated β-cell destruction (unpublished data), and therefore the induction of insulin-deficient diabetes through this method was not possible. Studies to test whether Lepr-b is required for the effects of leptin therapy are ongoing in our laboratory, and we are employing the use of pancreatectomy as an alternative method to induce insulin-deficient diabetes in Leprdb/db mice. Analysis of the downstream metabolic effects of leptin therapy in this thesis has revealed several key metabolic pathways that could play a causal role in leptin-induced glucose lowering. One potential mechanism is enhanced insulin sensitivity. Although studies claim that the effects of leptin are insulin-independent due to the lack of increased circulating insulin in response to leptin therapy (288,305,309), the potent insulin sensitizing action of leptin suggests that this may not be the case. To determine whether leptin reverses hyperglycemia through an insulin-independent pathway, leptin therapy must be tested in states of complete insulin deficiency. To this end, studies in our laboratory are currently being conducted in mice with congenital insulin deficiency (deletion of both insulin genes) to complement studies using an insulin antagonist. As a second potential mechanism, this study highlighted that the depletion of lipids or glycerol by leptin could mediate glucose lowering through diminishing hepatic glucose production. Studies that prevent the fall in circulating lipids/glycerol from the onset of leptin therapy could determine whether the depletion of these substrates is necessary for leptin’s anti-diabetic actions. Moreover, other strategies that reduce circulating lipids and/or glycerol such as thiazolidinedione treatment, HSL inhibitors, glycerol channel inhibitors or methods to selectively diminish adipose stores could be assessed for their ability to mimic the anti-diabetic action of leptin. Given that thiazolidinediones are already used clinically, this could reveal rapid novel strategies for the treatment of type 1 diabetes.  The activation of thermogenesis provides a third potential mechanism of leptin-induced glucose lowering. The anti-diabetic potential of increased BAT thermogenesis as a therapeutic strategy for diabetes and obesity is already being widely studied, and leptin is well known to induce BAT thermogenesis (124,215,216). Thus leptin-induced thermogenesis in BAT could activate an energy sink that depletes glucose, lipids, or substrates for endogenous glucose production. Indeed, ICV leptin has been shown to  150 induce BAT glucose uptake, and the expression of Glut4, and uncoupling protein 1 (Ucp1) in BAT of type 1 diabetic rodents (304,305,309,366). However, it was recently shown that activation of BAT thermogenesis by thyroid hormone or adrenergic agaonists, is not sufficient to lower blood glucose in type 1 diabetic rodents (366). Studies to better define the role of BAT thermogenesis in the anti-diabetic action of leptin therapy in rodent models of type 1 diabetes are ongoing in our laboratory. In light of the potential role of BAT or WAT in leptin’s therapeutic actions, the role of direct leptin receptor signalling in both of these tissues warrants investigation, and could be achieved through the Cre-lox method. Similarly, the indirect pathways through which leptin could activate thermogenic gene programs and adipose tissue depletion should be investigated. Since our study revealed that partial ablation of the sympathetic efferents does not blunt leptin action, alternative pathways should be considered, such as BAT thermogenesis induced by macrophage phenotypic switching (408). In addition to the studies needed to define the mechanism of action of leptin therapy in type 1 diabetes, the potential complications and risks of leptin therapy in type 1 diabetes need to be addressed. One particular risk that has been identified through the work in this thesis is the danger of hypoglycemia. Leptin has not been previously reported to induce hypoglycemia, and as such it has been suggested that the slow-acting glucose lowering action of leptin is safer than insulin (409). However, based on our studies, while acute leptin injection does not lower blood glucose substantially, the levels of leptin required to achieve improved metabolic control in mice with STZ-induced diabetes result in overt hypoglycemia during periods of fasting, and even during oral glucose challenge. Furthermore, the potent insulin-sensitizing action of leptin could increase the risk of insulin-induced hypoglycemia, as we have shown with ITTs. Thus the potential for hypoglycemia warrants rigorous investigation.  An additional concern, not addressed in this thesis, is the immune related actions of leptin on the underlying autoimmunity of type 1 diabetes (204). Treatment of young, pre-diabetic NOD mice with leptin accelerates the onset of insulitis, and diabetes (398), whereas NOD mice with a loss-of-function mutation in Lepr have suppressed insulitis and T-effector cell activation, resulting in reduced diabetes incidence (205,206). These data suggest that leptin therapy could have deleterious effects on autoimmune type 1 diabetes in humans. Therefore, leptin therapy could potentially worsen glycemic control by exacerbating β-cell destruction, or by stimulating inflammatory pathways resulting in deleterious metabolic consequences. Moreover, the immune effects of leptin therapy could negate the therapeutic  151 potential of leptin as an adjunct to strategies that replace or replenish patients’ β-cells, such as islet transplantation. However in contrast to studies in NOD mice, hyperleptinemia has been found to prevent the onset of virally induced autoimmune diabetes in the BB rat, and to prevent autoimmune attack of transplanted islets in this model (290). Likewise, leptin treatment was shown to confer clinical benefit to 2 patients with autoimmune lipodystrophy and type 1 diabetes without exacerbation of the underlying autoimmune disorder (401). Thus, additional studies of leptin therapy in autoimmune models of type 1 diabetes are needed to clarify the potential for immune related pitfalls of leptin therapy.  Owing to the pleiotropic nature of leptin, there are numerous anticipated benefits, but also potential risks of leptin therapy as an anti-diabetic agent that warrant further investigation in pre-clinical studies. The results of clinical trials for leptin in non-lipodystrophic patients with diabetes are eagerly awaited, and will inform research as to whether similar metabolic benefits are achieved, and whether the benefits outweigh the risks. Additionally, trials in patients with non-lipodystrophic diabetes have the advantage of a larger patient population, and thus could be extremely informative as to the effects and efficacy of leptin therapy as an anti-diabetic agent. If the benefits of leptin outweigh the risks, and the potential obstacles such as leptin resistance can be overcome, leptin therapy could be used as an effective adjuvant to other anti-diabetic therapies or even as a monotherapy for type 1 diabetes. In the event that leptin therapy fails to overcome the potential obstacles, or the risks outweigh the benefits, an understanding of the metabolic pathways and direct targets that mediate the anti-diabetic action of leptin could allow for the development of alternative strategies and therapeutic targets to treat diabetes. It is hoped that the pre-clinical studies of this thesis have contributed to the understanding of both the potential metabolic benefits, potential pitfalls, and the involved peripheral processes of leptin therapy as an anti-diabetic strategy, in order to improve the treatment of and prognosis for patients with diabetes.     152 BIBLIOGRAPHY 1. International Diabetes Federation. IDF Diabetes Atlas, 6 edn. Brussels, Belgium: International Diabetes Federation, 2013. Web. 6 Feb 2014. http://www.idf.org/diabetesatlas. 2. Karvonen M, Viik-Kajander M, Moltchanova E, Libman I, LaPorte R, Tuomilehto J. Incidence of childhood type 1 diabetes worldwide. 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