Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of leptin in the regulation of glucose homeostasis Levi, Jasna 2010

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2010_fall_levi_jasna.pdf [ 1.58MB ]
Metadata
JSON: 24-1.0071042.json
JSON-LD: 24-1.0071042-ld.json
RDF/XML (Pretty): 24-1.0071042-rdf.xml
RDF/JSON: 24-1.0071042-rdf.json
Turtle: 24-1.0071042-turtle.txt
N-Triples: 24-1.0071042-rdf-ntriples.txt
Original Record: 24-1.0071042-source.json
Full Text
24-1.0071042-fulltext.txt
Citation
24-1.0071042.ris

Full Text

  THE ROLE OF LEPTIN IN THE REGULATION OF GLUCOSE HOMEOSTASIS  by  Jasna Levi  B.Sc. (Hons) The University of British Columbia, 2007         THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Physiology)         THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)  June 2010    © Jasna Levi, 2010      ii  ABSTRACT The fat derived hormone leptin plays a crucial role in the normal maintenance of body weight and energy expenditure, as well as glucose homeostasis.  Low doses of exogenous leptin administered to leptin deficient ob/ob mice are able to reverse the hyperinsulinemia and hyperglycemia without altering body composition.  As leptin has the ability to directly suppress insulin secretion from ß-cells, we hypothesise that in the absence of leptin signalling, unregulated insulin secretion leads to hyperinsulinemia which in turn leads to increased adipogenesis and insulin resistance, ultimately culminating in the development of type 2 diabetes in this mouse model.  To test this hypothesis we induced an acute state of leptin deficiency with a PEGylated mouse leptin antagonist (PEG-MLA) to determine the hierarchy of leptin action.  Metabolic analysis by indirect calorimetry showed that PEG-MLA treatment resulted in increased food intake and respiratory quotient without altering body composition or energy expenditure.  These changes in energy balance were accompanied with increased fasting, and glucose stimulated insulin levels.  PEG-MLA treated mice also displayed decreased whole-body insulin sensitivity, elevated endogenous hepatic glucose production (HPG), and impaired insulin mediated suppressed of HPG as determined by euglycemic-hyperinsulinemic clamps. Overall, these findings demonstrate that leptin signalling is important in regulating insulin secretion, and that changes in insulin sensitivity occur prior to changes in body composition and energy expenditure in a state of acute leptin deficiency. It has been recently shown that the liver derived, leptin regulated insulin-like growth factor binding protein-2 (IGFBP-2) is responsible for the anti-diabetic effect of leptin in ob/ob mice.  We investigated the mechanism by which leptin regulates IGFBP-2 levels. ob/ob mice with attenuated hepatic leptin signalling or a subdiaphragmatic vagotomy were utilized to determine if leptin acts directly on the liver or centrally to increase plasma IGFBP- 2. Our results show that while leptin is able to increase plasma IGFBP-2 levels in ob/ob mice in a dose dependent manner, the mechanism does not involve heptic leptin signalling or vegal efferents and remains to be elucidated.   iii  TABLE OF CONTENTS  ABSTRACT....................................................................................................................... ii TABLE OF CONTENTS ................................................................................................ iii LIST OF TABLES .............................................................................................................v LIST OF FIGURES ......................................................................................................... vi LIST OF ABBREVIATIONS ....................................................................................... viii ACKNOWLEDGEMENTS ............................................................................................ ix INTRODUCTION..............................................................................................................1 Obesity .........................................................................................................................1 Type 2 Diabetes................................................................................................................2 Leptin .........................................................................................................................4 Leptin Receptor and Signalling........................................................................................5 Regulation of Body Weight and Energy Expenditure by Leptin .....................................9 Leptin and Lipid Metabolism.........................................................................................12 Leptin and Glucose Metabolism ....................................................................................14 THESIS INVESTIGATION ...........................................................................................19 METHODS AND MATERIALS ....................................................................................21 Animals .......................................................................................................................21 Lerpflox/flox Albcre ob/ob Mouse Line..............................................................................21 A-ZIP/F Mouse Line ......................................................................................................22 Energy Balance Physiology and Body Composition Measurements .............................23 Food intake .................................................................................................................23 Indirect calorimetry ....................................................................................................24 Activity levels.............................................................................................................25 Body composition.......................................................................................................25 Body Temperature Analysis and Cold Tolerance ..........................................................26 Implantation of Mini-Osmotic Pumps and Delivery of Peptides in vivo .......................26 Plasma Analyte Analysis................................................................................................27 PCR and RT-PCR Analysis............................................................................................28 Subdiaphragmatic Vagotomy.........................................................................................29 Verification of Vagotomy ..............................................................................................29 CCK experiment.........................................................................................................29 Gastric distension .......................................................................................................30 GLP-1 Bioassay..............................................................................................................30 CHO-Ob-Rb cells .......................................................................................................32 Stimulation of CHO-Ob-Rb cells with PEG-MLA and leptin ...................................32 Western blot analysis of phospho-STAT3 in simulated CHO-Ob-Rb cells...............33 Glucose and Insulin Tolerance Test ...............................................................................34 Arginine Stimulated Insulin, Glucagon and Total GLP-1 Secretion .............................34  iv  Hyperinsulinemic-Euglycemic Clamps..........................................................................35 Measurement of Hepatic Triglycerides and Cholesterol................................................36 Statistical Analysis .........................................................................................................36 RESULTS .........................................................................................................................37 STUDY 1: Acute Disruption of Leptin Signalling in vivo.............................................37 STUDY 2: Mechanism of Leptin Action in Regulation of Glucose Metabolism..........62 STUDY 3: Characterization of the Spontaneous Recovery of ob/ob Mice ...................89 DISCUSSION ...................................................................................................................94 Energy Homeostasis in a State of Acute Leptin Deficiency ..........................................95 Glucose Metabolism in a State of Acute Leptin Deficiency..........................................99 Dose Dependent Effect of Leptin on Glucose Homeostasis ........................................104 Mechanism by Which Leptin Increases Plasma IGFBP-2 Levels ...............................106 Basal GLP-1 Levels in ob/ob Mice and Effect of Leptin Treatment ...........................111 Transient Hyperglycemia in ob/ob Mice on a C57BL/6 Background .........................112 CONCLUSION AND FUTURE DIRECTIONS.........................................................118 REFERENCES...............................................................................................................120 APPENDIX.....................................................................................................................132 UBC Research Ethics Board Certificates of Approval ................................................132   v  LIST OF TABLES  Table 1: Lipid Analysis in PEG-MLA Treated Mice .......................................................61  Table 2: Metabolic Parameters of obese ob/ob and lipodystrophy AZIP/F1 mice...........75  Table 3: Metabolic Parameters in Female Leprflox/flox Albcre ob/ob Mice ........................82    vi  LIST OF FIGURES  Figure 1: Various leptin receptor isoforms and nature of mutations in rodent models of obesity. ..........................................................................................6  Figure 2: Leptin receptor signalling....................................................................................7  Figure 3: Control of food intake by leptin signalling in the arcuate neurons in the hypothalamus. ..............................................................................................10  Figure 4: LabMaster metabolic cages, an automated indirect calorimetry system...........24  Figure 5: A cell based luciferase reporter GLP-1 bioassay signalling pathway...............31  Figure 6: PEG-MLA does not activate Ob-Rb and the leptin signalling pathway in stably transfected CHO-ObRb cells. ........................................................39  Figure 7: Increased food intake and respiratory quotient in mice receiving PEG- MLA. ............................................................................................................40  Figure 8: Effects of PEG-MLA on spontaneous physical activity. ..................................42  Figure 9: Basal body temperature and cold tolerance in mice receiving PEG- MLA. ............................................................................................................43  Figure 10: Metabolic parameters in C57Bl/6 mice receiving 72 µg/day PEG- MLA. ............................................................................................................45  Figure 11: Metabolic parameters in C57Bl/6 mice receiving 36 µg/day PEG- MLA. ............................................................................................................47  Figure 12: Metabolic parameters in C57Bl/6 mice receiving 18 µg/day PEG- MLA. ............................................................................................................48  Figure 13: Oral glucose and i.p. arginine tolerance test in PEG-MLA treated mice. .............................................................................................................50  Figure 14: Effect of PEG-MLA on glucose homeostasis in the fasting state. ..................52  Figure 15: Fasting glucagon levels in PEG-MLA treated mice........................................53  Figure 16: PEG-MLA treated mice exhibit mild insulin resistance. ................................54  Figure 17: Blood glucose levels and glucose infusion rate during the euglycemic- hyperinsulinemic clamps..............................................................................56  Figure 18: Increased hepatic glucose production and decreased glucose infusion rate in PEG-MLA treated mice. ...................................................................57  Figure 19: PEG-MLA treated mice have elevated free fatty acid levels in the hyperinsulinemic state..................................................................................58  Figure 20: Plasma IGFBP-2 levels in PEG-MLA treated mice........................................59  Figure 21: PEG-MLA treatment does not alter hepatic triglyceride and cholesterol content........................................................................................60  Figure 22: Continuous leptin administration in ob/ob mice leads to reversal of hyperglycemia and hyperinsulinemia independently of weight loss. ..........63  Figure 23: Continuous leptin administration in ob/ob mice improves glucose tolerance and insulin sensitivity in a dose dependent manner. ....................64   vii  Figure 24: Continuous leptin administration in ob/ob mice leads to decreased total GLP-1 levels in a dose dependent manner. ..........................................67  Figure 25: Leptin treated ob/ob mice have glucose stimulated secretion of total GLP-1. ..........................................................................................................68  Figure 26: Total GLP-1 levels in response to acute leptin treatmen ob/ob and C57Bl/6 Mice. ..............................................................................................70  Figure 27: ob/ob mice have elevated active GLP-1 levels compared to wild-type controls. ........................................................................................................71  Figure 28: Total GLP-1 levels in response to fasting and re-feeding in ob/ob and C57Bl/6 Mice. ..............................................................................................73  Figure 29: Continuous leptin administration in ob/ob mice leads to increased plasma IGFBP-2 levels in a dose dependent manner. ..................................76  Figure 30: IGFBP-2 Administration to ob/ob mice. .........................................................78  Figure 31: Schematic of the leprflox and lepr∆17 gene and protein stuctures......................79  Figure 32: Recombination of the Leprflox allele in liver mediated by the Albcre transgene.......................................................................................................80  Figure 33: Leptin administration to Lerpflox/flox Albcre ob/ob mice leads to weight loss and reverses hyperglycemia and hyperinsulinemia. .............................82  Figure 34: Leptin increases plasma IGFBP-2 levels in Leprflox/flox Albcre+ ob/ob mice independently of hepatic leptin signalling...........................................83  Figure 35: Leptin treatment in ob/ob mice with a subdiaphragmatic vagotomy. .............85  Figure 36: Leptin treatment leads to increased IGFBP-2 levels in ob/ob mice with a subdiaphragmatic vagotomy......................................................................86  Figure 37: Subdiaphragmatic vagotomy inhibits CCK induced satiety in ob/ob mice and causes gastric distension. ..............................................................88  Figure 38: Transient hyperglycemia in ob/ob mice on a C57BL/6 background. .............90  Figure 39: Glucose tolerance and insulin sensitivity in young hyperglycemic and old euglycemic ob/ob mice. .........................................................................92  Figure 40: Glucagon levels during fasting and feeding in ob/ob mice. ............................93    viii  LIST OF ABBREVIATIONS AgRP   agouti-related protein ARC   arcuate nucleus BAT   brown adipose tissue CART   cocaine-and amphetamine-regulated transcript CCK    cholicystokinine db/db    diabetic leptin receptor deficient genotype DMN   dorsomedial nucleus EE   energy expenditure FFA   free fatty acid GLP-1   glucagon-like peptide 1 HGP   hepatic glucose production IGF   insulin-like growth factor IGFBP-2  insulin-like growth factor binding protein-2 i.p.   intraperitoneal ITT   insulin tolerance test JAK   janus kinase LH   lateral hypothalamus α-MSH  α-melanocyte stimulating hormone NPY   neuropeptide Y ob/ob   obese leptin deficient genotype OGTT   oral glucose tolerance test PBS   phosphate buffered saline PCR   polymerase chain reaction PEG   polyethylene glycol PEG-MLA  PEGylated Mouse Leptin Antagonist POMC   proopiomelanocortin PPAR-α  perioxisome proliferator-actviated receptor-α PPAR-γ  perioxisome proliferator-actviated receptor-γ PTP1B   protein tyrosine phosphatase-1B PVN   paraventricular hypothalamic nucleus RT-PCR  reverse transcriptase-polymerase chain reaction RQ   respiratory quotient SOCS-3  suppressor of cytokine signalling-3 STAT   signal transducer and activator of transcription STZ   streptozotocin TG   triglycerides TZD   thiazolidindiones UCP-1   uncoupling protein-1 VMN   ventromedial hypothalamic nucleus WHO   world health organization   ix  ACKNOWLEDGEMENTS I would like to thank Dr. Kieffer for the amazing opportunity of being a graduate student in his lab as well as for all his advice, guidance, and support throughout my time as a graduate student.  Special thanks to Dr. Scott Covey for the training and mentorship from my very first day in the lab as an undergraduate student, and all the way through graduate school.   I am also very grateful to the entire Kieffer lab for all their help, but above all, their friendship.  Not many people get to spend long days at work surrounded by friends, and I will always fondly remember my time in the lab.  I would especially like to thank my fellow graduate students; Frank, Cathy, Blair, Heather, and Gary. I thank you for your support as well as for always being willing to lend a helping hand with an experiment when one was needed.  It was amazing working with you and I look forward to collaborating with each one of you in the future! Finally, a special thanks to all my friends and family for their never-ending support in all aspects of life.  To my sister Nina, thank you for setting the bar high with your strong work ethic and drive to succeed. Bryan, thank you for always being there to cheer me up, and for always believing in me, I could not have done this without you.  All my achievements to date are thanks to my parents, who made countless sacrifices so that I could have endless opportunities made available to me.  Thank you for your encouragement and most importantly for making me believe anything was possible with hard work and effort.  It is with much gratitude that I dedicate this work to you.   1  INTRODUCTION  Obesity  The cause of obesity is the energy imbalance between calories consumed and calories expended.  In simple terms, obesity develops when energy intake exceeds energy expenditure.  The World Health Organization (WHO) defines obesity as “abnormal or excessive fat accumulation that may impair health.”  The most widely used system for assessing obesity is the body mass index (BMI) [1] as there is a high correlation of BMI with amount of body fat, which is in turn associated with the adverse effects of obesity. BMI is calculated by dividing the weight of an individual in kilograms by their height in meters squared, (kg/m2).  The WHO has classified a BMI from 18.5-24.9 as normal and healthy, and individuals with a BMI from 25.0-29.9, 30.0-39.9, and > 40 as overweight, obese, and morbidly obese, respectively.  In combination with BMI, other anthropometric methods such as the waist-to-hip ratio and waist circumference are also used to measure obesity [2, 3].  As central adiposity has been shown to strongly predispose individuals to certain disease states such as type 2 diabetes (T2D) and hypertension more so than subcutaneous adiposity [4], measures that spefically address fat distribution in combination with BMI measurnments may be better indicators of metabolic health [3]. A global shift in diet composed of foods high in fat and sugar content, combined with a sedentary lifestyle has caused obesity to reach epidemic proportions [5], and become a world wide health concern [6].  The WHO has estimated that in the year 2005, 1.6 billion adults were overweight, and that at least 400 million adults were obese worldwide.  The WHO also projects that by the year 2015, approximately 2.3 billion  2  adults will be overweight and more than 700 million will be obese.  This dramatic increase in obesity has become an enormous burden for many health care programs globally [7], as it is associated with increased morbidity and mortality due to hypertension, coronary heart disease [8], cancer [9], and T2D [10].  In fact, evidence supports a strong correlation between T2D and obesity [11], that individuals with a BMI >35 have a 20-fold increased risk of developing diabetes, relative to individuals with a BMI between 18.5 and 24.9 [12].  Type 2 Diabetes T2D is a metabolic disease and its incidence is growing at an alarming rate.  It has been estimated by the WHO that by 2025 the number of diabetic individuals will double to almost 300 million worldwide.  T2D is characterized by elevated blood glucose levels (≥ 7.0 mmol/L in the fasting state or ≥ 11.1 mmol/L 2 hours after ingestion of 75 g oral glucose load) [13] in the context of hyperinsulinemia and insulin resistance.  As the fundamental mechanism for maintaining glucose homeostasis is the rapid release of insulin (by the pancreatic ß-cells) and the action of insulin to stimulate glucose uptake in peripheral tissues [14], pathways that control insulin secretion and ß-cell mass are crucial in the development of T2D.  The pathophysiology is believed to involve peripheral insulin resistance which leads to compensatory changes, including increased ß-cell size/number and enhanced insulin secretion in order to maintain normal blood glucose levels [13].  When the ß-cells can no longer compensate for the insulin resistance, and insulin is no longer secreted at levels sufficient to maintain blood glucose levels within an appropriate range, T2D develops [15].  3  Despite the strong correlation between T2D and obesity [10, 12] (approximately 80% of subjects with T2D are obese), the molecular mechanisms that link the two are yet to be completely elucidated.  However, discoveries over the past several years have identified signalling molecules that affect food intake and that are critical for maintaining normal energy homeostasis.  Application of molecular genetics and rare genetic disorders giving rise to obesity in humans and rodents has been invaluable in deciphering the physiological origins for body weight regulation. Recessive mutations in the mouse genes obese (ob) and diabetes (db) result in both obesity and diabetes as part of a syndrome that resembles morbid obesity in humans [16, 17].  Mice with these recessive mutations weigh three times as much as their littermate controls and develop metabolic abnormalities similar to those seen in patients with T2D such as fasting hyperglycemia, hyperinsulinemia and insulin resistance [17].  Through parabiosis experiments, where the circulation of two mice is fused, it was predicted that the ob gene encoded a circulating factor that regulated body weight and energy expenditure, whereas the db gene encoded its receptor [17].  Fusing the circulation of a wild-type mouse and an ob/ob mouse caused the ob/ob mouse to lose weight and decrease food intake suggesting that the ob/ob mouse was missing a soluble factor that was present in the wild-type mouse.  This soluble factor was also present in the db/db mouse as indicated by the weight loss in the ob/ob mouse when fused to a db/db mouse, but the db/db mouse was obviously resistant to its affects due to its phenotype.  A parabiosis experiment on a db/db mouse and a wild-type mouse had no effect on the db/db mouse, but caused the wild-type mouse to die of starvation, suggesting that the db/db mouse was not only resistant to this soluble factor’s effects, but overproduced it.  Positional cloning of the ob gene showed that it encoded a 16 kDa  4  circulating hormone named leptin [18, 19]; while the expression cloning technique revealed that the db gene encoded the receptor for the ob gene product [20]. The ob gene was found to be mutant in the ob/ob strain of mice where a nonsense mutation results in the synthesis of a truncated leptin protein that is not secreted into the circulation [18], while db/db mice express a non-functional leptin receptor [19].  As lack of leptin or leptin signalling results in both obesity and a T2D phenotype, these data suggest that leptin is potentially an important link in providing key insights into both of these disease states.  Leptin Leptin, an adipocyte derived hormone is predominantly expressed and secreted by adipoctyes [18, 19] but lower levels of expression can also be detected in the placenta, skeletal muscle, and the gastric epithelium [21-23].  Plasma circulating leptin levels correlate to total body fat mass and increase proportionally to body mass in both obese mice and humans, and decrease in both following weight loss [24, 25].  It is therefore thought that leptin serves as a feedback signal indicating the size of fat stores, with high levels of circulating leptin signalling the animal to decrease food intake and increase energy expenditure [24, 26].  Indeed, leptin replacement therapy to leptin deficient ob/ob mice leads to a dose dependent decrease in body weight and food intake but not in the db/db mouse which lacks a functional leptin receptor [26-28].  Furthermore, exogenous leptin is also able to normalize the diabetic symptoms in the ob/ob mouse, improving fasting hyperglycemia, hyperinsulinemia, and insulin resistance [26, 28].  An alternative view is that leptin functions as a signal of low energy stores; as low leptin levels can trigger an adaptive response to conserve energy by manifesting hyperphagia, decreasing  5  energy expenditure and as well as having adverse affects on reproduction [17].  Indeed, the ob/ob mouse is hyperphagic, and displays various characteristics of starvation such as decreased energy expenditure, and infertility [17] all of which can be reversed with leptin therapy [26, 29].  As there is evidence that leptin has a role as both a signal of nutritional deprivation and nutrient excess, a broader view holds that leptin is able to dynamically regulate appetite and energy expenditure to maintain body weight within a narrow range.  Leptin Receptor and Signalling With the exception of the ob/ob mouse [17], and rare human cases of leptin deficiency [30], leptin levels are actually high in most models of rodent and human obesity illustrating the prevalence and importance of leptin resistance in the pathogenesis of obesity [24, 25, 31].  These observations initiated the characterization of the mechanism of leptin signal transduction. With the technique of expression cloning, a high affinity leptin receptor (Ob-R) was identified and found to be a membrane spanning receptor related to the cytokine receptor family [20].  Further positional cloning of db, the Ob-R gene, showed that this gene encoded 5 alternatively spliced forms [20, 32].  The various isoforms of the leptin receptor, Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd and Ob-Re [32-34] (Figure 1A) are the products of the alternate splicing.  The long form of the leptin receptor, Ob-Rb, is believed to be essential for the physiological actions of leptin as it is the only isoform believed to be capable of intracellular signalling.  C57Bl/Ks db/db mice produce the Ob-Rb transcript with an insertion that prematurely terminates the intracellular domain resulting in the replacement of Ob-Rb with the OB-Ra isoform (Figure 1B).  All other Ob-R isoforms  6  remain intact in C57Bl/Ks db/db mice, yet a severe obese phenotype comparable to other strains of db/db mice that lack all Ob-R isoforms is observed.  This mutation establishes the Ob-Rb as crucial for initiating intracellular signal transduction and critical for leptin function and exerting the weight reducing effects of leptin [32, 33].              Figure 1: Various leptin receptor isoforms and nature of mutations in rodent models of obesity. There are at least five different isoforms of the leptin receptor in rodents. (A) All isoforms share identical extracellular and ligand-binding domains but they differ at the C terminus.  Four of the five have transmembrane domains, but only Ob-Rb encodes all protein motifs capable of activating the JAK–STAT signal transduction pathway.  The remaining isoform, Ob-Re, is truncated before the membrane-spanning domain and is secreted.  (B) Mutations in Ob-R lead to massive obesity in db mice and fa rats.  Most of the mutations affect all of the splice forms. WT,wild type.  Figure is adapted from Friedman and Halaas (1998) Nature and used with Nature’s Publishing Groups permission.   The Ob-Rb receptor is involved in signalling via the janus kinase/signal transducer and activator of transcription 3 kinase (JAK/STAT-3) pathway [20] (Figure 2).    7                 Figure 2: Leptin receptor signalling. Leptin binding to OB-Rb results in conformational changes and receptor oligomerization.  These events stimulate tyrosine phosphorylation and activation of JAK2 that is constitutively associated with the receptor.  JAK2 phosphorylates the intracellular domain of OB-Rb, especially tyrosines within the SHP2 and STAT3 binding sites. Binding of STAT3 to OB-Rb induces STAT3 tyrosine phosphorylation, dimerization, nuclear translocation, and induction of target genes.  These include socs3, whose product inhibits JAK-induced phosphorylation of OB-Rb.  Induction of JAK2 can also stimulate PI- 3K, possibly through the recruitment and phosphorylation of scaffolding proteins IRS-1/2. Activation of PI-3K can increase cell migration and invasion via the Rac/Rho pathways and stimulate the major growth/survival pathway Akt. Figure modified from [35]  Leptin binding to the Ob-Rb receptor initiates receptor homo-oligomerization followed by phosphorylation of tyrosine (tyr) residues of the receptor (tyr985 and tyr1138), catalyzed by JAK [36].  STAT-3 can then bind to the phosphorylated leptin receptors and are themselves phosphorylated on tyr residues by a separate activity of JAK [36].  This  8  causes the STAT-3 to dimerize, translocate to the nucleus, and bind to specific regulatory regions of DNA altering the expression of certain genes involved in food intake regulation and metabolic status [37] There are also a number of molecules that are involved in attenuating the leptin signal transduction pathway.  As leptin receptor signalling requires the phosphorylation of JAK2 and STAT-3 activation, molecules interfering with these processes have been implicated in the attenuation of the leptin signal transduction pathway.  Protein-tyrosine phosphatase 1B (PTP1B) has been shown to interact with and dephosphorylate JAK2 [38, 39] .  ob/ob mice lacking PTP1B have been shown to have an enhanced response to leptin mediated weight loss and markedly increased leptin-induced STAT3 phosphorylation [38] while wild-type mice deficient in PTP1B are resistant to obesity when fed a high-fat diet [40].  Leptin also has an effect on the expression of the suppressors-of-cytokine signalling-3 (SOCS-3) protein [41].  SOCS-3 is able to bind to the phosphorylated tyr985 of the OB-Rb, preventing OB-Rb mediated STAT-3 activation [42].  Peripheral leptin administration to ob/ob, but not db/db mice, rapidly induced SOCS-3 mRNA in areas of the hypothalamus expressing high levels of the leptin receptor long form [41].  In mammalian cell lines, SOCS-3 blocked leptin-induced signal transduction, and expression of SOCS-3 mRNA in the hypothalamic nuclei is increased in Ay/a mice, a model of leptin-resistant obesity [41].  These data highlight the role of SOCS-3 and PTP1B as potential mediators of leptin resistance in obesity.   9  Regulation of Body Weight and Energy Expenditure by Leptin Leptin action in the brain is believed to be essential for the ability of leptin to exert its effects on food intake and body weight regulation [43, 44].  Leptin receptors (Ob-Rb) are expressed at high levels in the hypothalamus [32, 37], and leptin administration results in a dose dependent activation of STAT3 specifically in the hypothalamus of wild-type and ob/ob but not db/db mice [45].  The Ob-Rb is specifically expressed within regions that are involved in the regulation of food intake and energy expenditure [46-48] .  In situ hybridization has shown that the hypothalamic expression of Ob-Rb is in specific nuclei known to be involved in the regulation of body weight and appetite, such as the arcuate nucleus (ARC), dorsomedial hypothalamic nucleus (DMN), paraventricular hypothalamic nucleus (PVN), ventromedial hypothalamic nucleus (VMN) and the lateral hypothalamus (LH) [46-49].  These hypothalamic nuclei express neuropeptides including neuropeptide Y (NPY) [50] and agouti-related protein (AgRP) [51] which stimulate food intake, and α-melanocyte stimulated hormone (α-MSH) and cocaine-and amphetamine-regulated transcript (CART) which suppress food intake [52, 53].  NPY/AgRP neurons are inhibited by leptin [54, 55], while POMC/CART neurons are activated by leptin [53] (Figure 3).        10           Figure 3: Control of food intake by leptin signalling in the arcuate neurons in the hypothalamus. Populations of first-order NPY/AGRP (green) and POMC/CART (red) neurons in the arcuate nucleus (ARC) are regulated by leptin and project to the PVN and to the LHA and PFA, which are locations of second-order hypothalamic neuropeptide neurons involved in the regulation of food intake and energy homeostasis.  Figure is adapted from Schwarts M.W. et al (2000) Nature and used with Nature’s Publishing Groups permission.  Although leptin has potent effects on food intake [26, 56], leptin deficiency and the resulting increased caloric intake is not the sole reason for the observed obesity in the ob/ob mouse.  Food restricted ob/ob mice still become obese [17], and ob/ob mice food restricted to the same levels that leptin treated ob/ob mice voluntary eat (pair-fed) display smaller decreases in body weight compared to leptin treatment [57].  Increased storage of calories in a setting of normal food consumption indicates that these mice have reduced energy expenditure. Components of energy expenditure include energy expenditure required for basic cellular function, physical activity and adaptive thermogenesis [58].  Adaptive thermogenesis is defined as heat production in response to environmental temperature and diet [58].  The ob/ob mice have been found to have decreased energy expenditure as  11  well as impaired thermogenesis and cold tolerance [26] and both conditions are reversible with leptin treatment [26].  Leptin has clear effects on energy expenditure [26, 59, 60] and it is thought to exert its effects at least in part through the autonomic nervous system [61].  Brown adipose tissue (BAT) and skeletal muscle play a major role in adaptive thermogenesis, and these tissues are highly innervated by the sympathetic nervous system.  Sympathetic outputs act on ß-adrenergic receptors, and treatment with their agonists leads to lipolysis and an increase in energy expenditure [62-64].  It has been shown that sympathetic outflow to BAT is decreased in ob/ob mice [65], and leptin treatment leads to a dose-dependent increase in sympathetic output to BAT, kidney and adrenal glands in normal mice but not in the obese Zucker rats which have a mutation in the leptin receptor [66]. Energy expenditure is measured by oxygen consumption from which the metabolic rate can be calculated.  Oxygen consumption or the metabolic rate have been found to increase following feeding [67, 68] whereas food restriction leads to a decrease in the metabolic rate [69].  This system, although counter-productive in the dieting state, is in place to maintain a narrow set point in bodyweight.  Leptin treatment to ob/ob mice is also able to prevent the fall in energy expenditure [26] usually associated with weight loss [69].  Although obese individuals have high circulating leptin levels and are resistant to its effects [25], and further exogenous leptin administration has minimal weight reducing effect, it may help dieters maintain their reduced body weight [70].  Weight loss in obese individuals in associated with a drop in leptin levels, which leads to increased food intake and decreased energy expenditure promoting a restoration of their original weight [24, 25, 69].  Leptin administration at low doses during dieting has been shown to  12  prevent these effects associated with falling leptin levels, and aid with weight loss during active dieting [70].  Therefore, leptin seems to maintain proper energy homeostasis by regulating both food intake as well as various components of energy expenditure.  Leptin and Lipid Metabolism Although leptin acts centrally to modulate sympathetic and neuroendocrine activity, there is also evidence leptin has direct actions on peripheral tissues to regulate lipid and glucose metabolism.  The Ob-Rb which is capable of signal transduction is expressed in a number of peripheral tissues and direct effects of leptin on fat [71], pancreatic ß-cells [72-74], hepatocytes [75, 76], and other cell types have been shown [77].  The absence of leptin signalling in the ob/ob and db/db models, not only leads to increased triglyceride (TG) stores in adipose tissue, but also spillover into other peripheral tissues such as the liver, skeletal muscle, and islets [17, 19].  It is this build-up of fat in non-adipose tissue that leads to the lipotoxicity which contributes to insulin resistance in the obese state [78].  Given the serious consequences of ectopic lipid accumulation in the absence of leptin signalling it has been suggested that leptin plays an important role as a regulator of lipid metabolism in both adipose and non-adipose tissue [78, 79].  Leptin treatment, more so than pair-feeding alone is able to rapidly decrease lipid levels in non-adipose tissue sites by decreasing TG formation, as well as by increasing oxidation of free fatty acids (FFA) [80]. The role of leptin in mediating biochemical pathways of FFA oxidation are in the process of being elucidated.  The nuclear hormone receptor, perioxisome proliferator- actviated receptor-α (PPAR-α), a regulator of many of the enzymes responsible for FFA  13  oxidation, is thought to be involved in coordinating the actions of leptin on FFA oxidation.  PPAR-α expression is induced by hyperleptinemia in wild-type but not fa/fa rats [81, 82] which lack a functional leptin receptor.  Leptin therapy in mice lacking PPAR-α results in a smaller reduction of fat depots and a smaller depletion of liver TG [83] compared to leptin treatment in wild-type mice, confirming that this is an important pathway of leptin mediated lipid metabolism.  The effect of leptin on lipid metabolism is not only a result of enhanced FFA oxidation but also due to decreased lipogenesis in response to leptin treatment [84].  ob/ob mice have enlarged, steatotic livers [85] which is partly a result of an increased rate of hepatic lipogenesis, and leptin treatment is able to normalize the hepatomegaly and associated elevations in hepatic TG levels [57]. Utilizing leptin resistant Zucker Diabetic Fatty ZDF rats, it has been demonstrated that improvement in insulin resistance through the removal of visceral fat did not change hepatic TG content [86], while leptin treatment did, dissociating the action of leptin from that of insulin.  This study by Fishman et al. [86] showed that leptin action on hepatic TG levels is independent of the improved hepatic insulin sensitivity that also results from leptin treatment [87]. Therefore, absence of leptin action is potentially a major contributor to the development of hepatic steatosis [86].  These findings highlight the role of leptin as an acute regulator of lipid and hepatic TG metabolism by modulating pathways of fatty acid biosynthesis and oxidation, and confining the storage of TG to adipose tissue. Paradoxically, elevated serum TG and FFA levels as well as hepatic steatosis are also associated with reduced fat mass observed in lipodystrophy [88, 89].  Lipodystrophy, a rare set of diseases characterized by a complete or near complete loss of adipose tissue  14  results in ectopic accumulation of TG in liver, muscle and other peripheral tissues [89, 90].  Animal  models of lipodystrophy, where transgenic over expression in adipose tissue of either the sterol regulatory element binding protein-1c (ap2-SREBP-1c mice) or the dominant negative protein A-ZIP/F (AZIP/F-1 mice), have a near complete absence of adipose tissue and display hepatic steatosis, insulin resistance and hyperglycemia [88, 91].  In the absence of adipose tissue, leptin levels and other adipocyte derived hormones are very low suggesting that the lack of one or more of these factors may contribute to the metabolic abnormalities observed in these mice.  Leptin administration at physiological doses to the ap2-SREBP-1c mice is able to normalize the hepatic steatosis and lipid levels while pair-feeding had much more modest effects [92].  Clinical data also show that leptin therapy in patients with generalized lipodystrophy improves lipid metabolism by reducing fasting TG, total cholesterol levels, and additionally improves hepatic steatosis [93-96].  These data suggest that the dysregulated lipid metabolism and hepatic steatosis in both rodent and human models of lipodystrophy are a result of leptin deficiency.  Leptin and Glucose Metabolism Leptin also has well documented effects on glucose metabolism [26, 27, 56, 73, 97].  Hyperinsulinemia and hyperglycemia are both corrected in the ob/ob mouse with leptin treatment even at doses which have no effect on food intake [26] and to a greater extent than observed in pair fed animals [98, 99].  The marked reduction in plasma insulin and glucose levels following leptin treatment suggests that leptin treatment not only results in a decrease of insulin levels but also improves insulin action in vivo.  There is considerable evidence that leptin exerts its insulin lowering effects by acting directly  15  on the pancreatic insulin producing ß-cells [72, 74, 100], and improves insulin sensitivity by direct action on hepatocytes [87].    Functional leptin receptors are expressed in hepatocytes [37, 48, 75] and direct action of leptin on hepatic tissue with regard to glucose metabolism regulating abilities has been observed by several groups [87, 101].  Using the hyperinsulinemia clamp technique, leptin treatment via mini-osmotic pumps for 8 days, resulted in a 52% increase in the rate of tissue glucose uptake compared to saline treated rats during physiological hyperinsulinemic conditions [102].  This improvement in peripheral insulin action was accounted for by an increase in glycogen synthesis [102].  Similarly, leptin was also able to increase insulin sensitivity by markedly enhancing the action of insulin to inhibit hepatic glucose production [102].  Exposure of human hepatic cells to leptin at concentrations comparable with those present in obese individuals caused down- regulation of gluconeogenesis [75], and significant reduction of glucose production from different gluconeogenic precursors was observed in leptin treated, isolated rat hepatoctyes [101]. Besides the ability of leptin to augment insulin sensitivity, leptin also plays an important role in the regulation of circulating insulin levels.  Leptin receptors are expressed in the pancreatic ß-cells [72-74] and to date it has been shown that leptin is able to directly suppress ß-cell secretion of insulin in perfused pancreas of ob/ob mice [74] as well as in isolated human islets [103] by interacting with ATP-sensitive K+ ion channels, [74] which are integral in controlling glucose stimulated insulin secretion. Leptin activates ATP-sensitive K+ channels which results in hyperpolarization of the ß- cell, thus inhibiting the secretion of insulin [74].  In addition to inhibiting insulin  16  secretion from ß-cells, leptin counters hyperinsulinemia by decreasing expression of preproinsulin mRNA in ob/ob islets [73].  Leptin also has similar effects in insulin gene expression in islets from human donors [103].  While leptin has the ability to decrease insulin mRNA expression, insulin is able to up regulate leptin gene expression [104]. This interaction between the two hormones founded the basis for the existence of the adipoinsular axis [100].  Physiologically, as insulin is a major adipogenic hormone, the adipoinsular axis where insulin stimulates adipogenesis and leptin production, and leptin inhibits insulin secretion, is important in the regulation of fat deposition. The existence and importance of the adipoinsular axis in the maintenance of glucose metabolism is exemplified by the existing literature on the leptin deficient ob/ob mouse.  This literature suggests that the observed obesity and insulin resistance in the ob/ob mouse model are secondary to the initial manifestation of hyperinsulinemia, which results from unregulated insulin secretion from ß-cells due to the lack of leptin signalling. The common belief is that the obesity displayed by the ob/ob mouse model is the cause of the hyperinsulinemia, the body’s attempt to compensate for worsening insulin resistance. The capacity of the islets to secrete insulin is exceeded at a certain threshold, at which point the hyperinsulinemia fails to compensate for the insulin resistance, and hyperglycemia and diabetes become fully evident.  This notion that the hyperinsulinemia is due to the obesity is challenged by several key observations.  When the sequential development of the metabolic characteristics of hyperinsulinemia, hyperglycemia, and obesity was examined in the ob/ob mouse model, the first abnormality observed was elevated insulin levels [105] prior to weaning.  The increased insulin levels were not preceded by hyperglycemia but were associated with mild hypoglycaemia [106].  These  17  results suggest that insulin resistance is not likely to be the primary metabolic abnormality in ob/ob mice as it is unlikely the insulin producing pancreatic ß-cells would overcompensate to produce a condition of hypoglycaemia, but develops later on.  Further evidence that hyperinsulinemia due to unregulated ß-cell insulin secretion precedes the onset of insulin resistance in the ob/ob mouse model is that no defect in insulin mediated glucose uptake in muscle was detected [105] suggesting that these mice are insulin sensitive at this stage.  The developmental stages of the disorder have also been examined in other models of animal obesity and diabetes.  In the leptin receptor deficient (db/db) mouse, primary hypersinsulinemia was also observed at 10 days old and was also associated with mild hypoglycaemia [107].  Another group also showed that young db/db mice had improved glucose disposal following an oral glucose gavage.  Glucose levels 30 minutes following the glucose gavage were lower compared to age-matched wild-type controls suggesting an excess of glucose stimulated insulin levels in the young db/db mice [108].  The observed hyperinsulinemia prior to development of obesity and insulin resistance is not confined to animal models, as human evidence also exists.  Alterations in insulin sensitivity preceded by hyperinsulinemia in humans have been observed; challenging the current dogma that obesity induced insulin resistance is fully responsible for the development of diabetes [109-111].  Therefore, it is reasonable to propose that a primary defect that results in unregulated insulin secretion leading to hyperinsulinemia is responsible for initiating the development of obesity which then in turn further augments insulin resistance and compensatory hyperinsulinemia, with diabetes the ultimate outcome.  18  While leptin is able to exert significant effects on glucose homeostasis by regulating insulin secretion and insulin sensitivity, there is also evidence that leptin plays a more specific role in glucose metabolism independently of insulin action.  Utilizing the hyperinsulinemic-euglycemic clamp technique in streptozotocin (STZ) treated, insulin deficient rats, leptin was able to restore euglycemia, improve post-absorptive glucose metabolism, independently of food intake [112].  Hyperleptinemia induced by adenoviral expression of leptin is also able to normalize uncontrolled diabetes in non-obese diabetic (NOD) mice, as well as in chemically induced (STZ or alloxan treated) models of type 1 diabetes [113].  In this model of STZ induced insulin deficiency, leptin therapy was also able to not only reverse hyperglycemia, but also normalize haemoglobin A1c levels [114]. Taken together, these observations show that leptin has potent effects on glucose homeostasis independent of its anorexic effects, and this is showcased by the ability of leptin to cure diabetes associated with a state of obesity, lipodystrophy, insulin resistance as well as insulin deficiency.  Elucidating the hierarchy of leptin action and the exact mechanisms of how leptin exerts it anti-diabetic effects may ultimately help lead to new ways to diagnose, treat and prevent metabolic diseases associated with leptin resistance, hyperinsulinemia, insulin resistance and obesity.   19  THESIS INVESTIGATION The aim of this thesis was to investigate the role of leptin deficiency in the development of diabetes and obesity in the ob/ob mouse model as well as to delineate the mechanism of action of leptin’s anti-diabetic effects.  In Study 1 we aimed to delineate the acute physiological response to leptin deficiency in an attempt to determine the hierarchy of leptin action by inducing a state of acute leptin deficiency in an otherwise normal animal.  A recently characterized leptin antagonist that retains equal binding affinity to the leptin receptor yet has abolished biological activity and acts as a potent competitive inhibitor was utilized.  We acutely impaired leptin action in vivo so that we could assess the impact it would have on glucose and energy homeostasis. The objective in Study 2 was to try and elucidate the mechanism by which leptin exerts its potent affects on glucose homeostasis.  It has been shown that insulin-like growth factor binding protein-2 (IGFBP-2), a predominantly liver derived protein, is downstream of leptin signalling and is at least partially responsible for the anti-diabetic effects of leptin.  We attempted to elucidate if leptin regulation of IGFBP-2 is due to direct effect of leptin on the liver or centrally mediated via vagal efferents.  Lastly, it is observed that the fasting hyperglycemia in the ob/ob mouse model on the C57BL/6 background is transient.  When the mice age to approximately 16-20 weeks their fasting blood glucose levels are comparable to wild-type controls. In Study 3 we attempted to characterize the transient hyperglycemia and delineate the changes that occur leading to the improvement of fasting blood glucose levels despite the severe obesity that persists.  20   This research highlights the role of dysregulated insulin secretion in the development of obesity and diabetes, and may present an important step in our understanding of the progression of these two metabolic disease states.  21  METHODS AND MATERIALS Animals Male C57BL/6 mice (stock no. 000664), ob/ob (stock no.000632) mice, and C57BL/6- Tg(Alb-cre)21Mgn (referred to as Albcre tg+) (stock no. 003574) were obtained from The Jackson Laboratory (Bar Harbor, ME).  Mice were housed with a 12h light-12h dark cycle and had ad libitum access to water and a standard chow diet (#5015 Lab Diet, St Louis, MO).  All procedures with animals were approved by the University of British Columbia Animal Care Committee and carried out in accordance with the Canadian Council on Animal Care guidelines.  Lerpflox/flox Albcre ob/ob Mouse Line C57BL/6 Albcre tg+ mice were mated with Leprflox/flox  on a 129 X FVB N6 background, a kind gift from Dr. Streamson C. Chua (Albert Einstein College of Medicine) to generate Leprflox/wt mice either with or without the Albcre transgene.  These offspring (≈50% C57BL/6, 50% FVB, and less than 1% 129) were further bred to generate Leprflox/flox Albre+ and Lerpflox/flox Albcre- mice.  To cross these mice onto an ob/ob background, Leprflox/flox mice either with or without the Albcre transgene were mated with ob/+ to generate Leprflox/+ Albcre- ob/+ and Leprflox/+ Albcre+ ob/+ mice.  These mice were then mated to produce offspring that were Leprflox/flox Albcre tg+ ob/+ and Leprflox/flox Albcre tg- ob/+ which were further mated to produce Leprflox/flox Albcre tg+ ob/ob mice and Leprflox/flox Albcre tg- ob/ob littermate controls for experiments. For all experiments littermate Leprflox/flox Albcre tg- ob/ob controls were compared with Leprflox/flox Albcre tg+  22  ob/ob mice in order to minimize differences in genetic background, which can affect the phenotype of leptin deficiency [115].  A-ZIP/F Mouse Line The A-ZIP/F transgenic mice express a dominant negative transcriptional regulator in an adipocyte specific manner, driven by 7.6 kb of the Fabp4 promoter [88].  The A-ZIP/F repressor heterodimerizes and inactivates members of the C/EBP and JUN families of B- ZIP transcriptional regulators [88].  A-ZIP/F are hemizigous animals that were raised by crossing hemizygous FVB/N A-ZIP males with wild type C57BL/6 females and were kindly provided by Dr. Fabio Rossi (University of British Columbia).  The resulting heterozygous offspring have virtually no white adipose tissue and reduced amounts of brown adipose tissue [88].  Although at birth their metabolic parameters are similar to those of wild type littermates, higher levels of insulin, glucose, free fatty acids and triglycerides are found in blood at week 4-5 resulting in a diabetic phenotype [88]. A-ZIP/F-1 mice are sensitive to cold temperature and display viscera enlargement, in particular liver hypertrophy due to fat accumulation (steatosis) [88].  All experiments were performed in the animal facility of the Biomedical Research Centre at the University of British Columbia, Canada, in conjunction with Dr. Dario Lamos (University of British Columbia).    23  Energy Balance Physiology and Body Composition Measurements We recorded energy balance and home-cage activity using an automated indirect calorimetry system (Figure 4) (LabMaster Cages, TSE-Systems, Bad Homburg, Germany).  The metabolic cages were designed to mimic a home cage environment and it was necessary to singly house the mice to obtain accurate measurements of the respiratory quotient (RQ), energy expenditure (EE), activity levels and food intake.  The metabolic cages were located at the Child and Family Research Institute (Vancouver B.C, Canada) animal facility, and their use was kindly provided by Dr. Gibson (University of British Columbia).  Mice were allowed to acclimatize for 72 hours to the metabolic cages prior to start of experiment and metabolic parameter measurements.  Following acclimatization, mice were implanted with mini-osmotic pumps (Alzet, Cupertino, CA) and then food intake, energy expenditure (EE), respiratory quotient (RQ), and activity were measured every 15 minutes for a total of 72 hours.  Food intake Food intake in Study 1 was monitored through weight sensors directly associated with food baskets.  Mice were acclimatized to the metabolic cages as well as being singly housed for 3 days prior to start of experiment.  The weight of the food in each metabolic cage was recorded automatically every fifteen minutes during the duration of the experiment.  Food intake was inferred from the recorded change in weight.  Food intake in Study 2 Mice were acclimatized to the condition of being singly housed for 3 days prior to food intake measurements.  Food in hopper and crumbs in the cage were weighed  24  every day for 4 days manually, and then the average food intake over the 4 days was calculated for each mouse before and during leptin treatment.            Figure 4: LabMaster metabolic cages, an automated indirect calorimetry system. Eight metabolic cages were housed in a temperature control unit where the ambient room temperature was maintained between 24ºC - 25ºC. O2 consumption and CO2 production for the indirect calorimetry measurement of RQ and EE were measured via an open circuit indirect calorimetry system with sensors sampling air from each cage every 15 minutes.  Indirect calorimetry O2 consumption and CO2 production were measured via an open-circuit indirect calorimetry system, with sensors sampling air from each cage once every fifteen minutes. RQ, an indicator of fuel metabolism, was calculated from the ratio of VCO2 (ml/hr) produced to VO2 (ml/hr) consumed [116]  .  EE per kilogram (kg) of lean body mass (kcal/kg/hr) was calculated from the equation: VO2 * [3.815 + 1.232 * RQ] * 0.001 / lean body mass (kg) [116].  25  Activity levels Infrared beams along the x, y, and z axis of metabolic cages collected data on activity levels. Activity was assayed by automatic recording of infrared beam breakage by animals traveling within their cages.  Repeated breakage of the same beam was defined as fine movement.  Consecutive breaking of adjacent beams was defined as locomotor activity.  Absolute number of beam breaks was recorded automatically [117].  Body composition Total body composition in Figure 6 was measured in live conscious animals using QMR technology, which distinguishes differential proton states between lipids, lean tissues, and free water (EchoMRI-100 Echo Medical Systems, Houston, TX) [118].  Conscious animals were placed individually in the QMR system for approximately one minute while the machine collected body composition data.  All other data on total body composition were measured in live conscious animals with a Bruker Biospec 70/30 7 Tesla MRI scanner (Bruker Biospin, Ettlingen, Germany).  NMR signal from the body was acquired with a quadrature volume RF coil tuned to 300 MHz.  The “free” water component corresponding to body fluids was typically less than 5% of the total signal.  The ratio of lean/fat tissue expressed as weight/weight was calculated from the NMR data as described in [119].    26  Body Temperature Analysis and Cold Tolerance Isoflurane-anesthetized mice were implanted subcutaneously with sterile temperature transponders (IPTT-300; Bio Medic Data Systems, Seaford, DE) through a ~ 3 mm incision in the intrascapular region. Transponders were implanted longitudinally in the subcutaneous space, parallel but to one side of the spine so as to not interfere with the animal’s movement.  Incisions were closed with sutures before the animals were allowed to recover from the anaesthesia.  Basal body temperature was assessed using a hand-held Pocket Scanner (DAS-5007; Bio Medic Data Systems, Seaford, DE) to read the temperature transponders in mice.  Temperature was recorded in the morning and evening on for the duration of the experiment.  For the cold tolerance testing, mice were singly housed in cages without bedding for the duration of the experiment.  Mice were housed at 4°C and body temperature was measured every 15 minutes for 1 hour.  Implantation of Mini-Osmotic Pumps and Delivery of Peptides in vivo PEGylated mouse leptin antagonist (PEG-MLA) (mutant L39A/D40A/F41A) was from PLR Laboratories (Rehovolt, Israel) and was reconstituted with sterile dH20.  Mouse recombinant leptin (mrLeptin) was from the National Hormone and Peptide Program (Torrance, CA) and was reconstituted with sterile phosphate buffered saline (PBS). Recombinant Mouse IGFBP-2 was from R&D Systems Inc. (Minneapolis, MN) and was reconstituted with sterile PBS.  Mini-osmotic pumps (Alzet, Cuperino CA), which provide a continous and controlled method of compund delivery by osmotic displacement [120] were utilized to deliver the various peptides. The mini-osmotic pumps consist of three concentric layers, an inner reservoir, an osmotic layer, and an outer rate controlling  27  semipermeable membrane.  The reservoir within the core of the pump is filled with the peptide solution, and when the pump is implated, water is absorbed through the outer membrane expanding the osmotic layer.  This expansion compresses the flexible impermeable reservoir, enabling the peptide solution to be released trhought the exit port at a controlled and predetermined rate [120]. The mini-osmotic pumps were loaded with peptide (3 mg/mL PEG-MLA, 0.125 mg/mL IGFBP-2, or various concentrations of rmLeptin to deliver the desired dose) or dH2O or PBS (control) and pre-equilibrated overnight at 37°C.  The following day the pumps were implanted subcutaneously in isoflurane-anesthetized C57Bl/6 mice following a 4 hour fast.  The wound was closed with sutures and the animal was allowed to recover on a warm heating pad.  Plasma Analyte Analysis Body weight, blood glucose, insulin, glucagon, leptin, IGFBP-2 and plasma lipids were typically measured following a 4 hour fast unless specified otherwise.  Blood glucose concentration was measured with a One Touch Ultra Glucometer (Life Scan Inc., Burnaby, Canada) from the saphenous vein.  Blood samples were collected via heparinized 70 μl microcapillary tubes and then expelled into microcentrifuge tubes and spun at 7000 rpm for 9 minutes at 4°C.  Plasma was stored at -20°C until assay time. Plasma insulin levels were measured by an Ultrasensitive Mouse Insulin ELISA (ALPCO, Salem, USA), glucagon by a Glucagon RIA Kit (Millipore Corporation, Billerica, USA), leptin levels were measured using a Mouse Leptin ELISA (Crystal Chem Inc, Downers Grove, USA), total GLP-1 levels were measured using the Meso Scale Kit (Meso Scale Diagnostics, LLC, Gaithersburg, MD) circulating IGFBP-2 levels  28  were measured by a Mouse/Rat IGFBP-2 ELISA (ALPCO, Salem, USA), plasma triglycerides by Serum Triglyceride Kit (Sigma-Aldrich, St. Louis, MO USA), and cholesterol by Cholesterol E Kit (Wako Chemical, Richmond VA).  Non-esterified fatty acids (free fatty acids, FFA) were measured by a NEFA HR2 Kit (Wako Diagnostics, Richmond VA).  PCR and RT-PCR Analysis Liver tissue was collected from mice and genomic DNA was extracted using DNeasy kits (Qiagen, Mississauga, Canada).  For the leprflox PCR reactions, the forward primer was ATG CTA TCG ACA AGC AGC AGA ATG ACG and the reverse primer was CAG GCT TGA GAA CAT GAA CAC AAC AAC.  The PCR products were analyzed on a 0.8% agarose gel and visualized with SYPBR® Safe (Invitrogen, Burlington, Canada). For the RNA extraction, liver tissue was immediately placed in RNAlater (Qiagen, Mississauga, Canada) and stored at -80°C.  Tissue was homogenized with an ultra turrax and purified using an RNeasy kit (Qiagen, Mississauga, Canada).  Reverse transcript reactions were performed with a poly T primer using a Superscript First-Strand Synthesis kit (Invitrogen, Burlington, Canada).  The generated cDNA was then used for PCR. Primer sequences for the PCR reaction were; forward primer sequence was TAT TCC CAT CGA GAA ATA TCA and the reverse primer sequence was AGG CTC CAA AAG AAG AGG ACC.  The PCR products were analyzed on a 2% agarose gel and visualized with SYBR® Safe (Invitrogen, Burlington, Canada).   29  Subdiaphragmatic Vagotomy Subdiaphragmatic vagotomies or sham surgeries were performed by Jackson Laboratory (Bar Harbor, ME) in 4-week-old ob/ob (stock no.000638) mice using the ventral abdominal approach.  Isoflurane-anesthetized mice were placed in dorsal recumbency and a 2.0 cm skin incision was made immediately caudal to the xiphoid process.  The underlying muscle was incised exposing the stomach.  A moistened gauze sponge was then used to position and hold the liver cranially as the stomach was retracted caudally. Adjacent to the esophagus a section of both the dorsal and ventral vagal trunks were excised cranial to the stomach.  In sham operated mice the vagus was exposed 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.  Verification of Vagotomy CCK experiment Six weeks post surgery and 3 weeks post leptin administration, the completeness of vagotomy was assessed.  The test was based on the satiety effect of cholecystokinin (CCK) which is known to be mediated by the vagus nerve [121].  Mice were fasted from 8 am to 8 pm and then injected i.p. with CCK octapeptide (26-33) (American Peptide, CA) or saline.  Ten minutes post injection, mice were individually placed into a cage that contained a pre-weighed amount of food (~25 g) and were allowed to feed in the dark for 30 minutes.  Mice were removed from the cage following 30 minutes of feeding and the remaining food and crumbs were weighed and subtracted from the initial weight of food placed in the cage.  30  Gastric distension At the end of the experiment, following an overnight fast (16 hours), mice were sacrificed and a digital image of the stomach was captured immediately following the tissue harvest to showcase the gastric distension present in the ob/ob mice that received a subdiaphragmatic vagotomy.  The wet weight of the stomach was then weighed using an analytical scale, after removing residual gastric contents [122].  GLP-1 Bioassay HEK-hGLP1R-Luc cells were derived from HEK293 cells, a transformed line derived from human embryonic kidney. HEK293 cells were first stably transfected with a construct expressing the human GLP-1 receptor under the control of the CMV promoter [123] resulting in the generation of HEK-hGLP-1R cells which were generously provided to us by Dr. Jesper Gromada (Novo Nordisk A/S, Denmark).  These cells were then transfected with a second construct, one that contained a cAMP responsive element driving luciferase expression (pHTS-CRE; Biomyx) by Corinna Lee (University of British Columbia). The resulting HEK-hGLP1R-Luc cell line was utilized as a bioassay (Figure 5).  These HEK293-hGLP-1R-Luc cells were plated in 96 well flat bottom white polystyrene plates (BD Falcon, Mississauga, ON) at a density of 7 x 104 cells/well and incubated overnight at 37°C in a 5% CO2 atmosphere. The next day, the medium was aspirated, cells were washed with PBS (Invitrogen, Burlington, ON), and 100 μl of sample (plasma diluted 1/3 in KRB or GLP-1 standards in KRB) was added.  Following a 5 hour incubation at 37°C in a 5% CO2 atmosphere, the samples were removed and wells washed with PBS (Invitrogen, Burlington, ON).  A luciferase assay was then performed  31  using the Bright-Glo luciferase assay kit (Promega, Madison, MI) according to the manufacturer’s instructions.  Luminescence was measured using a Tecan Infinite M1000 luminometer (Tecan, Männedorf, Switzerland).  GLP-1 levels in the samples were then calculated by extrapolating from the standard curve.    Figure 5: A cell based luciferase reporter GLP-1 bioassay signalling pathway. Summary of the key intracellular steps that ultimately result in the production of luciferase in the HEK293-hGLP-1R-Luc cell line. The HEK293-hGLP-1R-Luc cell line stably expresses a cAMP responsive element (CRE) luciferase reporter gene as well as the GLP-1 receptor. Stimulation of the G-protein coupled receptor (GPCR) activates adenylyl cyclase, resulting in the formation of cAMP. Binding of cAMP to the regulatory (R) subunit of protein kinase A (PKA) results in the release of the active catalytic (C) subunit. The active kinase then translocates to the nucleus and phosphorylates, and therefore activates, the nuclear transcriptional activator CREB promoting binding to the CRE located in the reporter gene. Addition of the substrate (luciferin) to cells results in the production of light, which can be quantified in a luminometer. The light output is proportional to the quantity of GLP-1 added. CREB = cAMP response element-binding.  Figure generated by Catherine Merchant (University of British Columbia).    32  PEG-MLA Action in vitro  CHO-Ob-Rb cells CHO-Ob-Rb cells (generously provided by Dr. Takashi Murakami; University of Tokushima), are a chinese hamster ovary (CHO) cell line stably transfected with the gene for the long form of the mouse leptin receptor (Ob-Rb).    CHO-Ob-Rb cells were cultured in Ham’s F-12 media (GIBCO; Grand Island, New York) (10% fetal bovine serum, 1X penicillin/streptomycin) and were seeded in 6-well plates at a density of 3.5 x 105 cells per well for the bioassay.  Stimulation of CHO-Ob-Rb cells with PEG-MLA and leptin One day post seeding under conditions stated above, cells were washed with PBS and then stimulated with PEG-MLA (250 ng/mL) or leptin (2.5, 25 or 250 ng/mL) for 10 minutes.  The cells were then washed with ice cold PBS (2 x 2 mL) and lysed with 0.25 mL ice cold lysis buffer (1% w/v Triton X-100 (VWR International), 20 mM Tris HCl pH 7.5 (Sigma-Aldrich; St. Louis, MO), 150 mM NaCl (Fisher Scientific; Ottawa, ON), 5 mM EDTA (Fisher Scientific; Ottawa, ON).  Immediately prior to use, 1X protease inhibitor cocktail (Sigma-Aldrich; St. Louis, MO) was added to the lysis buffer.  Cells were detached, by cell scraper, and transferred on ice to 1.5 mL RNase- and DNase- containing microcentrifuge tubes to achieve a final concentration of 50 μg/ml for RNase and DNase (Sigma-Aldrich; St.Louis, MO), which were incubated on a rotating platform for 1 hour and spun (10,000 x g) for 10 minutes at 4°C.  The supernatant was transferred to fresh microtubes after centrifugation.  Protein concentration was measured in a 96-well plate by BCATM (bicinchoninic acid) Protein Assay according to manufacturer’s Standard  33  Microplate protocol (Thermo Scientific; Rockford, IL).  To obtain absorbance readings at 562 nm, the Infinite® M1000 plate reader was used to scan the plate (TECAN; Switzerland).  Western blot analysis of phospho-STAT3 in simulated CHO-Ob-Rb cells Four times sample buffer, (30% glycerin (Fisher Scientific; Ottawa ON), 25 mM Tris- HCl pH 6.8, 8% SDS, 0.02% Bromophenol Blue (Sigma-Aldrich; St. Louis, MO)) was added to 8 μg protein to achieve 1X concentration of sample buffer.  The sample was heated at 85°C for 10 minutes and then centrifuged (15,000 x g) for 1 minute. The supernatant was loaded (25 μl) on a 8% polyacrylamide gel, which was run by electrophoresis, in 1X running buffer (25 mM Tris base (Fisher Scientific; Ottawa ON), 192 mM glycine (EMD Chemicals; Gibbstown, NJ)), at 45 V initially until the dye front reached the top of the separating gel, at which point the voltage was increased to 105 V until the dye front reached ~0.5 cm from the bottom of the gel.  When electrophoresis was completed, the gel was equilibrated in transfer buffer (20 mM Tris base, 150 mM glycine (EMD) Chemicals; Gibbstown, NJ), 20% methanol (Fisher Scientific; Ottawa, ON), 0.038% SDS) for 15 minutes at the end of which the transfer apparatus was assembled in the following order: black plate, foam pad, 2 pieces of whatman paper, gel, PVDF (polyvinylidene fluoride) transfer membrane (Bio-Rad; Hercules, CA), 2 pieces of whatman paper (Bio-Rad, Hercules, CA), foam pad, clear plate.  The transfer was done in ice cold transfer buffer at 100 V for 60 minutes.  When the transfer was finished, the membrane was incubated in blocking solution (5% BSA in TBST (1X TBS, 0.1% v/v Tween20 (VWR International), milli-Q water)) 30 minutes at room temperature.  Two quick and then 3 x 5 minute TBST washes with rocking were done after the membrane  34  was removed from the blocking solution.  The membrane was subsequently incubated with 12 ml 1:1000 rabbit α-pSTAT3 primary antibody (Cell Signalling Technology; Danvers, MA) diluted in 1X TBST with 5% (w/v) BSA. Then, the membrane was subjected to 2 x quick, 1 x 5 minute, 1x 8 minute, and 1 x 10 minute washes in TBST with rocking.  After the washes, the membrane was incubated with 12 mL 1:8000 donkey-α-rabbit HRP secondary antibody (GE Healthcare; Piscataway, NJ) diluted in 1X TBST with 5% (w/v) BSA. Following secondary antibody incubation, the membrane was washed 3 x 10 minutes in TBST with rocking and the ECLTM Western Blotting Detection Reagents (GE Healthcare; Piscataway, NJ) were added for 1 minute.  The membrane was then wrapped in saran wrap and set inside a film cassette for film exposure.  The film exposure time was 1 minute for pSTAT3.  For quantifying band density, films were analyzed using densitometry software (Eagle Eye; Stratagene).  Glucose and Insulin Tolerance Test Mice were fasted for 4 hours and glucose tolerance tests were performed by oral gavage of glucose (1.5 mg of glucose per g body weight of a 30% solution), or an intraperitoneal (IP) injection of 0.75 U/kg body weight of human synthetic insulin (Novolin ge, Novo Nordisk, Canada).  Arginine Stimulated Insulin, Glucagon and Total GLP-1 Secretion Following a 4 hour fast, mice were injected i.p. with 1.5 mg/kg L-Arginine Hydrochloride (Sigma-Aldrich, St Louis, MO).  Blood was sampled from the saphenous  35  vein for blood glucose measurements and also so that plasma could be obtained and measured for insulin and glucagon levels.  Hyperinsulinemic-Euglycemic Clamps Hyperinsulinemic-euglycemic clamps were performed as previously described [124]. Specifically, mice were fasted overnight for 16 hours and anaesthetized with acepromazine (5 mg/kg), midazolam (5 mg/kg) and fentanyl (0.25 mg/kg), initial dose by i.p. injection, top up doses every 40 minutes by subcutaneous injection.  Once fully immobilized from anaesthesia, the tail vein was cannulated and a 1 hour basal infusion of 3H-D-glucose (1.2 μCi/hr) was started to determine steady state tracer.  Duplicate blood samples (50 μl) were taken from the tail vein at the end of the basal period to measure fasting blood glucose levels and to obtain plasma for basal insulin measurements. Hyperinsulinemia was induced with a constant infusion of insulin (Novolin ge, Novo Nordisk, Canada) (6.8 mU/hour).  To maintain euglycemia (approximately 5.0 mM) a variable infusion of 12.5% D-glucose was started simultaneously.  Once euglycemia had been achieved, steady state was maintained for 45 minutes after which triplicate blood samples (50 μl) were taken from the tail vein for further analysis.  Animals were sacrificed by cervical dislocation and tissues were dissected and frozen in liquid nitrogen. Briefly, plasma insulin concentrations were measured as described above and plasma samples were counted using a Beckman LS6000IC scintillation counter after extraction by TCA precipitation.  Whole body glucose utilization (μmol/kg·min) was determined as the ratio of the specific activity of glucose to the rate of 3H-D-glucose appearance.  36  Subsequently, endogenous glucose production (μmol/kg·minute) could be calculated as the difference between whole body glucose uptake and exogenous glucose infusion.  Measurement of Hepatic Triglycerides and Cholesterol Triglycerides and cholesterol were measured by a modified protocol of [125].  Briefly, liver (~ 100 mg) was homogenized in 3 mL of chloroform:methanol (2:1) and extracted twice with water.  Five hundred μl of the organic layer was dried down under N2(g) and 10 μl of Thesit (Sigma-Aldrich, St Louis, MO) was added and mixed under N2(g).  Water (100 μl) was added and incubated at 37°C for 30 minutes with intermittent vortexing. Aliquots of 5 μl were assayed using the Serum Triglyceride Determination kit (Sigma- Aldrich, St Louis, MO) modified for a 96-well plate, calibrated with a trioleate (Sigma- Aldrich, St Louis, MO) standard curve.  The cholesterol assay was performed with a 20 μl aliquot using the Cholesterol E kit and standard (Wako Chemicals, Richmond, VA).  Statistical Analysis Data analysis was carried out using Sigma Plot 10.01 (Systat Software, Inc. San Jose, USA).  Data are represented as mean ± SEM and were analyzed using a one-tailed Student’s t-test or an ANOVA where appropriate.  Statistical significance was defined as P values < 0.05.  37  RESULTS  STUDY 1: Acute Disruption of Leptin Signalling in vivo To acutely disrupt leptin signalling action in vivo and create a state of leptin deficiency, we used a mutant leptin protein that contains three amino acid substitutions (L39A/D40A/F41A) relative to the wild-type leptin sequence.  This mutant leptin protein retains equal binding affinity to the Ob-Rb compared to a non-mutant form of leptin [126], but its biological activity is abolished, as it is not able to initiate the leptin signalling cascade [126].  Although a potent antagonist, in a previously published study, the short half-life of the antagonist necessitated administration of superphysiological doses in vivo to produce a clinical response and did not suffice to induce a true metabolic state of leptin deficiency [127].   To address this issue, the mouse leptin antagonist was mono-pegylated (contains an attachment of a 20 kDa polyethylene glycol (PEG) molecule) at the N-terminus [128], and will be referred to as PEGylated mouse leptin antagonist (PEG-MLA).  The single PEGylation increased the protein’s size to more than 70 kDa due to the enlarged hydrodramatic volume, resulting in an increased half-life of PEG-MLA (half-life in circulation after SC injection was over 20 hours) by reducing renal clearance [128].  Collectively, the efficacy of the PEG-MLA is attributed to better stability, greater protection against proteolytic degradation, and longer in vivo circulation and bioavailability due to decreased clearance by the kidneys [128].  Administration of this PEG-MLA to normal mice has been shown to induce a dramatic increase in food intake and weight gain by inducing a severe central and peripheral leptin deficiency [128].  38  PEG-MLA has no agonistic activity in vitro Leptin has been shown to exert its biological activity by binding to Ob-Rb leading to the activation of STAT-3 by inducing its phosphorylation.  To verify that the PEG- MLA compound did not display partial agonistic character, the ability of PEG-MLA or leptin to induce STAT-3 phophorylation was compared utilizing a cell line bio-assay.  A Chinese Hamster Ovary (CHO) cell line expressing the Ob-Rb was utilized.  The functionality of the Ob-Rb receptor isoform expressed in the CHO cell line utilized was previously evaluated by the measurement of STAT-3 phoshorylation in response to leptin [129].  CHO-Ob-Rb cells were stimulated with recombinant mouse leptin (0, 2.5, 25, and 250 ng/mL) or PEG-MLA (250 ng/mL) for 10 minutes and cell lysates were then analyzed by western blot using an anti-phospho-STAT3 antibody.  In leptin treated cells, phoshorylation of STAT-3 was increased in a dose dependent manner as expected (Figure 6) while treatment with PEG-MLA did not significantly induce phospho-STAT-3 compared to levels found in non-induced cells.  PEG-MLA action in vivo was examined next to characterize the physiological effects of the leptin antagonist in mice.  39   Figure 6: PEG-MLA does not activate Ob-Rb and the leptin signalling pathway in stably transfected CHO-ObRb cells.  CHO-ObRb cells were stimulated (10 minutes) by recombinant murine leptin (0, 2.5, 25, and 250 ng/mL) or 250 ng/mL PEG-MLA. Phosphorylated STAT3 (pSTAT3), which is a product of leptin signalling upon action of Ob-Rb by leptin, in the cell lysate was determined by western blot.  Primary antibody rabbit α-pSTAT3 (1:1000 dilution), secondary antibody donkey-anti-rabbit HRP (1:8000 dilution). Film exposure time was 1 minute.  Data represent the mean result of two independent experiments and are expressed as mean ± SD.   PEG-MLA treated mice have increased food intake and respiratory quotient compared to controls   As leptin has well defined effects on food intake and energy expenditure we first investigated the effect of the PEG-MLA on energy metabolism utilizing indirect calorimetric techniques.  Following a 72 hour acclimatization to the metabolic cages, mice were implanted subcutaneously with mini-osmotic pumps delivering 72 µg/day S TA T- 3 P ho sp or yl at io n (A rb itr ar y U ni ts ) 0 20 40 60 80 100 120 Leptin (ng/mL)              0       2.5     25     250      0 PEG-MLA (ng/mL)        0         0       0        0      250  40  PEG-MLA.  The mice were then returned to the metabolic cages and monitored for the 3 day duration of the pump.  Acute disruption of leptin signalling was associated with Time (hours) 0 10 20 30 40 50 60 Fo od  In ta ke  (g ) 0 2 4 6 8 10 Control PEG-MLA C A B W ei gh t G ai n (g ) 0.0 0.5 1.0 1.5 2.0 P=0.041 PEG-MLA ControlP   0.05< < Time (hours) 0 10 20 30 40 50 60 R es pi ra to ry  Q uo tie nt  0.70 0.75 0.80 0.85 0.90 0.95 1.00 P   0.05 D Time (hours) 0 10 20 30 40 50 60 En er gy  E xp en di tu re  (k ca l/k g/ hr ) 5 10 15 20 25 Control PEG-MLA Control PEG-MLA  Figure 7: Increased food intake and respiratory quotient in mice receiving PEG-MLA.  (A) Food intake (B) weight gain (C) respiratory quotient and (D) energy expenditure were recorded in 7-week-old male C57Bl/6 mice during 3 day (72 µg/day) delivery of the PEG- MLA via mini-osmotic pumps.  Subcutaneous implantation of pumps and start of delivery of PEG-MLA occurred 2 hours prior to start of recording of above measurements.  Shaded bars represent data collected during the dark phase.  Data are expressed as mean ± SEM, n = 4 *P < 0.05 compared to controls.        41  increased cumulative food intake (Figure 7A) which was evident on the second day of PEG-MLA treatment and continued to increase until the end of treatment.  Overall, the increase in food intake led to an increase in weight gain in the PEG-MLA treated mice compared to controls (1.3 ± 0.3 g vs. 0.8 ± 0.1 g P=0.041, respectively) (Figure 7B), as well as a significant increase in the respiratory quotient (RQ) on day 2 and 3 of PEG- MLA treatment (Figure 7C).  The augmented RQ is likely due to the increase in food intake and indicates increased utilization of glucose and protein for energy production over lipid oxidation.  Surprisingly, the energy expenditure of the PEG-MLA treated mice was comparable to that of controls (Figure 7D), even once normalized to lean body mass (data not shown).  It is possible that a decrease in energy expenditure was balanced out by increased glucose oxidation as exhibited by the increased RQ, leading to no change.  Decreased spontaneous activity in PEG-MLA treated mice  In parallel to the indirect calorimetry measurements described above, locomotor and ambulatory activity was monitored using infrared beam break systems (Figure 8A). PEG-MLA treated mice trended towards displaying decreased total locomotive activity during the last 24 hour of treatment, which was evident once cumulative beam breaks were observed in the last 12 hour light and 12 hour dark phase of PEG-MLA treatment but did not reach significance (cumulative beam breaks last 12 hour light phase of treatment P=0.130 cumulative beam breaks last 12 hour dark phase of treatment P=0.075) (Figure 8B).  Total locomotive activity was further dissected into ambulatory movement (Figure 8C).  The same trend of decreased activity was observed during the last 12 hour light and 12 hour dark phase of the PEG-MLA administration (cumulative beam breaks  42  last 12 hour light phase of treatment P=0.100, cumulative beam breaks last 12 hour dark phase of treatment P=0.062) (Figure 8D). A C D B To ta l L oc om ot or  A ct iv ity (B ea m  B re ak s/ ph as e) P=0.130 P=0.075 PEG-MLA Control 0 10000 20000 30000 40000 50000 60000 A m bu la to ry  M ov em en t (B ea m  B re ak s/ ph as e) P=0.100 P=0.062 PEG-MLA Control 0 10000 20000 30000 40000 Time (hours) 0 10 20 30 40 50 60 A m bu la to ry  M ov em en t (b ea m  b re ak s/ hr s) 0 2000 4000 6000 Time (hours) 0 10 20 30 40 50 60 To ta l L oc om ot iv e A ct iv ity (B ea m  B re ak s/ hr ) 0 2000 4000 6000 8000 10000 12000 Control PEG-MLA Control PEG-MLA Figure 8: Effects of PEG-MLA on spontaneous physical activity. Using an infrared beam break system, home cage-activity was monitored in 7-week-old male C57Bl/6 mice receiving 72 µg/day of PEG-MLA.  (A) Total locomotive activity during treatment and (B) cumulative total locomotive activity over the last 12 hour light/12 hour dark period measurements from the respective tracings. Total activity was further dissected into (C) ambulatory movement and (D) cumulative ambulatory movement over the 12 hour light/12 hour dark period measurements.  PEG-MLA treated mice in general appeared to be less active.  Subcutaneous implantation of pumps and start of delivery of PEG-MLA occurred 2 hours prior to start of recording of above measurements. Shaded bars present data collected during the dark phase. Data are expressed as mean ± SEM, n = 4.    43  Core body temperature and cold tolerance in PEG-MLA treated mice  Energy can also be expended by performing work or producing heat (thermogenesis).  Adaptive thermogenesis is an important component of energy expenditure and mice with a genetic defect in leptin or leptin receptor signalling (ob/ob and db/db mice respectively) have a lower core body temperature as well as cold intolerance [130] compared to wild-type mice.  To assess if PEG-MLA treatment resulted in changes in body temperature regulation we implanted subcutaneous temperature sensors in mice.  At the same time, osmotic pumps were implanted on the opposite side, delivering 72 µg/day of PEG-MLA or vehicle for 3 days.  Age-matched ob/ob mice were implanted with temperature sensors only so that we could compare the effects of an acute vs. congenital leptin deficiency on temperature regulation. B od y Te m pe ra tu re  (o C ) 25 30 35 40 45 * * * Control PEG-MLA ob/ob 1 2 3 Time (days) A B Time (min) 0 10 20 30 40 50 60 B od y Te m pe ra tu re  (o C ) 32 34 36 38 40 Control PEG-MLA ob/ob ** * * Figure 9: Basal body temperature and cold tolerance in mice receiving PEG-MLA. 7-week-old C57BL/6 mice were administered 72 µg/day for 3 days via mini osmotic pumps (A) Basal body temperature was measured twice a day (8 am and 6 pm) in mice housed individually at room temperature after 1, 2 and 3 days of PEG-MLA treatment.  As there was no difference between PEG-MLA treated mice and controls in body temperature in the AM and PM recordings, the 2 readings per day were averaged for each mouse. (B) Mice housed individually for 1 hour at 4°C on third day of PEG-MLA administration at 6 PM.  Data are expressed as mean ± SEM, n ≥ 6, *P < 0.05 compared to ob/ob mice.  44  Basal body temperature was measured every day (8 am and 6 pm) during the 3 days of PEG-MLA or vehicle treatment.  Overall, there was no difference in basal body temperature between PEG-MLA treated mice compared to controls (when measured in the morning or evening), (Figure 9A) while ob/ob mice had a significantly lower basal body temperature (Figure 9A).  After 3 days of PEG-MLA treatment (72 µg/day), the mice were exposed to an acute cold tolerance test at the start of their dark phase.  PEG- MLA treatment did not affect the ability of mice to maintain their body temperature in a 4°C setting (Figure 9B) in contrast to the ob/ob mice which displayed cold intolerance.  Characterizing the effect of PEG-MLA on glucose homeostasis in wild-type mice Next we examined if the changes in energy balance were accompanied by changes in glucose homeostasis.  Three day mini-osmotic pumps delivering 72 µg/day PEG-MLA or vehicle (H2O) were implanted subcutaneously following a 4 hour fast. During the 3 day administration of PEG-MLA (72 µg/day) body weight and fasting blood glucose remained comparable to vehicle treated controls (Figure 10A).  Although PEG- MLA treatment caused the mice to gain more weight, the weight gain did not lead to a significant difference in weight in PEG-MLA treated mice compared to controls (P=0.100).  Since body weight did not differ significantly between treatment groups, we further investigated body composition to determine if the lean:lipid mass ratio was altered.  The lean:lipid ratio was not different before or after treatment between the PEG- MLA and vehicle (P=0.390 vs. P=0.245 respectively) (Figure 10B), nor was there a significant difference when comparing values before and after treatment within the  45  groups.  Leptin has a known role as a negative regulator of insulin secretion from the pancreatic ß-cells Time (days) -6 -4 -2 0 2 4 6 B od y W ei gh t ( g) 12 14 16 18 20 22 24 Control PEG-MLA 72 ug/day Time (days) -6 -4 -2 0 2 4 6 B lo od  G lu co se  (m M ) 4 6 8 10 12 Control PEG-MLA * Time (days) -6 -4 -2 0 2 4 6 Fa st in g In su lin  (n g/ m L) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Control PEG-MLA * A B C D Le an :L ip id  M as s 0 2 4 6 8 10 12 Post-TreatmentPre-Treatment P=0.245 PEG-MLA Control P=0.390 PUMP PUMP PUMP     Figure 10: Metabolic parameters in C57Bl/6 mice receiving 72 µg/day PEG-MLA. Continuous administration of 72 µg/day of PEG-MLA for 3 days. (A) Body weight following a 4 hour fast (B) total body lean:lipid ratio 1 day before treatment and on day 3 of treatment immediately following pump removal (C) blood glucose levels following a  4 hour fast (D) plasma insulin following a 4 hour fast. Male C57Bl/6 mice were 7-weeks-old when pumps were implanted following a 4 hour fast on day 0 and removed on day 3 following data collection.  Data are expressed as mean ± SEM, n=6, *P < 0.05 compared to controls.   46  so fasting blood glucose and insulin levels were measured next.  Differences in fasting blood glucose levels between PEG-MLA treated and control mice were not detected during the entire treatment period (Figure 10C), although fasting insulin levels were significantly higher on day 3 of PEG-MLA treatment (Figure 10D).  Treated mice maintained normal fasting blood glucose levels throughout the study, although their insulin levels were 26 ± 0.9 % higher compared to controls (0.85 ± 0.09 ng/mL vs. 1.15 ± 0.07 ng/mL respectively, P=0.01) on day 3 of treatment following a 4 hour fast.  The increase in fasting insulin levels due to PEG-MLA treatment was transient as fasting insulin levels returned to normal after the mini-osmotic pumps delivering the PEG-MLA were surgically removed.  To determine if there was a dose dependent effect and if longer administration of a lower dose of PEG-MLA could have the same effects, 7 day mini-osmotic pumps delivering 36 µg/day PEG-MLA were implanted subcutaneously.  Surprisingly, the lower dose administered over a longer period of time led to a modest, although significant difference in body weight of PEG-MLA treated mice compared to controls (24.1 ± 0.28 g vs. 23.2 ± 0.36 g, P =0.04, respectively) on the last day of PEG-MLA treatment (Figure 11A).  Although the PEG-MLA treated mice weighed significantly more, this increase in body weight did not translate into a significant difference in body composition as determined by the lean:lipid ratio (5.8 ± 0.2 vs. 5.0 ± 0.3 respectively, P=0.08) (Figure 11B).  Blood glucose levels also remained unchanged between PEG-MLA treated mice and controls (Figure 11C) but analysis of plasma samples collected at time of body weight and blood glucose measurements revealed significantly increased fasting insulin levels (Figure 11D).  PEG-MLA treated mice had significantly higher fasting insulin  47  Time (days) -5 0 5 10 15 20 B od y W ei gh t ( g) 16 18 20 22 24 26 28 30 Control PEG-MLA * PUMP Time (days) -5 0 5 10 15 20 B lo od  G lu co se  (m M ) 0 2 4 6 8 10 12 14 Control PEG-MLA PUMP Le an :L ip id  R at io 0 2 4 6 8 P=0.08 PEG-MLA Control A B C D Time (days) -5 0 5 10 15 20 Fa st in g In su lin  (n g/ m L) 0.0 0.5 1.0 1.5 2.0 2.5 Control PEG-MLA * * * * PUMP  Figure 11:Metabolic parameters in C57Bl/6 mice receiving  36 µg/day PEG-MLA. Continuous administration of 36 µg/day of PEG-MLA for 7 days. (A) Body weight following a 4 hour fast (B) total body lean:lipid ratio on day 7 of treatment immediately following pump removal (C) blood glucose levels following a 4 hour fast (D) plasma insulin following a 4 hour fast. Male C57Bl/6 mice were 7-weeks-old when pumps were implanted subcutaneously following a 4hr fast on day 0 and removed on day 7 following data collection.  Data are expressed as mean ± SEM, n=6 *P < 0.05 compared to controls.   levels on day 4 and day 7 of treatment, and this increase in insulin levels persisted even 2 days after PEG-MLA treatment was discontinued by surgical removal of pumps.  Insulin levels returned to levels comparable to control (vehicle treated) mice by 6 days post treatment.  To determine if further extending the delivery period of the PEG-MLA and  48  using an even lower dose would still affect body weight and insulin levels 14 day mini- osmotic pumps delivering 18 µg/day PEG-MLA were implanted subcutaneously. Time (days) -5 0 5 10 15 20 25 B od y W ei gh t ( g) 0 10 20 30 Control PEG-MLA PUMP PUMP Time (days) -5 0 5 10 15 20 25 B lo od  G lu co se  (m M ) 0 2 4 6 8 10 12 14 * Control PEG-MLA A B C PUMP Le an :L ip id  R at io 0 2 4 6 8 P=0.20 PEG-MLA Control D Time (days) -5 0 5 10 15 20 Fa st in g In su lin  (n g/ m L) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Control PEG-MLA * PUMP     Figure 12:  Metabolic parameters in C57Bl/6 mice receiving 18 µg/day PEG-MLA. Continuous administration of 18 µg/day of PEG-MLA for 14 days. (A) Body weight following a 4 hour fast (B) total body lean:lipid ratio on day 14 of treatment immediately following pump removal (C) blood glucose following a 4 hour fast (D) plasma insulin following a 4 hour fast. Male C57Bl/6 mice were 7-weeks-old when pumps were implanted following a 4 hour fast on day 0 and removed on day 14 following data collection.  Data are expressed as mean ± SEM, n=6 *P < 0.05 compared to controls.     49  This dose for a duration of 14 days did not affect body weight (Figure 12A), body composition as determined by the lean:lipid ratio (Figure 12B), fasting blood glucose (Figure 12C) or insulin levels (Figure 12D).  Disruption of leptin signalling leads to increases glucose stimulated insulin secretion As the delivery of 72 µg/day of PEG-MLA to wild-type mice using the 3 day osmotic mini-pump was observed to have no effect on energy expenditure or change in body composition but alterations in glucose metabolism were observed in the form of elevated fasting insulin levels, this dose was further used to assess changes in carbohydrate metabolism following a 4 hour fast.  Since fasting insulin levels were significantly increased after 3 days of treatment with 72 µg/day PEG-MLA, we next sought to determine if these mice would have improved glucose tolerance due to their elevated insulin levels.  An OGTT was performed on day 3 of PEG-MLA administration following a 4 hour fast and showed that there was no significant difference in glucose disposal between the PEG-MLA treated mice and controls (Figure 13A).  Blood collected at various time points during the OGTT revealed that although there was no change in glucose disposal, the PEG-MLA mice had significantly higher glucose induced insulin       50  Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 5 10 15 20 25 30 Control PEG-MLA A B C Time (min) 0 10 20 30 40 50 60 In su lin  (n g/ m L) 0.0 0.5 1.0 1.5 2.0 Control PEG-MLA** * Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 0 2 4 6 8 10 12 14 16 18 Control PEG-MLA D In su lin  (n g/ m L) 0 1 2 3 4 5 P=0.471 G lu ca go n (p g/ m L) 0 20 40 60 P=0.356 E  Figure 13: Oral glucose and i.p. arginine tolerance test in PEG-MLA treated mice. 7-week-old C57Bl/6 mice received continuous administration of 72 µg/day of PEG-MLA for 3 days via continuous administration by subcutaneously implanted mini-osmotic pumps. Oral glucose tolerance test and i.p. arginine tolerance test performed on day 3 of PEG-MLA administration following a 4 hour fast.  Each test done in a different cohort of mice.  (A) Oral glucose tolerance (1.5 mg/g) test (B) glucose stimulated insulin secretion (C) i.p. arginine tolerance test (2 mg/g) (D) arginine stimulated insulin secretion and (E) arginine stimulated glucagon secretion at 7 minutes post i.p. arginine injection. Data are expressed as mean ± SEM, n=6, *P < 0.05 compared to controls.     51  release at t=0, 7, and 15 minutes following the glucose gavage compared to controls (1.00 ± 0.1 ng/mL vs. 0.67 ± 0.02 ng/mL, P=0.02 at t=0 minutes; 1.4 ± 0.2 ng/mL vs. 0.97± 0.11 ng/mL, P=0.05 at t=7 minutes; and 1.64 ± 0.13 ng/mL vs. 1.16 ±0.08 ng/mL, P=0.006 at t=15 minutes, respectively) (Figure 13B).  PEG-MLA treatment did not lead to altered arginine tolerance (Figure 13C) and arginine stimulated insulin (Figure 13D) and glucagon (Figure 13E) levels 7 minutes following an i.p. injection of L-arginine were comparable between PEG-MLA treated mice and controls.  PEG-MLA treatment in the fasting state leads to elevated insulin levels We next sought to determine if PEG-MLA treatment also had effects on insulin levels independent of food intake.  Mice were injected i.p. with a single dose of 300 µg of PEG-MLA immediately before the start of their dark phase and a fast was initiated. Following a 16 hour fast glucose homeostasis was assessed.  Blood was collected following the 16 hour fast to determine fasting insulin levels, and then mice were administered an oral glucose gavage to assess glucose tolerance and glucose stimulated insulin secretion.  Insulin levels following the prolonged fast were elevated approximately 2 fold in the PEG-MLA treated mice compared to controls (0.48 ± 0.15 ng/mL vs. 0.21 ± 07 ng/mL respectively, P=0.069) although significance was not reached (Figure 14A) and this was independent of body weight differences between groups (Figure 14B).  Although PEG-MLA resulted in elevated insulin levels, the difference in insulin levels did not lead to improved glucose tolerance (Figure 14C) nor did PEG-MLA treatment in the fasting state alter glucose stimulated insulin secretion (Figure 14D).   52  A B Time (min) 0 10 20 30 40 50 60 In su lin  (n g/ m L) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Control PEG-MLA B od y W ei gh t ( g) 0 10 20 30 P=0.06 PEG-MLA Control Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 5 10 15 20 25 Control PEG-MLA C D * * Fa st in g In su lin  (n g/ m L) 0.0 0.2 0.4 0.6 0.8 1.0 P=0.07 PEG-MLA Control    Figure 14: Effect of PEG-MLA on glucose homeostasis in the fasting state. 7-week-old C57Bl/6 mice were injected i.p. with 300 µg of the PEG-MLA at 5 pm and fasted for 16 hours upon which glucose homeostasis was assessed.  (A) Insulin levels (B) body weight (C) oral glucose tolerance test (1.5 mg/g) and (D) glucose stimulated insulin secretion.  Data are expressed as mean ± SEM, n=6 *P < 0.05 compared to controls.       53  PEG-MLA treatment does not alter fasting glucagon levels As leptin receptors are also found on glucagon producing α-cells and have an inhibitory effect on glucagon secretion in vitro [131], we hypothesized that disruption of leptin signalling in vivo could potentially lead to elevated glucagon levels.  Glucagon levels on day 3 of PEG-MLA (72 µg/day) administration via mini osmotic pumps were measured following a 4 hour fast and no difference between PEG-MLA and control treated mice was observed (32.8 ± 5.1 pg/mL vs. 28.9 ± 2 pg/mL respectively, P=0.25) (Figure 15).  G lu ca go n (p g/ m L) 0 10 20 30 40 50 60 P=0.247 PEG-MLA Control   Figure 15: Fasting glucagon levels in PEG-MLA treated mice. 7-week-old C57Bl/6 mice received continuous administration of 72 µg/day of PEG-MLA for 3 days via continuous administration by subcutaneously implanted mini-osmotic pumps.  Plasma glucagon levels on day 3 of PEG-MLA administration following a 4 hour fast.  Data are expressed as mean ± SEM, n=6.      54  Insulin sensitivity in PEG-MLA treated mice As the observed increase in glucose stimulated insulin secretion following PEG- MLA treatment did not lead to improved glucose tolerance, nor were glucagon levels elevated to counteract the increased insulin levels following a 4 hour fast, whole body insulin sensitivity was investigated next.  Alterations in insulin sensitivity could account for the normal fasting glucose and glucose tolerance despite elevated insulin levels in the fasting and post-prandial state.  To assess whole body insulin sensitivity, an i.p. ITT was performed (Figure 16). A B Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 2 4 6 8 10 12 Control PEG-MLA * * * * Time (min) 0 20 40 60 80 100 120 %  o f F as tin g G lu co se 0 20 40 60 80 100 120 Control PEG-MLA * * *   Figure 16: PEG-MLA treated mice exhibit mild insulin resistance. 7-week-old C57Bl/6 mice received continuous administration of 72 µg/day of PEG-MLA for 3 days via continuous administration by subcutaneously implanted mini-osmotic pumps.  Insulin tolerance test performed on day 3 of PEG-MLA administration following a 4 hour fast. (A) i.p. insulin tolerance test (0.75 U/kg) and (B) blood glucose levels expressed as percent of basal blood glucose levels.  Data are expressed as mean ± SEM, n=6 *P < 0.05 compared to controls.   55  As expected, following a bolus insulin injection (0.75 U/kg), insulin was able to lower blood glucose levels effectively in both groups at 10 and 20 minutes post injection, however blood glucose increased much more rapidly in the PEG-MLA treated mice resulting in significantly higher blood glucose levels at t=30, 60, 90, and 120 minutes post insulin injection (Figure 16A) (P=0.027 at t=30 minutes, P=0.001 at t=60 minutes, P=0.006 at t=90 minutes, and P=0.008 at t=120 minutes) suggesting the presence of insulin resistance.  This was also evident if the results were expressed as a percentage of basal blood glucose (Figure 16B).  Decreased hepatic insulin sensitivity in leptin antagonist treated mice To further investigate the insulin sensitivity of different tissues, we utilized the euglycemic-hyperinsulinemic clamps to study hepatic glucose output and glucose intake in PEG-MLA treated mice.  Mice were subcutaneously implanted with mini-osmotic pumps delivering 72 μg/day PEG-MLA and euglycemic-hyperinsulinemic clamps were performed on day 3 of PEG-MLA treatment following a 16 hour fast.  Once fully immobilized from anaesthesia, the tail vein was cannulated and a 1hour basal infusion of 3H-D-glucose (1.2 μCi/hour) was started to determine steady state tracer.  Duplicate blood samples (50 μl) were taken from the tail vein at the end of the basal period to measure fasting blood glucose levels and for basal insulin measurements. Hyperinsulinemia was induced with a constant infusion of insulin (6.8 mU/hour).  To maintain euglycemia (approx 5.0 mM) a variable infusion of 12.5% D-glucose was started simultaneously and blood glucose levels were continuously monitored (Figure 17A) and the rate of infusion of the 12.5% D-glucose (Figure 17B) adjusted until  56  euglycemic levels were reached.  Once euglycemia had been achieved, steady state was maintained for 45 minutes after which blood samples were obtained for analysis. A Time (min) 0 20 40 60 80 B lo od  G lu co se  (m M ) 2 3 4 5 6 Control PEG-MLA B Time (min) 0 20 40 60 80 G lu co se  In fu si on  R at e ( μl /h r) 0 50 100 150 200 Control PEG-MLA *** *  Figure 17: Blood glucose levels and glucose infusion rate during the euglycemic- hyperinsulinemic clamps.  7-week-old C57Bl/6 mice received continuous administration of 72 µg/day of leptin antagonist for 3 days.  Clamps performed on day 3 of PEG-MLA treatment following a 16 hour fast.  (A) Blood glucose levels during clamp, mice administered bolus dose of insulin (30 µl of 4 mU insulin) via tail cannula at t= -5minutes (and insulin was then continuously infused at a dose of 6.8 mU insulin/hour  (B) glucose infusion rate  (µl/hr of a 12.5% D-glucose solution) during clamp. Data are expressed as mean ± SEM, n=5, *P < 0.05 compared to controls.  Blood glucose levels in the basal state were not different between PEG-MLA treated and control mice as previously seen, and were maintained at comparable euglycemic levels (5.0 ± 0.2 mM vs. 4.7 ± 0.3 mM respectively, P=0.23) during the hyperinsulinemic state (Figure 18A).  Consistent with our previous findings, PEG-MLA treatment lead to increased insulin levels in the fasting state compared to controls (1.11 ± 0.14 ng/mL vs. 0.59 ± 0.06 ng/mL respectively, P=0.003) even after a prolonged 16 hour fast (Figure 18B).   57  A C B B lo od  G lu co se  (m M ) 0 2 4 6 8 Basal Clamp P=0.260 P=0.230 PEG-MLA Control ED H G P  ( μ m ol /m in  k g) 0 10 20 30 40 50 P=0.07 G lu co se  In fu si on  R at e  ( μ m ol /m in  k g) 0 10 20 30 40 50 60 P=0.04 %  S up pr es si on  o f H G P  ( μ m ol /m in  k g) 0 20 40 60 80 100 120 P=0.05 In su lin  (n g/ m L) 0 2 4 6 Basal Clamp P=0.003 P=0.147 . . .  Figure 18: Increased hepatic glucose production and decreased glucose infusion rate in PEG-MLA treated mice.  Hyperglycemic-euglycemic clamps performed in 7-week-old C57Bl/6 mice on day 3 of continuous administration of 72 µg/day of PEG-MLA following a 16 hour fast.  (A-B) Plasma glucose and insulin levels during basal and hyperglycaemic state of clamp (C) glucose infusion rate during the hyperinsulinemic state (D) hepatic glucose production (HGP) and (E) percentage suppression of HGP in response to hyperinsulinemic state.  Data are expressed as mean ± SEM, n ≥ 5.  During the clamp, hyperinsulinemic conditions resulted in an expected increase in insulin levels in both the PEG-MLA and control mice that were not significantly different (Figure 18B) (3.68 ± 0.24 ng/mL vs. 4.22 ± 0.49 ng/mL respectively, P=0.147).  This increase in insulin confirmed that a hyperinsulinemic state in the mice was achieved  58  during the clamp. The glucose infusion rate (GIR) required to maintain euglycemia in PEG-MLA treated mice was 50 ± 15% lower compared to control mice (Figure 18C, P=0.05), and PEG-MLA treated mice in the hyperinsulinemic state displayed hepatic glucose production (HGP) levels that were approximately 2 fold higher (P=0.07) compared to vehicle treated control mice (Figure 18D).  When compared to hepatic glucose production in the basal state (levels following a 16 hour fast) the hyperinsulinemic conditions were not able to suppress HGP as efficiently as in control treated mice.  Hyperinsulinemia suppressed hepatic glucose production by 90 ± 19% in controls (Figure 18E), while in PEG-MLA treated mice, hepatic glucose production was only suppressed by 39 ± 19% (P=0.05) (Figure 18E).  The hyperinsulinemia was also able to decrease free fatty acid (FFA) compared to the basal state (Figure 19) levels as expected, although the hyperinsulinemic  Fr ee  F at ty  A ci ds  (m M ) 0.0 0.2 0.4 0.6 0.8 1.0 Basal Clamp P=0.042 Control PEG-MLAP=0.397 A B %  B as al  F re e Fa tty  A ci ds 0 10 20 30 40 50 60 P=0.047  Figure 19: PEG-MLA treated mice have elevated free fatty acid levels in the hyperinsulinemic state.  7-week-old C57Bl/6 mice received continuous administration of 72 µg/day of PEG-MLA for 3 days and the hyperinsulinemic-euglycemic clamp studies were on day 3 following a 16 hour fast. (A) Free fatty acid levels in the basal and hyperinsulinemic state (B) free fatty acids during the hyperinsulinemic state expressed as a percentage of basal state.  Data are expressed as mean ± SEM, n ≥ 5.   59  state was not able to suppress FFA as efficiently in PEG-MLA treated mice compared to controls (P=0.047) (Figure 19B) further suggesting that PEG-MLA treatment leads to decreased hepatic insulin sensitivity. It has also been recently reported that the predominantly liver derived insulin-like growth factor binding protein-2 (IGFBP-2), is regulated by leptin and implicated in the action of leptin to suppress hepatic glucose production [132].  In addition to this finding, it has also been observed that there is a strong association between low IGFBP-2 levels and insulin resistance [133-135].  Consistent with these data, PEG-MLA treatment for 3 days (72 µg/day) resulted in a modest, although significant reduction in circulating plasma IGFBP-2 levels (Figure 20) compared to controls (264.5 ± 14.9 ng/mL vs. 318.8 ± 22.8 ng/mL respectively, P= 0.042) following a 4 hour fast.  Pl as m a IG FP B -2  (n g/ m L) 0 100 200 300 400 500 P=0.042 PEG-MLA Control  Figure 20: Plasma IGFBP-2 levels in PEG-MLA treated mice.  7-week-old C57BL/6 mice received continuous administration of 72 µg/day of PEG-MLA for 3 days via continuous administration by subcutaneously implanted mini-osmotic pumps.  Plasma IGFBP-2 levels on day 3 of PEG-MLA administration following a 4 hour fast.  Data are expressed as mean ± SEM, n=6.   60  Circulating plasma lipids and hepatic lipid content As lipid accumulation and steatosis have been reported to be closely associated with insulin resistance and T2D [136, 137] we investigated if PEG-MLA treated mice had altered hepatic lipid content.  After 3 days of continuous administration of 72 µg/day of PEG-MLA via mini-osmotic pumps, mice were fasted for 4 hour, blood was collected for circulating lipid level analysis and the liver was harvested for a hepatic lipid extraction to determine hepatic TG and cholesterol levels.  No difference was detected in hepatic TG (Figure 21A) and cholesterol (Figure 21B) content after 3 days of PEG-MLA treatment compared to controls.  Tr ig ly ce rid es  (n m ol es /m g tis su e) 0 10 20 30 40 P=0.354 PEG-MLA Control A B C ho le st er ol  (n m ol es /m g tis su e) 0 2 4 6 8 10 12 14 P=0.50 PEG-MLA Control   Figure 21: PEG-MLA treatment does not alter hepatic triglyceride and cholesterol content.  7-week-old C57BL/6 mice received continuous administration of 72 µg/day of PEG-MLA for 3 days via mini-osmotic pumps.  Tissue harvest performed after 3 days of treatment following a 4 hour fast. (A) Hepatic triglyceride levels (B) hepatic cholesterol levels.  Data are expressed as mean ± SEM, n = 6.    61  Although no changes were detected at the level of the liver, we also investigated circulating plasma lipid levels.  Fasting triglyceride (TG), cholesterol, and free fatty acid (FFA) levels were comparable to those of control mice (Table 1) following the same PEG-MLA dosing regime.  Table 1: aLipid Analysis in PEG-MLA Treated Mice  Control PEG-MLA Triglycerides (mM) 0.39 ± 0.03 (5) 0.45 ± 0.03 (5) Free Fatty Acids (mM) 0.72 ± 0.05 (6) 0.74 ± 0.08 (6) Cholesterol (mg/dL) 117.8 ± 6.8 (6) 115.0 ± 3.8 (6) aData collected following a 4 hour fast Data are expressed as mean ± SEM.              62  STUDY 2: Mechanism of Leptin Action in Regulation of Glucose Metabolism Long term administration of exogenous leptin at low doses to ob/ob mice has been shown to reverse the hyperinsulinemia and hyperglycemia displayed by this mouse model independently of changes in body weight [26, 97].  Leptin treatment is also able to acutely reverse the hyperinsulinemia and hyperglycemias before changes in food intake and body composition are observed [73].  As leptin plays an important role in regulating glucose homeostasis independently of its effects on body weight, a dose response of leptin administration in ob/ob mice was carried out to determine the physiological response to leptin in ob/ob mice receiving a constant infusion of leptin.  Following the dose response we investigated changes that were induced at doses that had maximal effect on glucose homeostasis independently of effect on body weight through plasma analysis.   Leptin treatment of ob/ob mice is able to correct hyperglycemia and hyperinsulinemia independently of body weight. Osmotic pumps were implanted subcutaneously in ob/ob mice delivering a continuous dose of 0, 0.2, 1, or 5 µg/day leptin for 28 days and body weight (Figure 22A), blood glucose (Figure 22B) and insulin levels (Figure 22C) were tracked following a 4 hour fast.  Mice receiving the lowest dose of 0.2 µg/day leptin continued to gain weight at a rate comparable to PBS treated ob/ob mice.  63  Time (days) 0 5 10 15 20 25 %  C ha ng e bo dy  w ei gh t -80 -60 -40 -20 0 20 40 A B C Time (days) -5 0 5 10 15 20 25 B lo od  G lu co se  (m M ) 0 5 10 15 20 25 0 10 20 30 40 50 Pl as m a In su lin  (n g/ m L) 0.2 μg/day Leptin 0 μg/day Leptin 1 μg/day Leptin 5 μg/day Leptin * C57Bl/6 * * 0 μg/day Leptin 0.2 μg/day Leptin 1 μg/day Leptin 5 μg/day Leptin C57Bl/6  Figure 22: Continuous leptin administration in ob/ob mice leads to reversal of hyperglycemia and hyperinsulinemia independently of weight loss.  ob/ob mice were treated with continuous leptin administration via mini-osmotic pumps.  28 day pumps were implanted subcutaneously on day 0 following a 4 hour fast for 28 days.  (A) Percent change in body weight during treatment (B) blood glucose levels following a 4 hour fast (C) insulin levels on day 27 of leptin treatment via mini osmotic pumps.  Male ob/ob mice were 7- weeks-old at time of pump implant.  C57Bl/6 male mice were untreated and age matched to ob/ob mice.  Data are expressed as mean ± SEM, n ≥7 , *P < 0.05 compared to controls.  Although this low dose had no effect on body weight or body weight gain, an improvement in fasting blood glucose was observed despite the fact that this dose had no effect on fasting insulin levels.  A higher dose of 1 µg/day leptin administered to the ob/ob mice did not result in weight loss, although body weight gain reached a plateau, and the ob/ob mice did not gain weight during the duration of leptin treatment.  The 1 µg/day dose also resulted in fasting blood glucose levels rapidly decreasing in a time dependent manner with the administration of leptin, normalizing to wild-type levels by day 20 of leptin treatment, and fasting insulin levels were normalized to levels of wild- type mice by day 28 of treatment.  As expected, the maximal effect was seen at the highest dose of leptin (5 µg/day) administered to the ob/ob mice leading to a 25%  64  decrease in body weight by 28 days, and blood glucose levels were comparable to wild- type controls by day 5 of leptin treatment.  Dose dependent effect of leptin in ob/ob mice on glucose tolerance and insulin sensitivity We next investigated if leptin treatment also had a dose dependent effect on glucose tolerance and insulin sensitivity.  The lowest dose of leptin replacement therapy to ob/ob mice (0.2 µg/day) resulted in a mild improvement in oral glucose tolerance (Figure 23A) independently of increased insulin sensitivity (Figure 23B) as assessed by an ITT. Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 10 20 30 40 0 μg/day Leptin 0.2 μg/day Leptin 1 μg/day Leptin 5 μg/day Leptin C57Bl/6 * A B Time (min) 0 20 40 60 80 100 120 %  B as al  B lo od  G lu co se 20 40 60 80 100 120 140 160 180 0 μg/day Leptin 0.2 μg/day Leptin 1 μg/day Leptin 5 μg/day Leptin C57Bl/6  Figure 23: Continuous leptin administration in ob/ob mice improves glucose tolerance and insulin sensitivity in a dose dependent manner.  ob/ob mice were treated with continuous leptin administration via 28 day mini-osmotic pumps implanted subcutaneously. (A) Oral glucose tolerance test (1.5 mg/g glucose) on day 22 of leptin administration and (B) insulin tolerance test (1 U/kg insulin i.p.) on day 15 of leptin administration following a 4 hour fast.  Male ob/ob mice were 7-weeks-old at time of pump implant.  C57Bl/6 male mice were untreated and age matched to ob/ob mice.  Data are expressed as mean ± SEM, n ≥ 7.   65  Improved insulin sensitivity was observed at the 1 µg/day dose (Figure 23B) in combination with a further improvement in oral glucose tolerance (Figure 23A).  The 5 µg/day dose had the most potent effect and was able to improve both oral glucose tolerance (Figure 23A) and insulin sensitivity (Figure 23B) to an extent that was comparable to wild-type mice. Once we determined that our dose response was able to segregate the effect of leptin on glucose homeostasis and body weight we went on to determine the mechanism of leptin action on glucose metabolism that was independent of weight loss inducing effects.  Blood samples collected from ob/ob mice receiving leptin treatment at the various doses were first analyzed for glucagon-like peptide (GLP-1) levels. GLP-1 has received a lot of attention as a diabetes therapeutic given its range of blood glucose lowering effects [138, 139].  GLP-1 is secreted from intestinal endocrine L-cells [140] in a nutrient dependent manner [140, 141] and has been found to regulate blood glucose levels directly by potently stimulating glucose dependent insulin secretion [142-144], as well as indirectly via the inhibitory effects on gastric emptying and hypothalamic feeding centers [145-147].  It has been found that GLP-1 levels are reduced in obesity  [148-150] and mice fed a  high-fat diet have a diminished GLP-1 response to oral glucose [151]. Since obesity is linked to dysregulated leptin signalling it was postulated that leptin may modulate GLP-1 secretion [151].  Functional leptin receptors are expressed by both rodent and human L-cells [151], and acute leptin treatment has been shown to stimulate GLP-1 secretion by 1.8 fold (2 hours post leptin injection) in ob/ob mice [151].  Long term adenoviral GLP-1 expression (rAd-GLP-1) in ob/ob mice results in long term amelioration of diabetes [152].  Since acute leptin treatment has been  66  shown to modestly increase GLP-1 levels in ob/ob mice, we sought to determine if long term leptin treatment in ob/ob mice resulted in higher levels which could then be potentially responsible for the anti-diabetic effects of leptin.  Leptin decreases GLP-1 levels in ob/ob mice in a dose dependent manner As ob/ob mice treated with a continuous dose of 1 µg/day leptin displayed normalized fasting blood glucose levels as well as improved glucose tolerance in conjunction with improved whole body insulin sensitivity independently of weight loss, we measured GLP-1 levels before, during, and after leptin treatment at this dose. Surprisingly, we saw that total GLP-1 levels in ob/ob mice and the effect of leptin on GLP-1 levels were in disagreement to what has been previously shown.  Although the current literature states that GLP-1 levels are decreased in obesity [149, 150, 153], we observed that ob/ob mice had 3-5 fold higher GLP-1 levels (Figure 24A,B) compared to wild-type mice of same age and background (~45-80 pg/mL vs. ~15 pg/mL, respectively). Although we observed a variation in GLP-1 levels between different cohorts of ob/ob mice of same age and background, GLP-1 levels were always significantly higher compared to wild-type controls.  We observed that leptin treatment (1 µg/day for 28 days) lead to a significant decrease in GLP-1 levels (Figure 24A).  On day 25 of treatment, leptin treated ob/ob mice had a 4-fold reduction in GLP-1 levels compared to PBS treated controls (16.7 ± 2.6 pg/mL vs. 80.8 ± 5.1 pg/mL respectively, P < 0.0001).  Upon surgical removal of pump and cessation of leptin therapy, GLP-1 levels increased to levels observed prior to leptin treatment, and comparable to controls (64.3 ± 6.1 pg/mL vs. 55.0 ± 3.5 pg/mL respectively, P=0.162).  67  The effect of leptin on GLP-1 levels was dose dependent (Figure 24B) as the highest dose of treatment (5 µg/day leptin) decreased total GLP-1 levels below levels observed in wild-type mice (8.3 ± 0.5 pg/mL vs. 19.0 ± 1.4 pg/mL, respectively P < 0.001) while the lowest dose of 0.2 µg/day of leptin shown to have an effect on fasting blood glucose had no effect on GLP-1 levels (32.8 ± 4.6 pg/mL vs. 36.9 ± 3.4 pg/mL, P=0.24). Time (days) 0 20 40 60 80 100 To ta l G LP -1  (p g/ m L) 20 40 60 80 100 PBS Leptin * * PUMP A B 0 15 30 45 To ta l G LP -1  (p g/ m L) 0.2 μg/day Leptin 0 μg/day Leptin 5 μg/day Leptin * * C57Bl/6  Figure 24: Continuous leptin administration in ob/ob mice leads to decreased total GLP- 1 levels in a dose dependent manner.  ob/ob mice were treated with continuous leptin administration via mini-osmotic pumps.  28 day pumps were implanted subcutaneously on day 0 following a 4 hour fast for 28 days. (A) Plasma total GLP-1 levels during leptin (1 µg/day) or PBS treatment, and (B) plasma total GLP-1 levels on day 18 of leptin treatment following a 4 hour fast.  Data in Panel A and B are from two separate cohorts of mice.  Male ob/ob mice were 7-weeks-old at time of pump implant.  C57Bl/6 male mice were untreated and age matched to ob/ob mice.  Data are expressed as mean ± SEM, n ≥7, *P < 0.05 compared to controls.  Leptin treatment improved glucose stimulated total GLP-1 secretion in ob/ob mice It has been previously shown that mice fed a high fat diet have a diminished GLP-1 response to oral glucose [151].  We sought to determine if this was also true of ob/ob mice and if leptin treatment would be able to potentiate GLP-1 secretion in response to glucose even though it had an inhibitory effect on total GLP-1 levels in the  68  fasting state in ob/ob mice.  On day 22 of leptin treatment (1 µg/day) in ob/ob mice, an OGTT was performed and blood was collected at various time points following glucose gavage to obtain plasma and determine glucose stimulated total GLP-1 secretion.  Leptin treated mice displayed improved glucose tolerance compared to PBS treated controls (Figure 25A).  Glucose stimulated GLP-1 secretion in PBS treated ob/ob mice was diminished (Figure 23B) while leptin treated ob/ob mice displayed glucose stimulated GLP-1 levels (Figure 25B).  Total GLP-1 levels increased 3 fold in response to glucose 7 minutes post glucose gavage compared to fasting levels, and returned to basal levels 120 minutes post glucose gavage in the leptin treated ob/ob mice. Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 10 20 30 40 PBS Leptin * * * * * * * A B Time (min) 0 10 20 30 40 50 60 To ta l G LP -1  (p g/ m L) 0 20 40 60 80 PBS Leptin * * * * Figure 25: Leptin treated ob/ob mice have glucose stimulated secretion of total GLP-1. ob/ob mice were treated with continuous leptin administration via mini-osmotic pumps implanted subcutaneously.  (A) Oral glucose (1.5 mg/kg glucose) tolerance test and (B) total GLP-1 levels in response to the oral glucose load on day 22 of leptin treatment (1µg/day) following a 4 hour fast.  Data are expressed as mean ± SEM, n ≥ 7, *P < 0.05 compared to PBS treated mice.     69  Acute effects of leptin treatment on GLP-1 in ob/ob and C57BL/6 mice As the effects of leptin on GLP-1 levels we observed were contradictory to those published by Anini et al. [151] we next investigated the effect of acute leptin treatment on total GLP-1 levels.  GLP-1 is derived from the processing of the proglucagon precursor by prohormone convertase 1/3 which results in generation of GLP-11-37 , GLP- 11-36amide, GLP-17-37 and GLP-17-36amide. The assay we were using to measure GLP-1 was able to pick up all these forms and therefore we were measuring total GLP-1 levels.  It is generally believed that the most bioactive forms of the GLP-1 peptide are GLP-17-36amide and GLP-17-37, as both have been shown to be equally potent in their insulinotropic activity [154].  Anine et al. showed that leptin had the ability to acutely increase GLP-17-36amide levels in ob/ob mice 2-fold, 120 minutes post leptin administration.  As they measured levels of the active form of GLP-1, we wanted to determine the acute effect of leptin on total GLP-1 (as our assay picked up all forms of GLP-1 generated in circulation). Following their protocol of leptin administration,  ob/ob and C57BL/6 mice were fasted for 4 hours and then injected i.p. with leptin (1 mg/kg) and blood was collected 2 hours and 24 hours following leptin administration (in the fasting state) to obtain plasma and then assay for total GLP-1 levels.  No effect on total GLP-1 levels was observed 2 hours (28.9 ± 3.3 pg/mL vs. 28.4 ±2.5 pg/mL, P=0.45) or 24 hours (20.6 ± 1.5 pg/mL vs. 18.0 ± 1.6 pg/mL, P=0.13) post leptin treatment in ob/ob mice compared to ob/ob mice receiving PBS (Figure 26A). On the other hand, wild-type mice receiving leptin had an acute and transient increase in total GLP-1 levels (Figure 26B).  Leptin was able to significantly increase total GLP-1 levels in wild-type mice compared to PBS treated controls (23.9 ± 3.3 pg/mL vs. 16.1 ± 1.7 pg/mL respectively, P=0.01 at t=2  70  hours).  This increase in total GLP-1 levels was transient as 24 hours post leptin treatment levels of total GLP-1 were comparable to pre-treatment levels (17.8 ± 1.6 pg/mL vs. 17.4 ± 1.4 pg/mL respectively, P=0.44) in the wild-type mice.  Overall, we could only partially duplicate the results published by Anini et al., as acute leptin treatment resulted in an acute and transient increase total GLP-1 levels in wild-type mice but had no effect on total GLP-1 levels in ob/ob mice. Time (hours) 0 4 8 12 16 20 24 To ta l G LP -1  (n g/ m L) 0 5 10 15 20 25 30 35 40 PBS Leptin Time (hours) 0 4 8 12 16 20 24 To ta l G LP -1  (n g/ m L) 0 5 10 15 20 25 30 35 40 PBS Leptin * A Bob/ob C57Bl/6  Figure 26: Total GLP-1 levels in response to acute leptin treatmen ob/ob and C57Bl/6 Mice.  Mice were fasted for 4 hours (t=0) and then injected i.p. with leptin (1 mg/kg). Plasma was then collected 2 hours and 24 hours post leptin injection.  Mice did not have access to food during the entire the experiment. (A) Total GLP-1 levels in ob/ob mice (B) and C57Bl/6 mice.  Mice were 12-weeks-old at time of experiment.  Data are expressed as mean ± SEM, n ≥ 9, *P < 0.05 compared to PBS treated mice.  Increased levels of bioactive GLP-1 levels in ob/ob mice compared to wild-type controls We also wanted to determine if the elevated GLP-1 levels in the basal state of ob/ob mice were of biologically active GLP-1.  Plasma was collected from untreated ob/ob and C57Bl/6 mice and assayed using a bioassay which was different from our  71  previous method of measuring GLP-1, as the bioassay is able to determine levels of biologically active GLP-1.  HEK-hGLP1R-Luc cells stably expressing the human GLP-1 receptor and a cAMP responsive element driving luciferase expression were stimulated with 100 μl of sample (plasma diluted 1/3) for 5 hours.  A luciferase assay was then performed.  The output of the assay luminescence is proportional to the concentration of biologically active GLP-1 in the plasma samples.  It was determined that ob/ob mice have significantly higher biologically active GLP-1 levels following a 4 hour fast compared to wild-type mice (36.3 ± 7.3 pM vs. 6.0 ± 1.2 pM respectively, P=0.0004) (Figure 27). G LP -1  (p M ) 0 10 20 30 40 50 P=0.0004 C57Bl/6 ob/ob  Figure 27: ob/ob mice have elevated active GLP-1 levels compared to wild-type controls.  Plasma samples collected from 35-week-old ob/ob mice and age-matched C57Bl/6 controls following a 4 hour fast.  Plasma samples assayed using a bioassay to determine bioactive GLP-1 plasma levels.  HEK-hGLP1R-Luc cells stably expressing the GLP-1 receptor and a cAMP responsive element driving luciferase expression were stimulated with 100 μl of sample (plasma diluted 1/3 in Krebs or GLP-1 standards in Krebs) was added. Following a 5 hour incubation, the samples were removed and wells washed with PBS. A luciferase assay was then performed using the Bright-Glo luciferase assay kit according to the manufacturer’s instructions. Luminescence was measured using a luminometer.  GLP-1 levels in the samples were then calculated by extrapolating from the standard curve.   72  Effect of prolonged fasting on GLP-1 levels in ob/ob and C57Bl/6 mice To further investigate the source of elevated total and biologically active GLP-1 levels in ob/ob mice, we sought to determine if elevated levels were simply due to the increased food intake of ob/ob mice compared to control mice, as a well known stimulus for GLP-1 release is ingestion of carbohydrates or fats [155, 156].  Untreated ob/ob and age matched C57Bl/6 mice were fasted short term (4 hours) and long term (16 hours) to determine if  there was a correlation between duration of food restriction  and GLP-1 levels.  Fasting and re-feeding had the expected effect on blood glucose levels (Figure 28A), as blood glucose was dramatically reduced by more than 50% in both ob/ob and wild-type mice following a 16 hour fast.  The prolonged 16 hour fast also had a significant  effect on total GLP-1 levels in the ob/ob mice reducing levels by ~25% compared to levels observed following a 4 hour fast (Figure 28B).  Total GLP-1 levels in wild-type mice were comparable during the 4 hour and 16 hour fast.  These data indicate that food intake does play a significant role in GLP-1 levels in the ob/ob mouse model.  A 2 hour re-feed period following the 16 hour fast induced a potent increase in total GLP-1 levels in both ob/ob and wild type mice further demonstrating that food consumption is a potent regulator of total GLP-1 secretion.  Overall, the effect of food intake on total GLP- 1 levels revealed that the elevated total and active GLP-1 levels in the ob/ob mice are most likely due to the hyperphagia.  Also, as the dose of leptin (0.2 µg/day) that was shown to improve fasting blood glucose and oral glucose tolerance had no effect on total GLP-1 levels we ruled out leptin regulation of total GLP-1 levels as being an important downstream effect of leptin action which was responsible for the potent anti-diabetic effect of leptin.  73  B lo od  G lu co se  (m M ) 0 10 20 30 C57BL/6 ob/ob P=0.36 P  0.01< P  0.01< A B 4 hr Fast 16 hr Fast Re-feed 4 hr Fast 16 hr Fast Re-feed To ta l G LP -1  (p g/ m L) 0 20 40 60 80 C57BL/6 ob/ob P=0.31 P  0.01< P  0.01< Figure 28: Total GLP-1 levels in response to fasting and re-feeding in ob/ob and C57Bl/6 Mice.  Mice were fasted for 4 or 16 hours and plasma was assayed for total GLP-1 levels. Following the 16 hr fast and plasma collection, mice were allowed to feed for 2 hours and then plasma was collected and assayed for total GLP-1 to measure fed levels. (A) Blood glucose levels and (B) total GLP-1 levels in 11-week-old mice.  Data are expressed as mean ± SEM, n ≥ 9.            74  Relationship between insulin-like growth factor binding protein-2 and leptin on glucose metabolism Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) play an important role in regulating glucose metabolism and ß-cell function [132, 157, 158]. IGF-1 specifically has been found to influence blood glucose concentrations directly by stimulating glucose uptake in target cells and indirectly by increasing the sensitivity of tissues to insulin [157].  Effects of IGFs are modulated by IGF binding proteins (IGFBPs) through their ability to sequester them, however, several reports demonstrate that the IGFBPs can activate cell surface receptors directly and stimulate cellular events in the absence of IGFs [159].  The presence of an Arg-Gly-Asp domain which can associate with cell surface integrins, is important in mediating these IGF-indpendent effects by the IGFBPs [159].  Recently, it has been demonstrated that integrin signalling can influence metabolic pathways, and that there is crosstalk between intergrin-receptor binding and insulin signalling [160, 161].  However, whether specific IGFBP-integrin interactions can modulate insulin signalling requires further study. IGF binding protein-2 (IGFBP-2) is the second most abundant plasma circulating IGFBP but its physiological role remained poorly understood until recently.  It was initially speculated that since IGFBP-2 is the principle binding protein secreted by differentiating white pre-adiopocytes [162] that it played a potential role in development of obesity.  Instead, mice expressing IGFBP-2 were protected from age-and diet induced glucose intolerance and displayed increased insulin sensitivity [158].  Low levels of IGFBP-2 are correlated with decreased insulin sensitivity and levels are also reduced in the presence of obesity.  Genome wide association studies have also identified a single  75  nucleotide polymorphism in IGFBP-2 associated with type 2 diabetes [133].  Overall, there is an inverse association of IGFBP-2 with adiposity-related insulin resistance. Recently published data show that administration of leptin to ob/ob mice at levels that have effects on glucose homeostasis independent of leptin’s effect on body weight potently upregulate IGFBP-2 gene expression in the liver [132], and increase circulating plasma IGFBP-2 levels.  To determine if IGFBP-2 could account for a portion of leptin’s anti-diabetic effects, Hedbacker et al. [132] went on to show administration of a recombinant IGFBP-2 expressing adenovirus to ob/ob mice had the same anti-diabetic effects of leptin in various mouse models of insulin resistance and insulin deficient diabetes.  These results, in addition to the potent increase of circulating IGFBP-2 protein by leptin to the ob/ob mice suggest that IGFBP-2 is regulated by, and downstream in the leptin signalling pathway.  To determine if IGFBP-2 levels were low in both an obese and non-obese mouse model of insulin resistance in the presence of leptin deficiency, we measured IGFBP-2 levels in obese diabetic ob/ob mice as well as AZIP/F-1 mice, a model of lipoatrophic diabetes.  Both mouse models displayed hyperglycemia, hyperinsulinemia, low to undetectable leptin levels, and significantly reduced IGFBP-2 levels compared to their littermate controls (Table 2). Table 2: aMetabolic Parameters of obese ob/ob and lipodystrophy AZIP/F1 mice  C57Bl/6 ob/ob  AZIP/F1 tg- AZIP/F1 tg+ Body Weight (g) 21.6 ± 0.1 (12) 41.9 ± 0.5 (15)* 27.8 ± 0.8  (3) 28.8 ± 1.3 (9) Blood Glucose (mM) 10.3 ± 0.4 (12) 19.3 ± 1.0 (15)* 8.4 ± 0.4 (3) 23.9 ± 1.8 (9) * Insulin (ng/mL) 0.97 ± 0.1 (12) 21.9 ± 1.7 (15)* 1.6 ± 0.5 (3) 31.9 ± 6.9 (5) * Leptin (ng/mL) 1.36 ± 0.2 (12) 0.1 ± 0.01 (13)* 4.54 ± 1.88 (3) 0.24 ± 0.1 (9)* IGFBP-2 (ng/mL) 291 ± 8 (12) 31.8 ± 2.5 (14)* 223.4 ± 19.7 (3) 93.5 ± 13.3 (9) * aData collected following a 4 hour fast Data are expressed as mean ± SEM *P < 0.05 vs. littermate controls   76  These data indicate that leptin deficiency and insulin resistance in both an obese and lean mouse model correlate with low plasma IGFBP-2 levels.  Leptin therapy leads to a dose dependent increase in circulating plasma IGFBP-2 levels We next investigated if the various leptin doses administered to ob/ob mice in our experiment induced changes in circulating IGFBP-2 levels.  Continuous leptin administration at doses of 0.2, 1, and 5 µg/day for 28 days resulted in a dose dependent increase of leptin levels in the leptin deficient ob/ob mice by day 28 of treatment (Figure 29A). 0 1 2 3 4 5 Le pt in  (n g/ m L) 0 μg/day Leptin 0.2 μg/day Leptin 1 μg/day Leptin 5 μg/day Leptin C57Bl/6 * * * * A 0 50 100 150 200 250 300 350 Pl as m a IG FB P- 2 (n g/ m L) 0 μg/day Leptin 0.2 μg/day Leptin 1 μg/day Leptin 5 μg/day Leptin C57Bl/6 * * * * B   Figure 29: Continuous leptin administration in ob/ob mice leads to increased plasma IGFBP-2 levels in a dose dependent manner.  ob/ob mice were treated with continuous leptin administration via mini-osmotic pumps.  28 day pumps were implanted subcutaneously on day 0 following a 4 hour fast for 28 days. (A) Leptin levels and (B) plasma IGFBP-2 levels on day 28 of leptin administration following a 4 hour fast.  Male ob/ob mice were 7- weeks-old at time of pump implant.  C57Bl/6 male mice were untreated and age matched to ob/ob mice.  Data are expressed as mean ± SEM, n ≥ 7 *P < 0.05 compared to controls.   77  This increase in leptin resulted in an increase in IGFBP-2 levels, in a dose dependent manner (Figure 29B).  All doses were able to increase IGFBP-2 levels significantly above levels found in untreated ob/ob mice, and the highest dose of 5 µg/day for 28 days induced IGFBP-2 levels comparable to those observed in wild-type mice.  IGFBP-2 replacement in ob/ob mice Hedbacker et al. [132]  utilized an adenoviral approach to deliver IGFBP-2 to mice, which resulted in circulating IGFBP-2 protein levels significantly higher than the level at which it normally circulates in the blood stream (at least 6000 ng/mL in Ad- IGFBP-2 treated mice vs. ~350 ng/mL in wild-type mice).  To determine if the effects they observed were only due to the pharmacological IGFBP-2 levels attained, we tried an approach of recombinant IGFBP-2 protein replacement therapy via mini-osmotic pumps aimed at producing physiological levels of IGFBP-2.  Osmotic pumps were implanted subcutaneously in ob/ob mice delivering 1500 ng/day recombinant mouse IGFBP-2 (American Peptide) for 7 days.  Body weight (Figure 30A), fasting blood glucose (Figure 30B) and fasting insulin levels (Figure 30D) remained the same between IGFBP-2 treated mice and vehicle treated controls.  There was also no improvement in glucose tolerance (Figure 30E) or insulin sensitivity (Figure 30F) in IGFBP-2 treated mice compared to controls.  The lack of improvement in any of the metabolic abnormalities observed in the ob/ob mouse by the IGFBP-2 therapy can be at least partially attributed to that fact that the method of delivery of IGFBP-2 was not able to increase plasma IGFBP-2 levels compared to pre-treatment levels (Figure 30C).  78  Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 5 10 15 20 25 30 35 Time (days) -5 0 5 10 B lo od  G lu co se  (m M ) 0 5 10 15 20 PBS IGFBP-2 PUMP Time (days) -5 0 5 10 B od y W ei gh t ( g) 20 30 40 50 60 70 PUMP Time (days) -4 -2 0 2 4 6 8 10 12 IG FB P- 2 (n g/ m L) 0 20 40 60 A B C PUMP Time (days) -4 -2 0 2 4 6 8 10 12 Fa st in g In su lin  (n g/ m L) 0 10 20 30 40 50 60 PUMP D E Time (min) 0 20 40 60 80 100 120 %  o f F as tin g G lu co se 0 50 100 150 200 F  Figure 30: IGFBP-2 Administration to ob/ob mice.  ob/ob mice were treated with continuous IGFBP-2 administration via mini-osmotic pumps.  8 day pumps were implanted subcutaneously on day 0 following a 4 hour fast for 8 days. IGFBP-2 (1500 ng/day) was administered to 10-week-old male ob/ob mice.  (A) Blood glucose (B) body weight (C) circulating plasma IGFBP-2 (D) and insulin levels following a 4 hour fast. (E) Oral glucose (1.5 mg/g) tolerance test on day 7 of pump and (F) insulin tolerance test  (1 U/kg i.p. insulin) on day 8 of pump following a 4 hour fast. Data are expressed as mean ± SEM, n ≥ 5.   79  Leprflox/flox Albcre tg+ ob/ob mice have attenuated hepatic leptin receptors   In order to examine whether leptin regulates IGFBP-2 as a result of direct effects on the liver, ob/ob mice with tissue specific attenuation of leptin receptor signalling were utilized.  Mice containing the leprflox/flox gene were crossed with the Albcre tg+ mice to generate Leprflox heterozygous offspring which were then mated to ob/+ and then to each other to produce Leprflox/flox  ob/ob and Leprflox/flox Albcre tg+ ob/ob mice.  In the Albcre tg+ mice, the cre transgene contains a nuclear localization sequence and is under the control of a 2.34 kb mouse albumin enhancer/promoter (Jackson Laboratory, Bar Harbour, ME) conferring hepatic specific attenuation of the leptin receptor [163, 164].  Liver specific gene recombination was achieved using the Cre-loxP system where the Leprflox/flox mice have loxP sites flanking exon 17 of the leptin receptor gene (lepr) (Figure 31A) [165, 166].  The long signalling form of the leptin receptor (Ob-Rb)  Figure 31: Schematic of the leprflox and lepr∆17 gene and protein stuctures. (A) Genomic structure of the lepr gene, where in Leprflox/flox  Albcre tg+ ob/ob mice excision of exon 17 is generated by cre mediated recombination at loxP sites resulting in lepr∆17 . (B) The protein structure of leprflox is equivalent to the wild type isoform b of the leptin receptor containing the Box 1 and 2 motifs required for signal transduction.  The lepr∆17  lacks the Box 1 and 2 motif and is devoid of signalling function. Figure generated by Dr. Scott Covey (University of British Columbia). A B  80   contains the Box 1 and 2 motifs which are required for the signalling ability of the leptin receptor (Figure 31B).  The Box 2 motif specifically contains residues tyr985 and tyr1138, sites of JAK mediated tyr phosphorylation [167] which lead to STAT3 and STAT5 activation upon leptin binding [37].  Through cre mediated recombination at the loxP sites, exon 17 is excised, generating an altered 3’ terminus with a premature stop codon due to a frame shift mutation that results from the excision (Figure 31B) [166].  This altered 3’ terminus no longer contains the Box 1 and Box 2 motifs which are required for the signal transduction effects of the receptor.  Mice that are homozygous for the lepr∆17 gene are deficient in leptin mediated phosphorylation of the STAT proteins [168]. To confirm excision of exon 17 at the genomic level, genomic DNA was harvested from liver of Leprflox/flox and Leprflox/flox Albcre tg+ mice and used in a PCR reaction with primers flanking the location of the loxP sites (Figure 32A).   Figure 32: Recombination of the Leprflox allele in liver mediated by the Albcre transgene. (A) Genomic DNA was extracted from liver tissue of Leprflox/flox Albcre- ob/ob and  Leprflox/flox Albcre tg+ ob/ob mice and used as a template for the PCR reaction of the Leprflox allele (B) RT-PCR confirming transcript of leptin receptor isoform b in liver.  RNA was extracted from liver tissue of Leprflox/flox ob/ob mice and used as a template for the RT reaction to generate cDNA. The cDNA was then used as template for the PCR reaction of the Leprflox exon 17.   B  81  The PCR amplification from mice carrying the Albcre tg + produced a band at ~950 bp corresponding to the expected product from the Lepr∆17 allele, along with a faint band observed at ~1369 bp corresponding to the Leprflox  allele.  The 1369 bp faint band is likely due to the endothelial and Kupffer cells found in hepatic tissue that don’t express albumin specific transcription factors and don’t express the cre transgene so excision of exon 17 does not occur.  In liver, the cre-loxP method still reflects a high degree of excision of the Leprflox allele since the liver is comprised primarily of hepatocytes. Genomic DNA from mice not harbouring the Albcre transgene resulted in a larger product of ~1369 bp being amplified, which is consistent with the predicted size of the product from the Leprflox allele in the absence of the cre transgene.  It has been previously published that the leptin receptor is expressed in hepatocytes [37, 48, 75, 87].  RT-PCR was performed to confirm expression of the wild type and attenuated hepatic leptin receptor at the transcript level from Leprflox/flox and Leprflox/flox Albcre tg+ mice (Figure 32B).  RNA was extracted from liver of Leprflox/flox and Leprflox/flox Albcre tg+ mice and used as a template for the RT reaction.  The resulting cDNA was then used as a template for the PCR reaction using primers that flank exon 17.  Mice not carrying the cre transgene resulted in a PCR amplified product of expected size of ~ 343 bp.  A shift in molecular weight was seen for the excision of exon 17 in the presence of cre recombination occurring in Albcre tg+ mice with a  ~ 267 bp product being amplified. Besides showing excision of exon 17 at the transcript level, these results confirm the expression of leptin receptor mRNA in the liver.  The Lerpflox/flox Albcre+ ob/ob mice and their littermate controls not carrying the cre transgene were obese, hyperglycaemic, and hyperinsulinemia and did not significantly differ from each other (Table 3).  82  Table 3: aMetabolic Parameters in Female Leprflox/flox Albcre ob/ob Mice  Leprflox/flox Albcre- ob/ob Leprflox/flox Albcre+ ob/ob Body Weight (g) 45.9 ± 2.1 (8) 44.2 ± 2.5 (5) Blood Glucose (mM) 17.2 ± 2.2 (8) 20.2 ± 2.9 (5) Insulin (ng/mL) 9.0 ± 1.1 (8) 10.7 ± 3.1  (5) aData collected following a 4 hour fast Data are expressed as mean ± SEM  Leptin administration in Lepflox/flox Albcre+ ob/ob mice During the 14 day subcutaneous leptin administration (5 µg/day) via min-osmotic pumps, both Leprflox/flox Albcre+ ob/ob and Leprflox/flox Albcre- ob/ob mice lost weight equally (Figure 33A), and normalization of fasting blood glucose (Figure 33B) and insulin levels (Figure 33C) was observed by day 5 of treatment. (Figure 34B). Time (days) 0 20 40 B od y W ei gh t ( g) 20 30 40 50 60 70 80 Lepr flox/flox Albcre- ob/ob Leprflox/flox Albcre+ ob/ob A B Time (days) 0 20 40 B lo od  G lu co se  (m M ) 0 5 10 15 20 25 Lepr flox/flox Albcre- ob/ob Leprflox/flox Albcre+ ob/ob Time (days) 0 10 20 30 In su lin  (n g/ m L) 0 2 4 6 8 10 12 14 16 18 Leprflox/flox Albcre- ob/ob Leprflox/flox Albcre+ ob/ob C PUMP PUMP PUMP    Figure 33: Leptin administration to Lerpflox/flox Albcre ob/ob mice leads to weight loss and reverses hyperglycemia and hyperinsulinemia.  Female Leprflox/flox Albcre+ ob/ob mice were treated with continuous leptin (5 μg/day) administration via mini-osmotic pumps.  14 day pumps were implanted subcutaneously on day 0 following a 4 hour fast.  (A) Body weight (B) blood glucose, and (C) insulin levels following a 4 hour fast. Data are expressed as mean ± SEM, n ≥ 5.   83  Plasma IGFBP-2 levels were measured next to determine if leptin administration would result in an increase in circulating plasma IGFBP-2 levels in the absence of hepatic leptin signalling.  Plasma leptin levels were below detection before exogenous administration by the mini-osmotic pumps and leptin levels increased in a time-dependent manner during exogenous delivery (Figure 34A). Time (days) 0 5 10 15 20 Le pt in  (n g/ m L) 0 5 10 15 20 Leprflox/flox Albcre- ob/ob Leprflox/flox Albcre+ ob/ob * * * * * A PUMP Time (days) 0 10 20 30 IG FB P- 2 (n g/ m L) 0 100 200 300 400 * * B PUMP  Figure 34: Leptin increases plasma IGFBP-2 levels in Leprflox/flox Albcre+ ob/ob mice independently of hepatic leptin signalling.  Female Leprflox/flox Albcre+ ob/ob mice were treated with continuous leptin (5 μg/day) administration via mini-osmotic pumps.  14 day pumps were implanted subcutaneously on day 0 following a 4 hour fast. (A) Leptin levels and (B) plasma IGFBP-2 levels following a 4 hour fast.  Data are expressed as mean ± SEM, n ≥ 5, *P < 0.05 compared to littermate controls.  Leprflox/flox Albcre+ ob/ob mice had significantly higher leptin levels on day 2, 7, 9, 12 and 14 of leptin administration compared to the Leprflox/flox Albcre- ob/ob mice.    IGFBP-2 levels were equally low in the Leprflox/flox Albcre+ ob/ob and Leprflox/flox Albcre- ob/ob mice prior to leptin administration, and leptin was able to increase levels equally in both groups of mice.  IGFBP-2 levels increased in a time-dependent manner, and decreased to levels prior to leptin administration by 16 days post leptin withdrawal.  These data  84  indicate that hepatic leptin signalling is not required for leptin to increase circulating plasma IGFBP-2. Another potential mechanism of leptin regulation of circulating IGFBP-2 levels is that leptin regulates IGFBP-2 levels indirectly by activating central nervous system (CNS) efferent signals from the brain to the liver.  ob/ob mice with subdiaphragmatic vagotomies were utilized next to explore this hypothesis and determine if interrupting the efferent and afferent signalling of the vagus nerve would abolish the ability of leptin to increase circulating plasma IGFBP-2 levels.  Effect of vagotomy on metabolic parameters in ob/ob mice To investigate the role of vagal efferents on the ability of leptin to regulate plasma IGFBP-2 levels, ob/ob mice with a subdiaphragmatic vagotomy and sham surgery controls were purchased from Jackson Laboratories (Bar Harbor).  Mice that received subdiaphragmatic vagotomy weighed significantly less than sham-operated mice (Figure 35A), and consumed less food compared to sham operated controls (7.4 ± 0.2 g vs. 8.7 ± 0.2 g respectively, P<0.001) (Figure 35B).  Blood glucose levels following a 4 hour fast trended towards being higher in vagotomized mice compared to sham-operated mice (Figure 35C) and were accompanied by significantly lower insulin levels in the basal state compared to sham controls (7.2 ± 0.8 ng/mL vs. 14.7 ± 0.7 ng/mL respectively, P<0.0001 ) (Figure 35D).  Leptin treatment via mini-osmotic pumps (5 µg/day for 14 days) on day 0 following a 4 hour fast resulted in weight loss, decreased food intake, and normalization of fasting blood glucose (8.1 ± 0.3 mM vagotomy vs. 8.0 ± 0.5 mM sham surgery, P=0.45) and insulin levels (2.66 ± 0.29 ng/mL vagatomy vs.  85  3.01 ± 0.26 ng/mL sham surgery, P=0.19) equally in both vagotomized and sham- operated mice. Time (days) 0 10 20 30 B od y W ei gh t ( g) 10 20 30 40 50 Sham Surgery Vagotomy A B Time (days) 0 10 20 B lo od  G lu co se  (m M ) 0 10 20 30 Sham Surgery Vagotomy * C D In su lin  (n g/ m L) 0 5 10 15 20 Basal Leptin P    0.001 Sham Surgery Vagotomy < P=0.191 Fo od  In ta ke  (g ) 0 2 4 6 8 10 12 Basal Leptin P    0.001 Sham Surgery Vagotomy < P=0.264 PUMP PUMP  Figure 35: Leptin treatment in ob/ob mice with a subdiaphragmatic vagotomy. Male ob/ob mice received a subdiaphragmatic vagotomy or sham surgery at 3.5-weeks-old. At 6-weeks-old mini-osmotic pumps were implanted delivering leptin (5 μg/day) for 14 days. (A) Body weight (B) food intake (C) blood glucose, and (D) insulin levels following a 4 hour fast on day 0 (basal) and day 5 (leptin) of leptin treatment.  Data are expressed as mean ± SEM, n=9 *P < 0.05 compared to ob/ob mice that received sham surgery.    86  Effect of subdiaphramatic vagotomy on leptin regulation of IGFBP-2 levels Plasma leptin levels were measured next to confirm administration via the mini- osmotic pumps.  Leptin administration resulted in leptin levels increasing equally in both groups (4.37 ± 0.64 ng/mL vagotomy vs. 3.67 ± 0.37 ng/mL sham surgery, P=0.18) (Figure 36A).  To determine if subdiaphramatic vagotomy abolished the ability of leptin to increase IGFBP-2, plasma IGFBP-2 levels were measured.  IGFBP-2 levels were low (32.4 ± 1.8 ng/mL vagatomy vs. 29.8 ± 0.13 ng/mL sham surgery, P=0.16) in both vagotomized and sham-operated mice prior to leptin treatment (day 0) and increased equally in response to leptin administration on day 5 of leptin treatment (231.3 ± 18.5 ng/mL vagotomy vs. 207.8 ± 21.0 ng/mL sham surgery, P=0.213) (Figure 36B). Le pt in  (n g/ m L) 0 1 2 3 4 5 6 Basal Leptin Sham Surgery Vagotomy P=0.175 P=0.184 Pl as m a IG FB P- 2 (n g/ m L) 0 50 100 150 200 250 300 Basal Leptin Sham Surgery Vagotomy P=0.213 P=0.260 BA  Figure 36: Leptin treatment leads to increased IGBP-2 levels in ob/ob mice with a subdiaphragmatic vagotomy.  Male ob/ob mice received a subdiaphragmatic vagotomy or sham surgery at 3.5-weeks-old.  At 6-weeks-old mini-osmotic pumps were implanted delivering leptin (5 μg/day) for 14 days. (A) Leptin levels and (B) plasma IGFBP-2 levels following a 4 hour fast on day 0 (Basal) and day 5 of leptin treatment (Leptin).  Data are expressed as mean ± SEM, n=9.     87  This ability of leptin to increase IGFBP-2 levels independently of vagal efferents suggests that leptin does not act via a mechanism involving the vagus nerve to increase circulating plasma IGFBP-2 levels.  Verification of subdiaphragmatic vagotomy procedure To be able to conclude that vagal efferents were not necessary for leptin to increase   IGFBP-2 levels in ob/ob mice, we assessed the completeness of the subdiaphragmatic vagotomy.  Food intake analysis in response to CCK was performed as the satiety effects of CCK are mediated by vagal afferents [121].  Food intake was significantly decreased by 26% in CCK injected (4 μg/kg, 30 min) sham operated mice compared to saline treated controls (0.36 ± 0.02 g vs. 0.49 ± 0.02 g respectively, P=0.003) (Figure 37A).  Subdiaphragmatic vagotomy abolished the satiety effect of CCK, as there was no difference observed between food intake in the vagotomized mice that received CCK compared to the vagotomized mice that were saline treated (0.40 ± 0.03 g vs. 0.41 ± 0.03 g respectively, P=0.44) (Figure 37A).  Furthermore we also measured stomach weight to verify vagotomy as previously reported [122].  Empty stomach weight of vagotomized ob/ob mice showed a 1.75-fold increase (0.39 ± 0.02 g vs. 0.22 ± 0.01 g respectively, P<0.0001) (Figure 37B) compared to that of sham operated controls.  Gastric distension was also observed in the vagotomized ob/ob mice (Figure 37C) (following a 16 hour fast) at time of tissue harvest.     88      Figure 37: Subdiaphragmatic vagotomy inhibits CCK induced satiety in ob/ob mice and causes gastric distension.  CCK was administered 8 weeks post surgery and 3 weeks post leptin administration. (A) Food intake 30 minutes post CCK administration (4 μg/kg i.p.) following a 16 hour fast (B) empty stomach weight at time of tissue harvest (C) images of full stomach at time of tissue harvest following a 16 hour fast.  Data are expressed as mean ± SEM, n ≥ 8.         Fo od  In ta ke  (g /3 0 m in ) 0.0 0.2 0.4 0.6 0.8 Sham Vagotomy P=0.003 Saline CCK P=0.438 A B C St om ac h W ei gh t ( g) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 P   0.0001 Sham Vagotomy <  89  STUDY 3: Characterization of the Spontaneous Recovery of ob/ob Mice The ob/ob mouse is frequently used as a model for T2D, as in addition to its obesity, these mice exhibit a diabetes-like syndrome of hyperglycemia, glucose intolerance, and elevated plasma insulin levels [17]. The manifestation of the diabetic syndrome however, seems to be highly dependent on the genetic background [169]. ob/ob mice on the C57BL/6 background display transient hyperglycemia that subsided when mice age to 16-20 weeks [170] while when on a C57BKS background, ob/ob mice become severely diabetic due to regression of islets and succumb to an early death [169].  Spontaneous recovery from fasting hyperglycemia in ob/ob mice at 22 weeks of age To determine the cause of this remission of hyperglycemia, we sought to characterize this spontaneous recovery of ob/ob mice on the C57BL/6 background.  ob/ob mice were tracked for various metabolic parameters starting from 4-weeks-old all the way to 50-weeks-old.  These metabolic parameters included body weight (Figure 38A) body composition determined by the lean:lipid ratio (Figure 38B), blood glucose (Figure 38C ) and insulin levels (Figure 38D) following a 4 hour fast.  The ob/ob mice remained hyperglycemic compared to wild-type controls until approximately 20-weeks of age. Between 20 and 25-weeks of age, fasting blood glucose levels of ob/ob mice started to decrease, and by 25 weeks of age, the fasting blood glucose of ob/ob mice was comparable to that of wild-type mice (Figure 38C).  At the time of remission to euglycemia, body weight still continued to increase (Figure 38A), and there was no change in fasting insulin levels (Figure 38D).   90  Time (weeks) 10 20 30 40 B lo od  G lu co se  (m M ) 0 5 10 15 20 25 C57Bl/6 ob/ob Time (weeks) 10 20 30 40 B od y W ei gh t ( g) 0 20 40 60 80 100 C57Bl/6 ob/ob A B C D Le an :L ip id  M as s 0 2 4 6 42-week-old10-week-old P    0.001< ob/ob C57Bl/6 P    0.001< Time (weeks) 10 20 30 40 Fa st in g In su lin  (n g/ m L) 0 5 10 15 20 25 30 35 40 C57Bl/6 ob/ob ** * ** * * *   Figure 38: Transient hyperglycemia in ob/ob mice on a C57BL/6 background. Male ob/ob mice were tracked from 4-weeks-old to 42-weeks-old. (A) Body weight (B) body composition presented at lean:lipid ratio (C) blood glucose levels, and (D) insulin levels following a 4 hour fast. Data are expressed as mean ± SEM, n ≥ 9 *P < 0.05 compared to C57BL/6 mice.      91  Old euglycemic ob/ob mice have a modest improvement in glucose tolerance and insulin sensitivity Although insulin levels remained constant throughout our study in the ob/ob mice, we further examined if the fasting euglycemia in the aged ob/ob mice was accompanied by improvement in glucose disposal and insulin sensitivity.  As expected, the young hyperglycemic ob/ob mice were severely glucose intolerant following an OGTT as blood glucose levels 120 minutes following the glucose gavage were still significantly elevated compared to wild-type controls (22.0 ± 1.9 mM vs. 9.6 ± 0.4 mM respectively, P<0.001) (Figure 39A). Once the mice became euglycemic, glucose tolerance was re-examined and we discovered that there was an improvement in glucose disposal.  An OGTT revealed that although blood glucose levels peaked higher compared to wild-type controls (28.3 ± 1.8 mM vs. 19.6 ± 1.1 mM respectively, P< 0.001) 20 minutes post glucose gavage, by 90 minutes following the glucose administration, blood glucose levels had returned to basal levels and were comparable to wild-type controls (13.5 ± 2.7 mM vs. 13.9 ± 1.8 mM respectively, P=0.45).  An ITT in young hyperglycemic ob/ob mice showed, as expected, that they were insulin resistant, as an i.p. injection of insulin had no glucose lowering effect (Figure 39B).  Once insulin sensitivity was revisited in the older, euglycemic ob/ob mice, it was discovered there was a modest improvement in insulin sensitivity.  At 60 minutes post i.p. insulin injection blood glucose levels were 75.3 ± 13.5% of basal blood glucose levels in the old euglycemic ob/ob mice, compared to the young hyperglycemic ob/ob mice which had blood glucose levels 96.2 ± 5.7% of basal levels, P=0.07, at the same time point (Figure 39C).  92  B Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 0 5 10 15 20 25 30 10-wk-old C57Bl/6 10-wk-old ob/ob 46-wk-old C57Bl/6 46-wk-old ob/ob * * * * * # # * * # # # C * * * * * * # # # # Time (min) 0 20 40 60 80 100 120 %  o f F as tin g G lu co se 0 50 100 150 200 250 300 10-wk-old C57Bl/6 10-wk-old ob/ob 46-wk-old C57Bl/6 46-wk-old ob/ob Time (min) 0 20 40 60 80 100 120 B lo od  G lu co se  (m M ) 10 20 30 40 9-wk-old C57Bl/6 9-wk-old ob/ob 40-wk-old C57Bl/6 40-wk-old ob/ob * *## * * * * * # A Figure 39: Glucose tolerance and insulin sensitivity in young hyperglycemic and old euglycemic ob/ob mice.  All experiments done following a 4 hour fast. (A) OGTT (1.5 mg/g glucose) (B) ITT (1 U/kg i.p. insulin) and (C) percent of fasting blood glucose following i.p insulin injection. Data are expressed as mean ± SEM, n ≥ 9 *P < 0.05 compared to C57BL/6 mice, # P < 0.05 young ob/ob mice compared to old ob/ob mice.  Old euglycemic ob/ob mice remain hyperglucagonemic  As diabetes is also associated with hyperglucagonemia which contributes to the hyperglycemia observed in T2D [171], we next investigated if alteration in glucagon levels were responsible for the age induced euglycemia. We measured glucagon levels at the various ages and various nutritional states.  Young ob/ob mice were hyperglycemic following a 4 and 16 hour fast and in the random fed state (Figure 40A) and this hyperglycemia was accompanied by hyperglucagonemia (Figure 40B).  As the mice aged, they became euglycemic not only following a 4 and 16 hour fast, but also during the random fed state.  Even though the old, euglycemic mice now had significantly lower blood glucose levels compared to their littermate controls (Figure 40C) following a 4 hour fast (9.1 ± 0.9 mM vs. 11.2 ± 0.5 mM respectively, P=0.02) as well as in the random fed state (7.9 ± 0.6 mM vs. 11.0 ± 0.5 mM respectively, P<0.001), these mice still remained hyperglucagonemic, with glucagon levels at least 2 fold higher than wild-type  93  controls during the various fasting times as well as during random feeding (Figure 40D). Therefore, euglycemia was present in the old ob/ob mice, irrespective of the hyperglucagonemia which remained consistent throughout the aging process in the fasted and fed state. YO U N G O LD 4 hr Fast 16 hr Fast 4 hr Fast 4 hr Fast16 hr Fast 16 hr FastRandom B lo od  G lu co se  (m M ) 0 5 10 15 20 25 30 C57BL/6 ob/ob P=0.02 P=0.23 P  0.01< B lo od  G lu co se  (m M ) 0 5 10 15 20 25 30 C57BL/6ob/ob P  0.01< P  0.01< P  0.01< 4 hr Fast 16 hr Fast Random A C G lu ca go n (p g/ m L) 0 50 100 150 200 C57BL/6 ob/ob P  0.01< P=0.12 P  0.01< Random G lu ca go n (p g/ m L) 0 50 100 150 200 C57BL/6 ob/ob P  0.01< P  0.01 P  0.01 < < B D Random  Figure 40: Glucagon levels during fasting and feeding in ob/ob mice.  (A) Blood glucose levels and (B) glucagon levels following a 4 and 16 hour fast and during random feeding (10- week-old) mice. (C) Blood glucose levels and (D) glucagon levels following a 4 and 16 hour fast and during random feeding in old (41-week-old) mice. Data are expressed as mean ± SEM, n ≥ 9.    94  DISCUSSION The discovery of leptin, an adipocyte derived hormone that functions as an afferent signal in a negative –feedback loop to maintain appropriate levels of adipose mass [18] and energy expenditure, has advanced our understanding of the specific mechanisms that underlie the regulation of energy balance.  Mutations in leptin, or leptin receptor, in both animals [17] and humans [30, 172, 173] are associated with profound metabolic abnormalities which include obesity, reduced energy expenditure, dyslipidemia, as well as alteration in glucose homeostasis including hyperinsulinemia [93] and insulin resistance [93].  This wide range of phenotypic abnormalities observed in the absence of leptin action, and their reversibility by leptin replacement therapy [26, 27, 174], provides compelling evidence for the existence of multiple physiological action of the hormone.  As the most common alteration in energy balance, obesity, is tightly associated with insulin resistance, the current dogma is that alterations in glucose homoeostasis often develop secondary to the obesity induced insulin resistance [175]. Although changes in food intake and body adiposity can clearly affect insulin sensitivity in peripheral tissues, several observations suggest leptin regulation of glucose homeostasis occurs independently of its effects on food intake.  Impaired glucose metabolism is characteristic of genetic models of leptin deficiency regardless of whether the animals are obese (ob/ob mice) [17, 18] or lean (models of lipodystrophy) [88, 176] and leptin treatment corrects these metabolic abnormalities via a mechanism independent of food intake or body weight changes [26, 54, 92, 97].  Although these finding point to an important role of leptin in glucose homeostasis in each of these syndromes, the contribution of leptin deficiency relative to other associated features of these conditions is  95  unclear and it has been difficult to delineate the precise physiological response to a leptin deficiency in normal animals without these confounding alterations.  With this in mind, we set out to characterize the specific biological response to an acute leptin deficiency in a normal animal, resulting from the attenuation of endogenous leptin signalling.  To achieve this, we utilized a potent leptin antagonist (PEGylated mouse leptin antagonist, PEG-MLA) which acts as a competitive inhibitor of leptin [128].  We wanted to evaluate the importance of endogenous leptin action on body weight, energy homeostasis and glucose metabolism, as well as determine the hierarchy of leptin action on energy and glucose homeostasis.  By inducing a state of acute leptin deficiency in otherwise normal animals, we investigated the progression of metabolic abnormalities in our model of acute leptin deficiency compared to those observed in an animal model of congenital leptin deficiency (ob/ob mice).  Energy Homeostasis in a State of Acute Leptin Deficiency A congenital leptin (ob/ob mice) or leptin signalling (db/db mouse) deficiency results in obesity, hyperphagia, as well as decreased energy expenditure [17].  These metabolic alterations are due to specifically the absence of leptin action, as leptin replacement therapy has been shown to potently induce weight loss and decrease food intake [26, 97], while increasing energy expenditure [26, 60].  Therefore, the effects of an acute disruption of leptin signalling on appetite, body composition and energy balance were examined first using indirect calorimetry.  This approach allowed us to examine defined parameters of energy homeostasis such as EE and RQ.  We observed that mice receiving delivery of PEG-MLA (72 µg/day) had a significant increase in food intake by  96  the second day of treatment, which persisted until the end of PEG-MLA administration. The increase in food intake was also an indication that our mode of PEG-MLA delivery was working and that we had induced a state of leptin deficiency due to the well defined effects of leptin action (or lack thereof) on food intake [17, 27, 169].  In combination to the increased food intake we also observed that the RQ in the PEG-MLA treated mice was significantly higher compared to control mice.  The RQ is defined as the volume of CO2 produced divided by volume of O2 consumed, where the relative amount of O2 consumed versus CO2 produced depends on the substrate.  When carbohydrates are metabolized, the theoretical RQ is 1.0, for protein it is 0.83, and for fat, it is 0.70 [177]. The increase in RQ suggested that the PEG-MLA treated mice were switching their energy partitioning from fat oxidation to carbohydrate metabolism.  This is in agreement with data that showed ob/ob mice have increases RQ [60] and the fact that a single dose of leptin is able to markedly reduce the RQ [60], forcing energy partitioning to increased fat oxidation [60].  While ob/ob mice have significantly reduced EE, surprisingly PEG- MLA treatment had no effect on EE during the 3 day period of treatment.  Leptin treatment in wild-type mice has been shown to prevent the fall in energy expenditure observed during starved states (food restriction) to further conserve energy [178] but in contrast, in free-feeding mice, even a high dose of leptin treatment did not change daily energy expenditure above base line values [178].  It is possible that because PEG-MLA treatment only resulted in a modest increase of food intake that the PEG-MLA treated mice were not yet in state of perceived starvation like the the ob/ob mice, and alteration in energy expenditure to conserve energy were not yet necessary.  Perhaps if PEG-MLA  97  treatment was continued for a longer period of time, alterations in energy expenditures would have been observed. Physical activity is also an important component of energy expenditure, and it has been shown that administration of leptin to leptin deficient animals elevates spontaneous physical activity [179].  Utilizing the beam break system of the metabolic cages, we were able to assess spontaneous activity in the PEG-MLA treated mice.  No significant differences were observed in total locomotor activity or ambulatory movement, although in the last 12 hour light phase and 12 hour dark phase, there was a trend that PEG-MLA treated mice were less active compared to controls.  These data imply that physical activity is not acutely regulated by leptin, as it has been shown that longer treatment with a leptin antagonist (L39A/D40A/F41A)  in rats leads to decreased activity as determined by voluntary wheel running [180].  Using an adeno-associated virus (rAAV)-mediated gene delivery to centrally over express a leptin antagonist in rats, resulted in reduced free-wheel running by up to 40%, but this effect was only seen 30 days after viral vector administration [180].  Therfore, as no significant difference was reached in physical activity in PEG-MLA treated mice compared to control indicating that spontaneous physical activity along with energy expenditure is not high up in the hierarchy of leptin action. Abnormal thermoregulation and adaptive thermogenesis is found in congenital leptin deficiency, at least in part due to decreased activation of leptin sensitive sympathetic nerve innervations of brown adipose tissue [130, 181].  A key mediator of thermogenic adaptation is thought to be uncoupling protein 1 (UCP-1) which is expressed almost exclusively in the mitochondria of brown adipose tissue (BAT) where it uncouples  98  cellular ATP synthesis such that heat is generated [182, 183].  Leptin administration to mice results in increased levels of UCP-1 mRNA and protein expression [184].  In UCP- 1 ablated mice, leptin looses its thermogenic effect [184], indicating that BAT is essential for leptin induced thermogenesis. As adaptive non-shivering thermogenesis has effects on energy balance [185, 186] during dietary challenges, we investigated if an acute leptin deficiency would affect thermoregulation.  No difference was observed in our PEG-MLA treated mice compared to controls in regards to the ability to retain normal body temperature nor was cold intolerance induced (Figure 9).  Untreated ob/ob mice observed in parallel to the PEG- MLA treated mice, as an internal control of the experimental technique, displayed lower basal body temperature and cold intolerance compared to normal mice in accordance to previously published data [26, 181].  The fact that the acute leptin deficiency we induced in normal mice did not lead to altered thermogenesis can be potentially explained by the duration of the leptin deficient state.  Although UCP-1 mRNA levels can be altered within hours (i.e. acute cold exposure), a long time delay of up to weeks has been shown to take place where alterations in UCP-1 mRNA amount result in corresponding changes in amount of UCP-1 protein.  Therefore, if lack of leptin signalling did have an effect on UCP-1 expression levels, more time would be necessary for this to change to result in altered UCP-1 protein levels and potentially dysregulated thermoregulation.  Overall, the effect of the acute leptin deficiency induced in normal mice on energy homeostasis was modest.  An increase in food intake did lead to a significantly higher weight gain in PEG-MLA treated mice, although this weight gain of ~0.5 grams did not result in significantly heavier mice compared to controls, nor did PEG-MLA  99  treatment alter body composition as assessed by the lean:lipid mass ratio.  The increase in RQ suggested in the acute state of leptin deficiency mice were initiating a switch in the metabolism of energy source, from fat oxidation to carbohydrate metabolism.  Energy expenditure and thermoregulation remained unchanged in the PEG-MLA treated mice. As our model of leptin deficiency did not result in severe obesity, hyperphagia and reduced EE as observed in congenital leptin deficiency animal models, we went on to complete our characterization of the physiological response to an acute leptin deficiency with regard to glucose metabolism without these confounding metabolic alterations observed in the congenital model of leptin deficiency.  Glucose Metabolism in a State of Acute Leptin Deficiency Significantly increased fasting insulin levels were observed on day 3 of PEG- MLA treatment (72 µg/day) (Figure 10) as well as on day 4, and 7 of PEG-MLA treatment with the lower 36 µg/day dose (Figure 11).  In the lower dose of 36 µg/day PEG-MLA for 7 days, we also observed that insulin levels remained significantly elevated 3 days after cessation of PEG-MLA treatment by surgical removal of pump (Figure 11B).   These results that disrupted leptin signalling in vivo in normal mice resulted in increased insulin levels is in agreement with previous studies that reported that ß-cell specific attenuation of leptin signalling in vivo leads to hyperinsulinemia in mice [187, 188] as well as evidence that direct leptin action on ß-cells is involved in suppression of insulin secretion [74].  The functioning OB-Rb is expressed on ß-cells and leptin has been shown to suppress insulin secretion by activating ATP-sensitive K+ channels causing hyperpolarizing of the ß-cell, suppressing insulin secretion [74, 103].  100  These results suggest that PEG-MLA was able to prevent the binding of endogenous leptin to OB-Rb on ß-cells so that endogenous leptin was no longer able to curtail insulin secretion.  Increased glucose stimulated insulin secretion was also observed in PEG-MLA treated mice (Figure 13B) suggesting that leptin signalling is also important in regulating insulin secretion in both the fasting and post-prandial state.  These observations indicated that in normal mice, one of the important physiological actions of leptin is to directly regulate insulin secretion. Although PEG-MLA treatment resulted in significantly higher insulin levels, fasting blood glucose levels as well glucose disposal following an oral glucose gavage remained unchanged in PEG-MLA treated mice.  Recently it has been shown that the OB-Rb is expressed on pancreatic α-cells and leptin has inhibitory actions on glucagon secretion in vitro [131].  We therefore measured glucagon levels following PEG-MLA treatment to determine if lack of leptin signalling resulted in elevated glucagon levels in conjunction with elevated insulin levels, which could potentially explain why we saw no change in fasting blood glucose levels.  Glucagon levels were measured following a 4 hour fast on the 3rd day of PEG-MLA treatment (72 µg/day), under the same conditions where insulin levels were significantly elevated in PEG-MLA treated mice.  Glucagon levels in PEG-MLA treated mice were not elevated and were comparable to the vehicle treated controls.  This can be potentially explained by other counter-regulatory measures that inhibit glucagon secretion in the presence of high blood glucose since the 4 hour fast was not sufficient enough to induce glucagon production.  The finding that leptin has the ability to regulate glucagon secretion from α-cells in vivo was under conditions of low glucose levels [131].  Further studies looking at glucagon levels following longer fasting  101  times, when blood glucose levels are lower would be informative.  Therefore, the maintenance of normal fasting glucose and glucose disposal in the presence of elevated insulin levels did not results from elevated glucagon levels.  Leptin not only regulates glucose homeostasis through direct effects on the ß-cell to regulate insulin secretion, but by also augmenting peripheral insulin sensitivity.  It has been previously reported that insulin resistance is a prominent feature in animal models of leptin deficiency and that leptin has insulin sensitizing effects [27, 174].  More specifically, leptin has been shown to regulation insulin sensitivity via the insulin receptor substrate-phosphatidylinositol-3-OH kinase (IRS-PI3K) pathway [189, 190]. Hypothalamic leptin action is also associated with increase insulin signal transduction via the PI3K pathway in liver but not muscle [191].  Recently it has also been shown that leptin deficiency plays a key role in the pathogenesis of insulin resistance [190].  German and colleagues demonstrated that leptin replacement therapy at physiological levels (leptin levels comparable to plasma leptin in non-diabetic animals) was able to prevent insulin resistance in STZ treated rats by a mechanism that was unrelated to food intake or body weight changes [190].  Consistent with these data that leptin plays an important role in regulating insulin sensitivity, treatment with PEG-MLA (72 µg/day for 3 days) and the disruption of leptin signalling resulted in whole body insulin resistance as determined by an ITT.  Leptin deficiency is not only associated with decreased whole body insulin resistance but hepatic insulin resistance which is results in increased hepatic glucose production (HGP) in ob/ob mice [132].  We examined the affect of acute leptin deficiency on hepatic insulin resistance by utilizing the hyperinsulinemic-euglycemic  102  clamp technique [192].  The hyperinsulinemic-euglycemic clamp technique is the gold standard for the evaluation of insulin sensitivity and allows for measurement glucose metabolism and insulin sensitivity of individual organs [193]. PEG-MLA treatment (72 µg/day, 3 days) resulted in a significantly lower glucose infusion rate required to maintain euglycemia during the hyperinsulinemic clamp compared to control treated mice, indicating whole body insulin resistance and corroborating our previous results from the ITT.  We also observed that inhibition of HGP during the hyperinsulinemic state was significantly decreased in the PEG-MLA treated mice compared to control mice (Figure 18), suggesting hepatic insulin resistance.  These data provide complimentary findings to the study by Rossetti et al. [194].  They showed that leptin action was able to acutely regulate peripheral and hepatic insulin action [194].  In weight matched rats, short term administration of  leptin (6 hours) during a hyperinsulinemic-euglycemic state resulted in enhanced ability of insulin to suppress HGP by decreasing gluconeogenesis [194]. To further determine if PEG-MLA augments insulin sensitivity and alters insulin stimulated uptake of glucose in other tissues (i.e. muscle, fat), mice were administered a radiolabelled glucose analog isotope (14C labeled 2-deoxyglucose) during the last 30 minutes of the hyperinsulinemic clamp.  The use of the this radiolabelled glucose analog allows for quantification of glucose uptake by tissues, as this compound it taken up by the tissue but unlike glucose, it is not further metabolized.  These data have yet to be analyzed but will provide further knowledge if an acute leptin deficiently leads to alteration of glucose uptake by liver, fat, and muscle.  103   Hepatic insulin resistance has also been implicated in hepatic TG accumulation and steatosis [136, 137].  To investigate if lipid accumulation due to the acute leptin deficiency was contributing to the hepatic insulin resistance present in PEG-MLA treated mice, heptic lipid content was measured.  A heptic lipid extraction was performed on livers from mice receiving PEG-MLA at the same dose and duration that resulted in peripheral insulin resistance and reduced hepatic insulin sensitivity.  The hepatic lipid extraction reveled that TG and cholesterol levels were comparable to control treated mice.  It has been previously shown that leptin action in the hypothalamic actuate nucleus is able to improve hepatic insulin action via a mechanism involving the hepatic vagus nerve [189, 191].  As PEG-MLA treatment has been shown to induce potent peripheral as well as central leptin deficiency in vivo [128] we hypothesize that the induced central leptin deficiency is responsible for the alterations in hepatic insulin sensitivity as hepatic lipid accumulation did not occur in the time frame of our study. Taken together, these findings demonstrate that leptin signalling is important in regulating insulin secretion in the fasting and post-pyramidal state.  Leptin also has direct effects on the liver to augment insulin sensitivity and regulate hepatic glucose production as the acute leptin deficiency in normal mice resulted in increased HGP and hepatic insulin resistance.  The alterations  prior to changes in body composition and energy expenditure indicate that hyperinsulinemia and insulin resistance can develop in the absence of obesity and that leptin has an important physiological role in regulating glucose metabolism independently of its effect on energy homeostasis.   104  Dose Dependent Effect of Leptin on Glucose Homeostasis The importance of leptin action on glucose homeostasis and metabolism has been demonstrated by ability of leptin to reverse hyperglycemia and increase insulin sensitivity in obese [27, 97] and lean [92, 176] models of T2D independently of effects on food intake and body weight [54, 57, 92].  We carried out a leptin dose response in ob/ob mice to determine the specific doses required to correct the different abnormalities observed in ob/ob mice such as the obesity, hyperglycemia, hyperinsulinemia, glucose intolerance, and insulin resistance.  Leptin was delivered continuously for a period of 28 days via mini-osmotic pumps that were implanted subcutaneously in ob/ob mice.  The maximum effect on metabolic parameters was achieved with the highest dose of leptin (5 µg/day), which resulted in 25% loss of initial body weight as well as reversed all examined metabolic abnormalities in the ob/ob mice. Treatment resulted in fasting blood glucose, insulin levels, glucose tolerance and insulin sensitivity comparable to levels observed in normal wild-type animals were observed. The 1 µg/day dose resulted in improvements of all metabolic parameters we assess without the associated effect of weight loss (weight gain reached a plateau during the first couple of days of treatment).  We observed that the lowest dose of leptin (0.2 µg/day) had no effect on body weight or weight gain, insulin levels or insulin sensitivity, but was able to improve the hyperglycemia as well as glucose tolerance in the ob/ob mice.  Although we did not measure food intake, we speculate that food intake remained unchanged as well with the 0.2 µg/day leptin dose, as these mice continued to gain weight throughout the duration of leptin delivery at the same rate as PBS treated ob/ob mice.  Therefore, with the use of 0.2 µg/day leptin we were able to segregate the effects of leptin on glucose homeostasis and body weight regulation.  These  105  data indicate that a hierarchy of leptin action exists and is constistant with previous work showing acute leptin treatment in the fasting state has glucose lowering effects independently of food intake and body weight [73]. Recently it has been published that the anti-diabetic effects of leptin that are independent of leptin induced weight loss, are mediated at least in part by IGFBP-2 [132].  Leptin infusion that corrects the hyperglycemia and insulin resistance in ob/ob mice at doses that do not result in weight loss potently increases IGFBP-2 gene expression and circulating IGFBP-2 protein levels suggested that this induction of IGFBP-2 was responsible for the effect of leptin on glucose metabolism [132]. Over- expression of IGFBP-2 in the liver of ob/ob mice, its normal site of expression [195, 196], using a recombinant adenovirus construct resulted in the amelioration of diabetes [132] confirming the above hypothesis. The adenoviral approach  used by Hedbacker et al. [132] to deliver IGFBP-2 to ob/ob mice, resulted in pharmacological levels of circulating IGFBP-2 protein levels which were 20 fold higher than normal measured levels in wild-type mice (6000 ng/mL in Ad-IGFBP-2 treated mice vs. ~350 ng/mL in wild-type mice).  To determine if similar anti-diabetic effects of IGFBP-2 would be observed delivering IGFBP-2 at physiological doses, we implanted mini-osmotic pumps subcutaneously in ob/ob mice delivering 1500 ng/day recombinant mouse IGFBP-2 for 7 days.  At this dose and mode of delivery we did not see any significant effect of recombinant mouse IGFBP-2 on fasting blood glucose and fasting insulin levels.  The ob/ob mice were also equally glucose intolerant and insulin resistant after 7 days of IGFBP-2 treatment compared to PBS treated controls. The lack of improvement in any of the metabolic abnormalities observed in the ob/ob  106  mouse by IGFBP-2 replacement therapy can be attributed to that fact that we were unable to increase circulating plasma IGFBP-2 above basal levels.  There could be several explanations for this.  Previous reports state that the IGFBP-2 protein has numerous disulfide bonds [197] making it difficult to produce as a recombinant protein in sufficient quantities to assess its in vivo function.  It is possible that the recombinant IGFBP-2 protein was not biologically active as disulfide linkages of cysteines are important for correct folding and maintenance of three-dimension structure and can affect the ability of a protein to exert its biological action.  The half-life of IGFBP-2 has also been determined to be in the order of 1-2 hours [198] so there is a chance that the rate of clearance was faster than the rate of administration.  It is also possible that there was technical problem in our mode of delivery.  There could have been a malfunction in the mini-osmotic pumps and the lack of increase in IGFBP-2 levels because our system of peptide administration failed to deliver the peptide.  Mechanism by Which Leptin Increases Plasma IGFBP-2 Levels Leptin treatment has also been shown to significantly increase IGFBP-2 levels in ob/ob mice [132].   To determine the effect our leptin dose response in ob/ob mice had on IGFBP-2 levels, plasma samples were assayed for IGFBP-2.  In accordance with data published by Hedbacker et al. we saw that even at the lowest dose of leptin (0.2 µg/day), which only had effects on glucose homeostasis and not body weight, leptin was able to significantly increase IGFBP-2 levels compared to PBS treated mice.  The effect of leptin on circulating levels of IGFBP-2 was dose dependent, and the highest dose of leptin treatment (5 µg/day) was able to increase plasma IGFBP-2 to levels found in normal,  107  wild-type mice.  These data further establish the role of leptin in regulating plasma IGFBP-2 levels but the mechanism for this induction still remained unknown. In order to examine whether leptin regulates IGFBP-2 as a result of direct effects on the liver, ob/ob mice with tissue specific attenuation of leptin receptor signalling in the liver were utilized.  These Leprflox/flox Albcre+ ob/ob and their littermate controls not carrying the cre transgene (Lerpflox/flox Albcre- ob/ob mice) were obese, hyperglycaemic, hyperinsulinemic, and had undetectable leptin levels as well as low plasma IGFBP-2 levels comparable to normal ob/ob mice on the C57BL/6 background.  Leptin was administered to these mice via mini-osmotic pumps delivering a continuous dose of 5 µg/day to determine if IGFBP-2 levels would increase in the absence of hepatic leptin signalling.  Plasma analysis of IGFBP-2 levels following leptin administration showed that hepatic leptin signalling was not necessary for leptin to increase circulating IGFBP-2 levels.  Plasma IGFBP-2 levels increased in time dependent manner in both Leprflox/flox Albcre+ ob/ob mice and their littermate controls, and levels returned to normal following cessation of leptin treatment.  There were even several time points where Leprflox/flox Albcre+ ob/ob had significantly higher IGFBP-2 levels compared to controls.  This observation can be potentially explained by the fact that the Leprflox/flox Albcre+ ob/ob mice also had significantly higher circulating leptin levels during leptin treatment.  The reason for the statistically different circulating leptin levels in the two groups remains unclear as the dose of leptin administration and method of delivery was same for both groups of mice. We speculate that perhaps lack of hepatic leptin signalling in the Leprflox/flox Albcre+ ob/ob mice alters the clearance rate of leptin.  Overall, our data show that leptin does not regulate IGFBP-2 levels as a direct result of hepatic leptin signalling.  108  Alternately, available evidence supports a theory that leptin can act centrally to activate efferent signals to the liver, which results in an increase of circulating IGFBP-2 levels [91].   In the leptin deficient lipodystrophy mice, intracerebroventricular delivery of leptin is able to up-regulate IGFBP-2 gene expression in the liver [91].  Also consistent with a central site of action for leptin, a brain specific knockout of the leptin receptor is able to recapitulate the ob/ob phenotype [43], whereas a mouse model with a disruption in peripheral leptin signalling which retains intact leptin receptors in the brain is without ob/ob associated metabolic abnormalities [199].   These data indicate the central leptin signalling plays a significant role in mediating the effect of leptin on energy homeostasis and glucose metabolism.  To investigate if leptin acts centrally and signals to the periphery to increase plasma IGFBP-2 levels via efferent signals, we utilized ob/ob mice with a subdiaphragmatic vagotomy which were purchased from Jackson labs (Bar Harbor, ME).  Before leptin administration was started, we noted some key difference in the ob/ob mice with the subdiaphragmatic vagotomy compared to the sham operated controls.  The vagotomised mice weighed significantly less, as well as consumed less food compared to sham operated control.  These differences in body weight can be explained at least in part by the difference in food intake.  The vagotomised ob/ob mice also had significantly lower fasting insulin levels and higher fasting blood glucose levels compared to the sham operated controls.  These observations can be explained by the important role of the vagus nerve in the regulation of blood glucose levels through innervations of the liver [200] and pancreas [201].  Vagal efferent activation elicits the release of acetylcholine at the level of the ß-cell which has stimulatory effects on insulin secretion [202].   Therefore, the absence of vagal innervations in the vagotomised mice is  109  probably the main contributor for the lower insulin levels observed compared to sham operated controls.  It has also been shown that a vagotomy results in an increase in HGP [200, 203], so it is likely that this effect contributes to the severity of the hyperglycemia observed in the vagotomised mice, in addition to the lower insulin levels.  Despite the initial difference in the vagotomised and sham operated mice, continuous leptin treatment via mini-osmotic pumps (5 µg/day) was able to induce weight loss, reduce food intake, and decrease fasting blood glucose and insulin levels to the same extent in both vagotomised and sham operated mice.  Leptin administration also resulted in equal levels of circulating leptin in both vagotomised and sham operated mice.  Moreover, we observed that leptin administration was able to increase circulating IGFBP-2 levels in both the vagotomised and sham operated mice to an equal extent.  These data indicate that leptin does not act centrally to signal to the liver via vagal efferents to increase IGFBP-2 production and circulating levels.  We tested the completeness of the vagotomy by treating mice with CCK, a gut derived hormone with well established effects on satiety mediated by vagal afferents [121].  CCK administered to the sham operated mice was able to significantly reduce food intake compared to saline treated controls and this satiety inducing effect of CCK was abolished in the vagotomised mice.  We observed no difference between food intake in the vagotomized mice that received CCK compared to the vagotomized mice that were saline treated (Figure 37A).  We further assessed the vagotomy procedure by measuring stomach weight and gastric distension as a vagotomy abolishes the cues of satiety in response to gastric distension [122].  Upon tissue harvest, extensive gastric distension and  110  increase in stomach weight was observed in the vagotomised mice compared to sham operated controls providing further evidence that the surgical procedure was successful.  The experiments designed to determine the mechanism of leptin action to increase plasma IGFBP-2 levels were successful in eliminating various pathways, but did not lead to the determination of a specific mode of action of how leptin administration is able to increase plasma IGFBP-2 levels.  Our data shows that leptin does not act directly on the liver via hepatic leptin signalling to increase circulating plasma IGFBP-2 levels.  Further investigation of a central leptin action also showed that even when the communication between the brain and liver is severed by a subdiaphragmatic vagotomy, leptin was still able to increase IGFBP-2 levels.  Therfore the specific mechanim of how leptin increases circulating plasma IGFBP-2 levels remains to be elucidated.  Future studies that can shed light on peripheral versus central action of leptin in the regulation of increased expression and circulation of IGFBP-2 are required.  Although we showed that leptin doesn’t act on liver directly to increase IGFBP-2 levels, its possible it could have peripheral effects by acting on other tissues to induce IGFBP-2 gene expression and secretion.  It has been recently reported that IGFBP-2 is expressed by subcutaneous adipocytes of obese children [204] and expression of IGFBP-2 in vitro occurs during adipogenesis [162].   It would be informative to measure IGFBP-2 gene expression following leptin administration in adiopcytes of ob/ob mice to determine if fat contributes to the increase in circulating IGFBP-2 levels.  To further examine the central effects of leptin on regulating plasma IGFBP-2 levels, leptin treatment of mice that lack neuronal leptin signalling (ie ObRSynI KO mice) [43] would help determine if a signal to increase IGFBP-2 is initiated centrally by leptin.  If leptin treatment in the ObRSynI KO  111  mice does not lead to increased plasma IGFBP-2 levels that would confirm that central leptin action is important in increasing IGFBP-2 levels and further investigation of how these central signals are mediated to the periphery could be investigated. Thus a better understanding of the source or sources of IGFBP-2 production and IGFBP-2 regulation will add significant information in the search of the exact mechanism of leptin regulation of circulating IGFBP-2.  Basal GLP-1 Levels in ob/ob Mice and Effect of Leptin Treatment Although low levels of GLP-1 in a setting of obesity and diabetes has been reported [148-150], results from our leptin dose response study showed that the ob/ob mouse, a rodent model of obesity and T2D, had significantly higher, biologically active GLP-1 levels which were significantly lowered following leptin treatment.  We hypothesized that this increase in GLP-1 levels in the normal ob/ob mouse prior to the leptin treatment was due to the hyperphagic nature of ob/ob mice [17, 169] since nutrient intake is potent stimulus for the increase of GLP-1 levels in the circulation [155, 156]. We tested our hypothesis by fasting the ob/ob mice short term and long term to determine if food restriction would have a GLP-1 lowering effect.  Indeed, while fasting had minimal effects on GLP-1 levels in wild-type mice, GLP-1 levels in ob/ob mice were significantly decreased following a 16 hour fast, when compared to levels after a short term fast of 4 hours.  Our theory that food intake is responsible for the elevated GLP-1 levels in the ob/ob mice is further supported by our experiment that showed a 2 hour period of feeding following a 16 hour fast resulted in increased GLP-1 levels in both ob/ob and wild-type mice that were even higher than the GLP-1 levels in ob/ob mice after  112  a 4 hour fast (Figure 25).  Therefore, the elevated GLP-1 levels we observed in 4 hour fasted ob/ob mice can also be obtained by wild-type mice following feeding, to an even greater extent.  The fact that the ob/ob mice are in a perceived state of starvation continuously consuming food is the likely explanation for the constant elevation of GLP- 1 levels in the normal untreated ob/ob mice.  These high GLP-1 levels in ob/ob mice were also reduced by exogenous leptin treatment, in a dose dependent manner.  It is also highly likely that the effect of leptin on GLP-1 levels was secondary to the well known effect of leptin in decreasing food intake [27, 28].  The low dose of leptin that had no effect on weight loss or weight gain, and therefore we assume no effect on food intake, had no lowering effect on GLP-1 levels in the ob/ob mice.  The 1 µg/day dose of leptin that prevented weight gain possibly due to alterations in food intake, and the 5 µg/day dose that induced significant weight loss both resulted in lower GLP-1 levels that were omparable to wild-type mice.  Therefore, we conclude that the of elevated GLP-1 levels in the ob/ob mice as well as the ability of leptin to decrease levels is secondary to the food intake observed in ob/ob mice and the effect of leptin to reduce it.   Transient Hyperglycemia in ob/ob Mice on a C57BL/6 Background We tracked young, hyperglycemic ob/ob male mice on the C57BL/6 background to characterize the transient hyperglycemia [170, 205] and determine alteration in other metabolic parameters such as glucose tolerance and insulin sensitivity as the mice aged. These ob/ob mice were hyperglycemic compared to wild-type controls until approximately 20 weeks of age when some animals in the cohort became euglycemic. By 26 weeks of age, the average blood glucose of the ob/ob mice being tracked was not  113  significantly different from the wild-type controls.  This remission of hyperglycemia was also associated with decreased urine output, as their cages no longer needed to be changed multiple times a week. Glucose tolerance and insulin sensitivity performed when the ob/ob mice were young and hyperglycaemic showed that these mice were glucose intolerant and insulin resistant.  Re-assessment of glucose tolerance and insulin sensitivity when the mice were older and euglycemic revealed that there was a modest improvement in both glucose tolerance and insulin sensitivity.  Another interesting observation of this study was not only that these mice became euglycemic with age despite continued weight gain, but that their blood glucose continued to improve so that by the end of the study, these ob/ob mice had significantly lower fasting and random fed glucose levels than their age-matched wild-type controls despite their severe obesity (Figure 40).  By 40 weeks of age the euglycemic ob/ob mice weighed twice as much compared to their younger hyperglycaemic state and had a 2-fold decrease in their lean:lipid ratio as determined by NMR.  Overall, besides their severe obesity, the ob/ob mice become euglycemic and had a modest improvement in glucose tolerance and insulin sensitivity without any signs of deteriorating health. Remission from hyperglycemia in ob/ob mice on the C57BL/6 background is hypothesized to correlate with a compensatory hyperplasia and hypertrophy of the pancreatic ß-cells [169].  This resultant hyperinsulinemia is then assumed to account for the remission of hyperglycemia after approximately 16 weeks of age [169].  In our tracking of ob/ob mice from a young hyperglycemic age to the older euglycemic state, we observed that insulin levels in 10 week old hyperglycemic mice were comparable to levels observed in the 35 week old euglycemic mice (Figure 38D).  Therefore, these data  114  suggest that alterations in insulin levels are not responsible for the transient hyperglycemia observed in this mouse model. Besides elevated insulin levels and insulin resistance, ob/ob mice are also hyperglucagonemic [206].  This excess of glucagon in the setting of insulin resistance contributes to the development of hyperglycemia in the ob/ob mouse model [206], and accumulating evidence supports a pathophysiological role of glucagon in the development and progression of diabetes [171].  The role of glucagon in diabetes progression is also supported by studies that showed that by antagonizing the action of glucagon with glucagon receptor antagonist, or reducing glucagon receptor expression with antisense oligonucleotides results in amelioration of diabetes in rodent models [207, 208].  We observed that in the young hyperglycemic animals both basal and fed glucagon levels were inappropriately elevated.  We hypothesized that if glucagon levels decreased as the ob/ob mice aged due to α-cell exhaustion, that the resulting decrease in glucagon levels could be a potential contributor to the reversal of hyperglycemia in ob/ob mice. However, analysis of glucagon levels revealed that the transient hyperglycemia was not due to alteration of glucagon levels as the mice aged, as plasma glucagon concentrations were inappropriately raised irrespective of age or nutritional status.  Although our attempt to characterize the spontaneous recovery of the ob/ob mouse on the C57BL/6 background did not produce a mechanism for the improvement of glucose levels and mild improvement in glucose tolerance and insulin sensitivity, future directions are proposed that can be investigated with available plasma and tissues collected from these old, severely obese, euglycemic mice.  115    A literature search on animal models of increased obesity that paradoxically lead to improvement in glucose metabolism and insulin sensitivity revealed several interesting findings.  Adiponectin, an adipocyte derived protein, has been found to modulate a number of metabolic processes including glucose metabolism regulation and fatty acid catabolism [209].  Over-expression of adiponectin in ob/ob mice resulted in a novel animal model of severe obesity with an improved metabolic profile [210].  It was the ability of adiponectin to expand adipose tissue preventing ectopic lipid accumulation that resulted in the normalization of all the metabolic abnormalities observed in the ob/ob mouse model.  Adiponectin levels were only increased 2-3 times above baseline, indicating that levels within the physiological range were sufficient for this effect of fat mass expansion.  Interestingly, the obesity in the ob/ob mouse is of hypertrophic- hyperplastic type, where both the size and number of adipocytes are increased [211], which is in contrast to most other rodent models where adipocyte enlargement is responsible for the massive fat stores [169].  Is therefore possible, that levels of adiponectin increase with age, leading to the continued weight gain where the increase in fat mass is actually beneficial in the ob/ob mice, leading to the observed improvement in fasting blood glucose levels, and modest improvements in glucose tolerance and insulin sensitivity.  Therefore, it would be worthwhile to measure plasma adiponectin levels in young hyperglycemic and old euglycemic mice to help determine if alterations in adiponectin levels correlate to the observed transient hyperglycemia in the ob/ob mice on the C57BL/6 background. An increase in fat mass and weight gain associated with increased insulin sensitivity and reversal of diabetic symptoms is also observed with the use of a relatively  116  new class of oral anti-diabetic drugs, thiazoledinediones (TZDs).  TZDs are a group of agonists for the peroxisome proliferator-activator receptor-γ (PPAR-γ).  PPAR-γ is a ligand-activated transcription factor that is preferentially expressed in adipose tissue and is involved in lipid metabolism [212], as well as adipocyte differentiation.  The mechanism of action in the treatment of T2D is increased insulin sensitivity due to markedly reducing plasma FFA and TG prior to decreasing blood glucose levels, which only occurs 8 to 12 weeks into treatment [213].  Interestingly, a side effect of these TZD drugs is significant weight gain.  The weight gain mechanism is not entirely clear, but it’s presumed to be due to an increase in fat deposition, an inherent mechanism of PPAR-γ action [213].  Although the weight gain is a drawback of treating T2D with TZDs, it is suggested that the increase in fat is re-distributed in a favourable direction (visceral to subcutaneous) resulting in increased insulin sensitivity [213]. Natural agonists of PPAR-γ exist which include fatty acids that are either absorbed from the diet or partially metabolized compounds that also originate from the diet, as well as fatty acids that are generated from de novo lipogenesis [214].  It has also been found that PPAR-γ mRNA expression is increased in ob/ob mice [215].  These two observations suggest that perhaps activation of PPAR-γ activity by endogenous FFA ligands is initiated during a rapid weight gain phase leading to the distribution of ectopic fat to be re-distributed to the adipose tissue.  The result of this fat re-distribution could then potentially lead to increased insulin sensitivity in the liver or skeletal muscle. This effect of fat re-distribution would take weeks to be observed, as seen in TZD treatment in diabetic patients taking 8-12 weeks to exert maximal anti-diabetic effects. The idea that PPAR-γ is activated by endogenous agonist, initiating changes in fat re-  117  distribution over a course of weeks is also consistent with the observation that ob/ob mice become euglycemic as they age to approximately 20 weeks.  Analysis of plasma samples from young hyperglycemic ob/ob mice and old euglycemic ob/ob mice for plasma FFA and TG levels, as well as comparison of lipid accumulation in skeletal muscle, liver, and islets, in young and old ob/ob mice, will provide further insight if this is a potential mechanism for the reversal of diabetes in severely obese ob/ob mice. The re-distribution of adipose tissue by activation of PPAR-γ by endogenous FFAs could also result in changes in IGFBP-2 levels in ob/ob mice.  PPAR-γ is part of the adipocyte differentiation program that matures pre-adipocytes into fat cells [216, 217], and it has been shown that IGFBP-2 is secreted by differentiating white pre- adipocytes [162].  An increase in pre-adipoctye differentaion could then potentially lead to an increase in plasma IGFBP-2 levels.  IGFBP-2 has been implicated as an important factor in glucose homeostasis and insulin sensitivity [132, 158] and could therefore be a potential mediator of improved fasting glucose levels in the ageing ob/ob mice. Analysis of plasma samples for IGFBP-2 levels in young hyperglycemic and old euglycemic ob/ob mice would reveal if an increase of plasma IGFBP-2 levels is observed as the ob/ob mice age and potentially contributes to the resolution of diabetes in the old ob/ob mice.      118  CONCLUSION AND FUTURE DIRECTIONS The findings in this thesis highlight the physiological role of leptin in regulating insulin secretion, insulin sensitivity and glucose homeostasis, and help delineate the potential sequence of events in metabolic diseases associated with leptin resistance.  We induced a state of acute leptin deficiency in an otherwise normal animal to study the hierarchy of leptin action on energy.  Our results show that in the absence of leptin signalling, insulin levels were increased significantly in the fasting and post-prandial state compared to control treated mice.  These changes were accompanied with a reduction in whole-body insulin sensitivity as well as hepatic insulin resistance indicated by increased levels of HGP in the hyperinsulinemic state.  These metabolic alterations occurred prior to changes in body weight, body composition, and energy expenditure as assessed by indirect calorimety.  Overall, these data indicate that the action of leptin on glucose homeostasis and regulation of insulin secretion is high in the hierarchy of physiological leptin function.  Further studies to determine the effects on glucose homeostasis resulting from a peripheral and/or central leptin deficiency would provide further insight on direct leptin targets important in glucose homeostasis.  Administration of the PEG-MLA by an intracerebroventricular route would indicate the importance in acutely disrupting central vs. peripheral signalling. The important anti-diabetic effect of leptin has been shown to be exerted at least in part through the regulation of IGFBP-2 levels.  Our data show that while leptin treatment to ob/ob mice is able to potently increase circulating IGFBP-2 levels in a dose dependent manner, the mechanism does not involve hepatic leptin signalling or vagal efferents.  Further studies aimed at better understanding of the source IGFBP-2  119  production and IGFBP-2 regulation will add significant information in the search of the exact mechanism of leptin regulation of circulating IGFBP-2.    120  REFERENCES 1. Brown, C.D., et al., Body mass index and the prevalence of hypertension and dyslipidemia. Obes Res, 2000. 8(9): p. 605-19. 2. Janssen, I., P.T. Katzmarzyk, and R. Ross, Body mass index, waist circumference, and health risk: evidence in support of current National Institutes of Health guidelines. Arch Intern Med, 2002. 162(18): p. 2074-9. 3. Janssen, I., et al., Body mass index and waist circumference independently contribute to the prediction of nonabdominal, abdominal subcutaneous, and visceral fat. Am J Clin Nutr, 2002. 75(4): p. 683-8. 4. Kissebah, A.H. and G.R. Krakower, Regional adiposity and morbidity. Physiol Rev, 1994. 74(4): p. 761-811. 5. Bloom, S.R., et al., The obesity epidemic: pharmacological challenges. Mol Interv, 2008. 8(2): p. 82-98. 6. Kopelman, P.G., Obesity as a medical problem. Nature, 2000. 404(6778): p. 635- 43. 7. Ryan, J.G., Cost and policy implications from the increasing prevalence of obesity and diabetes mellitus. Gend Med, 2009. 6 Suppl 1: p. 86-108. 8. Sowers, J.R., Obesity as a cardiovascular risk factor. Am J Med, 2003. 115 Suppl 8A: p. 37S-41S. 9. Hjartaker, A., H. Langseth, and E. Weiderpass, Obesity and diabetes epidemics: cancer repercussions. Adv Exp Med Biol, 2008. 630: p. 72-93. 10. Pradhan, A., Obesity, metabolic syndrome, and type 2 diabetes: inflammatory basis of glucose metabolic disorders. Nutr Rev, 2007. 65(12 Pt 2): p. S152-6. 11. Colditz, G.A., et al., Weight as a risk factor for clinical diabetes in women. Am J Epidemiol, 1990. 132(3): p. 501-13. 12. Field, A.E., et al., Impact of overweight on the risk of developing common chronic diseases during a 10-year period. Arch Intern Med, 2001. 161(13): p. 1581-6. 13. Diagnosis and classification of diabetes mellitus. Diabetes Care, 2005. 28 Suppl 1: p. S37-42. 14. Pessin, J.E. and A.R. Saltiel, Signaling pathways in insulin action: molecular targets of insulin resistance. J Clin Invest, 2000. 106(2): p. 165-9. 15. Bell, G.I. and K.S. Polonsky, Diabetes mellitus and genetically programmed defects in beta-cell function. Nature, 2001. 414(6865): p. 788-91. 16. Friedman, J.M., et al., Molecular mapping of the mouse ob mutation. Genomics, 1991. 11(4): p. 1054-62. 17. Coleman, D.L., Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia, 1978. 14(3): p. 141-8. 18. Zhang, Y., et al., Positional cloning of the mouse obese gene and its human homologue. Nature, 1994. 372(6505): p. 425-32. 19. Friedman, J.M. and J.L. Halaas, Leptin and the regulation of body weight in mammals. Nature, 1998. 395(6704): p. 763-70. 20. Tartaglia, L.A., et al., Identification and expression cloning of a leptin receptor, OB-R. Cell, 1995. 83(7): p. 1263-71. 21. Masuzaki, H., et al., Nonadipose tissue production of leptin: leptin as a novel placenta-derived hormone in humans. Nat Med, 1997. 3(9): p. 1029-33.  121  22. Bado, A., et al., The stomach is a source of leptin. Nature, 1998. 394(6695): p. 790-3. 23. Wang, J., et al., A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature, 1998. 393(6686): p. 684-8. 24. Maffei, M., et al., Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med, 1995. 1(11): p. 1155-61. 25. Considine, R.V., et al., Serum immunoreactive-leptin concentrations in normal- weight and obese humans. N Engl J Med, 1996. 334(5): p. 292-5. 26. Pelleymounter, M.A., et al., Effects of the obese gene product on body weight regulation in ob/ob mice. Science, 1995. 269(5223): p. 540-3. 27. Campfield, L.A., et al., Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science, 1995. 269(5223): p. 546-9. 28. Halaas, J.L., et al., Weight-reducing effects of the plasma protein encoded by the obese gene. Science, 1995. 269(5223): p. 543-6. 29. Chehab, F.F., M.E. Lim, and R. Lu, Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet, 1996. 12(3): p. 318-20. 30. Farooqi, I.S., et al., Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J Clin Invest, 2002. 110(8): p. 1093-103. 31. Hamilton, B.S., et al., Increased obese mRNA expression in omental fat cells from massively obese humans. Nat Med, 1995. 1(9): p. 953-6. 32. Lee, G.H., et al., Abnormal splicing of the leptin receptor in diabetic mice. Nature, 1996. 379(6566): p. 632-5. 33. Chen, H., et al., Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell, 1996. 84(3): p. 491-5. 34. Chua, S.C., Jr., et al., Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin) receptor. Science, 1996. 271(5251): p. 994-6. 35. Garofalo, C. and E. Surmacz, Leptin and cancer. J Cell Physiol, 2006. 207(1): p. 12-22. 36. Myers, M.G., Jr., Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog Horm Res, 2004. 59: p. 287-304. 37. Ghilardi, N., et al., Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci U S A, 1996. 93(13): p. 6231-5. 38. Cheng, A., et al., Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B. Dev Cell, 2002. 2(4): p. 497-503. 39. Cook, W.S. and R.H. Unger, Protein tyrosine phosphatase 1B: a potential leptin resistance factor of obesity. Dev Cell, 2002. 2(4): p. 385-7. 40. Elchebly, M., et al., Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science, 1999. 283(5407): p. 1544-8. 41. Bjorbaek, C., et al., Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell, 1998. 1(4): p. 619-25.  122  42. Bjorbak, C., et al., SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J Biol Chem, 2000. 275(51): p. 40649-57. 43. Cohen, P., et al., Selective deletion of leptin receptor in neurons leads to obesity. J Clin Invest, 2001. 108(8): p. 1113-21. 44. Kowalski, T.J., et al., Transgenic complementation of leptin-receptor deficiency. I. Rescue of the obesity/diabetes phenotype of LEPR-null mice expressing a LEPR-B transgene. Diabetes, 2001. 50(2): p. 425-35. 45. Vaisse, C., et al., Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet, 1996. 14(1): p. 95-7. 46. Mercer, J.G., et al., Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett, 1996. 387(2-3): p. 113-6. 47. Elmquist, J.K., et al., Leptin activates distinct projections from the dorsomedial and ventromedial hypothalamic nuclei. Proc Natl Acad Sci U S A, 1998. 95(2): p. 741-6. 48. Fei, H., et al., Anatomic localization of alternatively spliced leptin receptors (Ob- R) in mouse brain and other tissues. Proc Natl Acad Sci U S A, 1997. 94(13): p. 7001-5. 49. Elmquist, J.K., C.F. Elias, and C.B. Saper, From lesions to leptin: hypothalamic control of food intake and body weight. Neuron, 1999. 22(2): p. 221-32. 50. Stanley, B.G., et al., Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Peptides, 1986. 7(6): p. 1189-92. 51. Rossi, M., et al., A C-terminal fragment of Agouti-related protein increases feeding and antagonizes the effect of alpha-melanocyte stimulating hormone in vivo. Endocrinology, 1998. 139(10): p. 4428-31. 52. Cone, R.D., et al., The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation. Recent Prog Horm Res, 1996. 51: p. 287-317; discussion 318. 53. Kristensen, P., et al., Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature, 1998. 393(6680): p. 72-6. 54. Schwartz, M.W., et al., Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes, 1996. 45(4): p. 531-5. 55. Stephens, T.W., et al., The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature, 1995. 377(6549): p. 530-2. 56. Harris, R.B., Acute and chronic effects of leptin on glucose utilization in lean mice. Biochem Biophys Res Commun, 1998. 245(2): p. 502-9. 57. Levin, N., et al., Decreased food intake does not completely account for adiposity reduction after ob protein infusion. Proc Natl Acad Sci U S A, 1996. 93(4): p. 1726-30. 58. Lowell, B.B. and B.M. Spiegelman, Towards a molecular understanding of adaptive thermogenesis. Nature, 2000. 404(6778): p. 652-60. 59. Mistry, A.M., A.G. Swick, and D.R. Romsos, Leptin rapidly lowers food intake and elevates metabolic rates in lean and ob/ob mice. J Nutr, 1997. 127(10): p. 2065-72.  123  60. Hwa, J.J., et al., Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice. Am J Physiol, 1997. 272(4 Pt 2): p. R1204-9. 61. Scarpace, P.J., et al., Leptin increases uncoupling protein expression and energy expenditure. Am J Physiol, 1997. 273(1 Pt 1): p. E226-30. 62. Davies, J.I., In vitro regulation of the lipolysis of adipose tissue. Nature, 1968. 218(5139): p. 349-52. 63. Galton, D.J. and G.A. Bray, Studies on lipolysis in human adipose cells. J Clin Invest, 1967. 46(4): p. 621-9. 64. Landsberg, L., M.E. Saville, and J.B. Young, Sympathoadrenal system and regulation of thermogenesis. Am J Physiol, 1984. 247(2 Pt 1): p. E181-9. 65. Bray, G.A. and D.A. York, The MONA LISA hypothesis in the time of leptin. Recent Prog Horm Res, 1998. 53: p. 95-117; discussion 117-8. 66. Haynes, W.G., et al., Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest, 1997. 100(2): p. 270-8. 67. Levine, J.A., N.L. Eberhardt, and M.D. Jensen, Leptin responses to overfeeding: relationship with body fat and nonexercise activity thermogenesis. J Clin Endocrinol Metab, 1999. 84(8): p. 2751-4. 68. Sims, E.A. and E. Danforth, Jr., Expenditure and storage of energy in man. J Clin Invest, 1987. 79(4): p. 1019-25. 69. Leibel, R.L., M. Rosenbaum, and J. Hirsch, Changes in energy expenditure resulting from altered body weight. N Engl J Med, 1995. 332(10): p. 621-8. 70. Rosenbaum, M., et al., Low dose leptin administration reverses effects of sustained weight-reduction on energy expenditure and circulating concentrations of thyroid hormones. J Clin Endocrinol Metab, 2002. 87(5): p. 2391-4. 71. Huan, J.N., et al., Adipocyte-selective reduction of the leptin receptors induced by antisense RNA leads to increased adiposity, dyslipidemia, and insulin resistance. J Biol Chem, 2003. 278(46): p. 45638-50. 72. Kieffer, T.J., R.S. Heller, and J.F. Habener, Leptin receptors expressed on pancreatic beta-cells. Biochem Biophys Res Commun, 1996. 224(2): p. 522-7. 73. Seufert, J., T.J. Kieffer, and J.F. Habener, Leptin inhibits insulin gene transcription and reverses hyperinsulinemia in leptin-deficient ob/ob mice. Proc Natl Acad Sci U S A, 1999. 96(2): p. 674-9. 74. Kieffer, T.J., et al., Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells. Diabetes, 1997. 46(6): p. 1087-93. 75. Cohen, B., D. Novick, and M. Rubinstein, Modulation of insulin activities by leptin. Science, 1996. 274(5290): p. 1185-8. 76. Ceddia, R.B., et al., Modulation of insulin secretion by leptin. Gen Pharmacol, 1999. 32(2): p. 233-7. 77. Kim, Y.B., et al., In vivo administration of leptin activates signal transduction directly in insulin-sensitive tissues: overlapping but distinct pathways from insulin. Endocrinology, 2000. 141(7): p. 2328-39. 78. Unger, R.H., Lipotoxic diseases. Annu Rev Med, 2002. 53: p. 319-36. 79. Shulman, G.I., Cellular mechanisms of insulin resistance. J Clin Invest, 2000. 106(2): p. 171-6.  124  80. Shimabukuro, M., et al., Direct antidiabetic effect of leptin through triglyceride depletion of tissues. Proc Natl Acad Sci U S A, 1997. 94(9): p. 4637-41. 81. Zhou, Y.T., et al., Role of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells. Proc Natl Acad Sci U S A, 1998. 95(15): p. 8898-903. 82. Zhou, Y.T., et al., Reversing adipocyte differentiation: implications for treatment of obesity. Proc Natl Acad Sci U S A, 1999. 96(5): p. 2391-5. 83. Lee, Y., et al., PPAR alpha is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue. Proc Natl Acad Sci U S A, 2002. 99(18): p. 11848-53. 84. Bryson, J.M., et al., Leptin has acute effects on glucose and lipid metabolism in both lean and gold thioglucose-obese mice. Am J Physiol, 1999. 277(3 Pt 1): p. E417-22. 85. Koteish, A. and A.M. Diehl, Animal models of steatosis. Semin Liver Dis, 2001. 21(1): p. 89-104. 86. Fishman, S., et al., Resistance to leptin action is the major determinant of hepatic triglyceride accumulation in vivo. FASEB J, 2007. 21(1): p. 53-60. 87. Lam, N.T., et al., Leptin increases hepatic insulin sensitivity and protein tyrosine phosphatase 1B expression. Mol Endocrinol, 2004. 18(6): p. 1333-45. 88. Moitra, J., et al., Life without white fat: a transgenic mouse. Genes Dev, 1998. 12(20): p. 3168-81. 89. Shimomura, I., et al., Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy. Genes Dev, 1998. 12(20): p. 3182-94. 90. Colombo, C., et al., Transplantation of adipose tissue lacking leptin is unable to reverse the metabolic abnormalities associated with lipoatrophy. Diabetes, 2002. 51(9): p. 2727-33. 91. Asilmaz, E., et al., Site and mechanism of leptin action in a rodent form of congenital lipodystrophy. J Clin Invest, 2004. 113(3): p. 414-24. 92. Shimomura, I., et al., Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature, 1999. 401(6748): p. 73-6. 93. Ebihara, K., et al., Efficacy and safety of leptin-replacement therapy and possible mechanisms of leptin actions in patients with generalized lipodystrophy. J Clin Endocrinol Metab, 2007. 92(2): p. 532-41. 94. Oral, E.A., et al., Leptin-replacement therapy for lipodystrophy. N Engl J Med, 2002. 346(8): p. 570-8. 95. Petersen, K.F., et al., Leptin reverses insulin resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest, 2002. 109(10): p. 1345-50. 96. Park, J.Y., et al., Type 1 diabetes associated with acquired generalized lipodystrophy and insulin resistance: the effect of long-term leptin therapy. J Clin Endocrinol Metab, 2008. 93(1): p. 26-31. 97. Harris, R.B., et al., A leptin dose-response study in obese (ob/ob) and lean (+/?) mice. Endocrinology, 1998. 139(1): p. 8-19. 98. Attig, L., et al., Early postnatal leptin blockage leads to a long-term leptin resistance and susceptibility to diet-induced obesity in rats. Int J Obes (Lond), 2008. 32(7): p. 1153-60.  125  99. Weigle, D.S., et al., Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J Clin Invest, 1995. 96(4): p. 2065-70. 100. Kieffer, T.J. and J.F. Habener, The adipoinsular axis: effects of leptin on pancreatic beta-cells. Am J Physiol Endocrinol Metab, 2000. 278(1): p. E1-E14. 101. Ceddia, R.B., et al., Acute effects of leptin on glucose metabolism of in situ rat perfused livers and isolated hepatocytes. Int J Obes Relat Metab Disord, 1999. 23(11): p. 1207-12. 102. Barzilai, N., et al., Leptin selectively decreases visceral adiposity and enhances insulin action. J Clin Invest, 1997. 100(12): p. 3105-10. 103. Seufert, J., et al., Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus. J Clin Endocrinol Metab, 1999. 84(2): p. 670-6. 104. Kolaczynski, J.W., et al., Acute and chronic effects of insulin on leptin production in humans: Studies in vivo and in vitro. Diabetes, 1996. 45(5): p. 699-701. 105. Genuth, S.M., R.J. Przybylski, and D.M. Rosenberg, Insulin resistance in genetically obese, hyperglycemic mice. Endocrinology, 1971. 88(5): p. 1230-8. 106. Dubuc, P.U., The development of obesity, hyperinsulinemia, and hyperglycemia in ob/ob mice. Metabolism, 1976. 25(12): p. 1567-74. 107. Coleman, D.L. and K.P. Hummel, Hyperinsulinemia in pre-weaning diabetes (db) mice. Diabetologia, 1974. 10 Suppl: p. 607-10. 108. Chick, W.L., R.L. Lavine, and A.A. Like, Studies in the diabetic mutant mouse. V. Glucose tolerance in mice homozygous and heterozygous for the diabetes (db) gene. Diabetologia, 1970. 6(3): p. 257-62. 109. Le Stunff, C. and P. Bougneres, Early changes in postprandial insulin secretion, not in insulin sensitivity, characterize juvenile obesity. Diabetes, 1994. 43(5): p. 696-702. 110. Sigal, R.J., et al., Acute postchallenge hyperinsulinemia predicts weight gain: a prospective study. Diabetes, 1997. 46(6): p. 1025-9. 111. Odeleye, O.E., et al., Fasting hyperinsulinemia is a predictor of increased body weight gain and obesity in Pima Indian children. Diabetes, 1997. 46(8): p. 1341- 5. 112. Chinookoswong, N., J.L. Wang, and Z.Q. Shi, Leptin restores euglycemia and normalizes glucose turnover in insulin-deficient diabetes in the rat. Diabetes, 1999. 48(7): p. 1487-92. 113. Yu, X., et al., Making insulin-deficient type 1 diabetic rodents thrive without insulin. Proc Natl Acad Sci U S A, 2008. 105(37): p. 14070-5. 114. Wang, M.Y., et al., Leptin therapy in insulin-deficient type I diabetes. Proc Natl Acad Sci U S A. 107(11): p. 4813-9. 115. Haluzik, M., et al., Genetic background (C57BL/6J versus FVB/N) strongly influences the severity of diabetes and insulin resistance in ob/ob mice. Endocrinology, 2004. 145(7): p. 3258-64. 116. Ravussin, E., et al., Determinants of 24-hour energy expenditure in man. Methods and results using a respiratory chamber. J Clin Invest, 1986. 78(6): p. 1568-78. 117. Pfluger, P.T., et al., Simultaneous deletion of ghrelin and its receptor increases motor activity and energy expenditure. Am J Physiol Gastrointest Liver Physiol, 2008. 294(3): p. G610-8.  126  118. Tinsley, F.C., G.Z. Taicher, and M.L. Heiman, Evaluation of a quantitative magnetic resonance method for mouse whole body composition analysis. Obes Res, 2004. 12(1): p. 150-60. 119. Kunnecke, B., et al., Quantitative body composition analysis in awake mice and rats by magnetic resonance relaxometry. Obes Res, 2004. 12(10): p. 1604-15. 120. Theeuwes, F. and S.I. Yum, Principles of the design and operation of generic osmotic pumps for the delivery of semisolid or liquid drug formulations. Ann Biomed Eng, 1976. 4(4): p. 343-53. 121. Smith, G.P., et al., Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat. Science, 1981. 213(4511): p. 1036-7. 122. Bluthe, R.M., et al., Vagotomy attenuates behavioural effects of interleukin-1 injected peripherally but not centrally. Neuroreport, 1996. 7(9): p. 1485-8. 123. Gromada, J., et al., Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK 293 cells induces cAMP-dependent activation of calcium- induced calcium release. FEBS Lett, 1995. 373(2): p. 182-6. 124. Voshol, P.J., et al., In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake. Diabetes, 2001. 50(11): p. 2585- 90. 125. Briaud, I., et al., Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes, 2001. 50(2): p. 315-21. 126. Salomon, G., et al., Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists. Protein Expr Purif, 2006. 47(1): p. 128-36. 127. Elinav, E., et al., Competitive inhibition of leptin signaling results in amelioration of liver fibrosis through modulation of stellate cell function. Hepatology, 2009. 49(1): p. 278-86. 128. Elinav, E., et al., Pegylated leptin antagonist is a potent orexigenic agent: preparation and mechanism of activity. Endocrinology, 2009. 150(7): p. 3083-91. 129. Roy, A.F., et al., Lack of cross-desensitization between leptin and prolactin signaling pathways despite the induction of suppressor of cytokine signaling 3 and PTP-1B. J Endocrinol, 2007. 195(2): p. 341-50. 130. Trayhurn, P., Thermoregulation in the diabetic-obese (db/db) mouse. The role of non-shivering thermogenesis in energy balance. Pflugers Arch, 1979. 380(3): p. 227-32. 131. Tuduri, E., et al., Inhibitory effects of leptin on pancreatic alpha-cell function. Diabetes, 2009. 58(7): p. 1616-24. 132. Hedbacker, K., et al., Antidiabetic effects of IGFBP2, a leptin-regulated gene. Cell Metab. 11(1): p. 11-22. 133. Cauchi, S., et al., Post genome-wide association studies of novel genes associated with type 2 diabetes show gene-gene interaction and high predictive value. PLoS One, 2008. 3(5): p. e2031. 134. Ruan, W. and M. Lai, Insulin-like growth factor binding protein: a possible marker for the metabolic syndrome? Acta Diabetol. 47(1): p. 5-14.  127  135. Martin, R.M., et al., Associations of adiposity from childhood into adulthood with insulin resistance and the insulin-like growth factor system: 65-year follow-up of the Boyd Orr Cohort. J Clin Endocrinol Metab, 2006. 91(9): p. 3287-95. 136. Samuel, V.T., et al., Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease. J Biol Chem, 2004. 279(31): p. 32345-53. 137. Cohen, P. and J.M. Friedman, Leptin and the control of metabolism: role for stearoyl-CoA desaturase-1 (SCD-1). J Nutr, 2004. 134(9): p. 2455S-2463S. 138. Ding, X., et al., Exendin-4, a glucagon-like protein-1 (GLP-1) receptor agonist, reverses hepatic steatosis in ob/ob mice. Hepatology, 2006. 43(1): p. 173-81. 139. Arulmozhi, D.K. and B. Portha, GLP-1 based therapy for type 2 diabetes. Eur J Pharm Sci, 2006. 28(1-2): p. 96-108. 140. Drucker, D.J., Glucagon-like peptides. Diabetes, 1998. 47(2): p. 159-69. 141. Kieffer, T.J. and J.F. Habener, The glucagon-like peptides. Endocr Rev, 1999. 20(6): p. 876-913. 142. Thorens, B., Expression cloning of the pancreatic beta cell receptor for the gluco- incretin hormone glucagon-like peptide 1. Proc Natl Acad Sci U S A, 1992. 89(18): p. 8641-5. 143. Weir, G.C., et al., Glucagonlike peptide I (7-37) actions on endocrine pancreas. Diabetes, 1989. 38(3): p. 338-42. 144. Kreymann, B., et al., Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet, 1987. 2(8571): p. 1300-4. 145. Gunn, I., et al., Central glucagon-like peptide-I in the control of feeding. Biochem Soc Trans, 1996. 24(2): p. 581-4. 146. Turton, M.D., et al., A role for glucagon-like peptide-1 in the central regulation of feeding. Nature, 1996. 379(6560): p. 69-72. 147. Flint, A., et al., Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans. J Clin Invest, 1998. 101(3): p. 515-20. 148. Vilsboll, T., et al., Reduced postprandial concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic patients. Diabetes, 2001. 50(3): p. 609-13. 149. Ranganath, L.R., et al., Attenuated GLP-1 secretion in obesity: cause or consequence? Gut, 1996. 38(6): p. 916-9. 150. Mannucci, E., et al., Glucagon-like peptide (GLP)-1 and leptin concentrations in obese patients with Type 2 diabetes mellitus. Diabet Med, 2000. 17(10): p. 713-9. 151. Anini, Y. and P.L. Brubaker, Role of leptin in the regulation of glucagon-like peptide-1 secretion. Diabetes, 2003. 52(2): p. 252-9. 152. Lee, Y.S., et al., Glucagon-like peptide-1 gene therapy in obese diabetic mice results in long-term cure of diabetes by improving insulin sensitivity and reducing hepatic gluconeogenesis. Diabetes, 2007. 56(6): p. 1671-9. 153. Verdich, C., et al., The role of postprandial releases of insulin and incretin hormones in meal-induced satiety--effect of obesity and weight reduction. Int J Obes Relat Metab Disord, 2001. 25(8): p. 1206-14. 154. Orskov, C., et al., Tissue and plasma concentrations of amidated and glycine- extended glucagon-like peptide I in humans. Diabetes, 1994. 43(4): p. 535-9.  128  155. Herrmann, C., et al., Glucagon-like peptide-1 and glucose-dependent insulin- releasing polypeptide plasma levels in response to nutrients. Digestion, 1995. 56(2): p. 117-26. 156. Greenberg, G.R., et al., Effect of total parenteral nutrition on gut hormone release in humans. Gastroenterology, 1981. 80(5 pt 1): p. 988-93. 157. Wheatcroft, S.B. and M.T. Kearney, IGF-dependent and IGF-independent actions of IGF-binding protein-1 and -2: implications for metabolic homeostasis. Trends Endocrinol Metab, 2009. 20(4): p. 153-62. 158. Wheatcroft, S.B., et al., IGF-binding protein-2 protects against the development of obesity and insulin resistance. Diabetes, 2007. 56(2): p. 285-94. 159. Jones, J.I., et al., Insulin-like growth factor binding protein 1 stimulates cell migration and binds to the alpha 5 beta 1 integrin by means of its Arg-Gly-Asp sequence. Proc Natl Acad Sci U S A, 1993. 90(22): p. 10553-7. 160. Bisht, B., H.L. Goel, and C.S. Dey, Focal adhesion kinase regulates insulin resistance in skeletal muscle. Diabetologia, 2007. 50(5): p. 1058-69. 161. Huang, D., et al., Reduced expression of focal adhesion kinase disrupts insulin action in skeletal muscle cells. Endocrinology, 2006. 147(7): p. 3333-43. 162. Boney, C.M., et al., Expression of insulin-like growth factor-I (IGF-I) and IGF- binding proteins during adipogenesis. Endocrinology, 1994. 135(5): p. 1863-8. 163. Postic, C., et al., Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem, 1999. 274(1): p. 305-15. 164. Pinkert, C.A., et al., An albumin enhancer located 10 kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes Dev, 1987. 1(3): p. 268-76. 165. Balthasar, N., et al., Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis. Neuron, 2004. 42(6): p. 983-91. 166. McMinn, J.E., et al., An allelic series for the leptin receptor gene generated by CRE and FLP recombinase. Mamm Genome, 2004. 15(9): p. 677-85. 167. Chua, S.C., Jr., et al., Fine structure of the murine leptin receptor gene: splice site suppression is required to form two alternatively spliced transcripts. Genomics, 1997. 45(2): p. 264-70. 168. Coppari, R., et al., The hypothalamic arcuate nucleus: a key site for mediating leptin's effects on glucose homeostasis and locomotor activity. Cell Metab, 2005. 1(1): p. 63-72. 169. Coleman, D.L., Diabetes-obesity syndromes in mice. Diabetes, 1982. 31(Suppl 1 Pt 2): p. 1-6. 170. Menahan, L.A., Age-related changes in lipid and carbohydrate metabolism of the genetically obese mouse. Metabolism, 1983. 32(2): p. 172-8. 171. Unger, R.H. and L. Orci, The essential role of glucagon in the pathogenesis of diabetes mellitus. Lancet, 1975. 1(7897): p. 14-6. 172. Clement, K., et al., A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature, 1998. 392(6674): p. 398-401. 173. Montague, C.T., et al., Congenital leptin deficiency is associated with severe early-onset obesity in humans. Nature, 1997. 387(6636): p. 903-8.  129  174. Halaas, J.L., et al., Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci U S A, 1997. 94(16): p. 8878-83. 175. Consensus Development Conference on Insulin Resistance. 5-6 November 1997. American Diabetes Association. Diabetes Care, 1998. 21(2): p. 310-4. 176. Gavrilova, O., et al., Leptin and diabetes in lipoatrophic mice. Nature, 2000. 403(6772): p. 850; discussion 850-1. 177. Ferrannini, E., The theoretical bases of indirect calorimetry: a review. Metabolism, 1988. 37(3): p. 287-301. 178. Doring, H., et al., Leptin selectively increases energy expenditure of food- restricted lean mice. Int J Obes Relat Metab Disord, 1998. 22(2): p. 83-8. 179. Choi, Y.H., et al., ICV leptin effects on spontaneous physical activity and feeding behavior in rats. Behav Brain Res, 2008. 188(1): p. 100-8. 180. Matheny, M., et al., Central overexpression of leptin antagonist reduces wheel running and underscores importance of endogenous leptin receptor activity in energy homeostasis. Am J Physiol Regul Integr Comp Physiol, 2009. 297(5): p. R1254-61. 181. Trayhurn, P. and W.P. James, Thermoregulation and non-shivering thermogenesis in the genetically obese (ob/ob) mouse. Pflugers Arch, 1978. 373(2): p. 189-93. 182. Golozoubova, V., et al., Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold. FASEB J, 2001. 15(11): p. 2048-50. 183. Nedergaard, J., et al., UCP1: the only protein able to mediate adaptive non- shivering thermogenesis and metabolic inefficiency. Biochim Biophys Acta, 2001. 1504(1): p. 82-106. 184. Commins, S.P., et al., Leptin selectively reduces white adipose tissue in mice via a UCP1-dependent mechanism in brown adipose tissue. Am J Physiol Endocrinol Metab, 2001. 280(2): p. E372-7. 185. Rothwell, N.J., M.J. Stock, and B.P. Warwick, The effect of high fat and high carbohydrate cafeteria diets on diet-induced thermogenesis in the rat. Int J Obes, 1983. 7(3): p. 263-70. 186. Stock, M.J., Gluttony and thermogenesis revisited. Int J Obes Relat Metab Disord, 1999. 23(11): p. 1105-17. 187. Covey, S.D., et al., The pancreatic beta cell is a key site for mediating the effects of leptin on glucose homeostasis. Cell Metab, 2006. 4(4): p. 291-302. 188. Morioka, T., et al., Disruption of leptin receptor expression in the pancreas directly affects beta cell growth and function in mice. J Clin Invest, 2007. 117(10): p. 2860-8. 189. Morton, G.J., et al., Leptin regulates insulin sensitivity via phosphatidylinositol-3- OH kinase signaling in mediobasal hypothalamic neurons. Cell Metab, 2005. 2(6): p. 411-20. 190. German, J.P., et al., Leptin Deficiency Causes Insulin Resistance Induced by Uncontrolled Diabetes. Diabetes. 191. German, J., et al., Hypothalamic leptin signaling regulates hepatic insulin sensitivity via a neurocircuit involving the vagus nerve. Endocrinology, 2009. 150(10): p. 4502-11.  130  192. DeFronzo, R.A., J.D. Tobin, and R. Andres, Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol, 1979. 237(3): p. E214-23. 193. Kim, J.K., Hyperinsulinemic-euglycemic clamp to assess insulin sensitivity in vivo. Methods Mol Biol, 2009. 560: p. 221-38. 194. Rossetti, L., et al., Short term effects of leptin on hepatic gluconeogenesis and in vivo insulin action. J Biol Chem, 1997. 272(44): p. 27758-63. 195. Rajaram, S., D.J. Baylink, and S. Mohan, Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocr Rev, 1997. 18(6): p. 801-31. 196. Schneider, M.R., et al., Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins. FASEB J, 2000. 14(5): p. 629-40. 197. Forbes, B.E., et al., Localization of an insulin-like growth factor (IGF) binding site of bovine IGF binding protein-2 using disulfide mapping and deletion mutation analysis of the C-terminal domain. J Biol Chem, 1998. 273(8): p. 4647- 52. 198. Young, S.C., M.V. Miles, and D.R. Clemmons, Determination of the pharmacokinetic profiles of insulin-like growth factor binding proteins-1 and -2 in rats. Endocrinology, 1992. 131(4): p. 1867-73. 199. Guo, K., et al., Disruption of peripheral leptin signaling in mice results in hyperleptinemia without associated metabolic abnormalities. Endocrinology, 2007. 148(8): p. 3987-97. 200. Pocai, A., et al., Hypothalamic K(ATP) channels control hepatic glucose production. Nature, 2005. 434(7036): p. 1026-31. 201. Zawalich, W.S., K.C. Zawalich, and H. Rasmussen, Cholinergic agonists prime the beta-cell to glucose stimulation. Endocrinology, 1989. 125(5): p. 2400-6. 202. Teff, K.L. and R.R. Townsend, Prolonged mild hyperglycemia induces vagally mediated compensatory increase in C-Peptide secretion in humans. J Clin Endocrinol Metab, 2004. 89(11): p. 5606-13. 203. Pocai, A., et al., A brain-liver circuit regulates glucose homeostasis. Cell Metab, 2005. 1(1): p. 53-61. 204. Claudio, M., et al., Adipocytes IGFBP-2 Expression in Prepubertal Obese Children. Obesity (Silver Spring). 205. Herberg, L., et al., Differences in the development of the obese-hyperglycemic syndrome in obob and NZO mice. Diabetologia, 1970. 6(3): p. 292-9. 206. Flatt, P.R., C.J. Bailey, and K.D. Buchanan, Regulation of plasma immunoreactive glucagon in obese hyperglycaemic (ob/ob) mice. J Endocrinol, 1982. 95(2): p. 215-27. 207. Johnson, D.G., et al., Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist. Science, 1982. 215(4536): p. 1115-6. 208. Liang, Y., et al., Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice. Diabetes, 2004. 53(2): p. 410-7. 209. Diez, J.J. and P. Iglesias, The role of the novel adipocyte-derived hormone adiponectin in human disease. Eur J Endocrinol, 2003. 148(3): p. 293-300.  131  210. Kim, J.Y., et al., Obesity-associated improvements in metabolic profile through expansion of adipose tissue. J Clin Invest, 2007. 117(9): p. 2621-37. 211. Johnson, P.R. and J. Hirsch, Cellularity of adipose depots in six strains of genetically obese mice. J Lipid Res, 1972. 13(1): p. 2-11. 212. Edvardsson, U., et al., Rosiglitazone (BRL49653), a PPARgamma-selective agonist, causes peroxisome proliferator-like liver effects in obese mice. J Lipid Res, 1999. 40(7): p. 1177-84. 213. Larsen, T.M., S. Toubro, and A. Astrup, PPARgamma agonists in the treatment of type II diabetes: is increased fatness commensurate with long-term efficacy? Int J Obes Relat Metab Disord, 2003. 27(2): p. 147-61. 214. Kliewer, S.A., et al., Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma. Proc Natl Acad Sci U S A, 1997. 94(9): p. 4318-23. 215. Memon, R.A., et al., Up-regulation of peroxisome proliferator-activated receptors (PPAR-alpha) and PPAR-gamma messenger ribonucleic acid expression in the liver in murine obesity: troglitazone induces expression of PPAR-gamma-responsive adipose tissue-specific genes in the liver of obese diabetic mice. Endocrinology, 2000. 141(11): p. 4021-31. 216. Rosen, E.D., et al., PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell, 1999. 4(4): p. 611-7. 217. Kersten, S., B. Desvergne, and W. Wahli, Roles of PPARs in health and disease. Nature, 2000. 405(6785): p. 421-4.    132  APPENDIX UBC Research Ethics Board Certificates of Approval   133  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0071042/manifest

Comment

Related Items