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Regulation of glucose and lipid metabolism by hepatic leptin signalling Huynh, Frank Khan 2012

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REGULATION OF GLUCOSE AND LIPID METABOLISM BY HEPATIC LEPTIN SIGNALLING  by  FRANK KHAN HUYNH B.Sc., The University of British Columbia, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Physiology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July 2012  © Frank Khan Huynh, 2012  Abstract Incidences of obesity and type 2 diabetes have risen worldwide at alarming rates. While there is an undeniable correlation between obesity and type 2 diabetes, a clear mechanistic link between these two conditions has not been fully elucidated. The adipocyte-derived hormone leptin may play a role linking obesity and type 2 diabetes. Leptin can regulate body weight through its effects on the brain to decrease food intake and increase energy expenditure, but these effects are disrupted in obesity. Interestingly, leptin also has effects on glucose and lipid metabolism independent of its effects on body weight, so it is possible that disrupted leptin signalling during obesity can also perturb glucose and lipid metabolism, leading to symptoms associated with type 2 diabetes. Since the liver plays a critical role in integrating and controlling glucose and lipid metabolism, it was hypothesized that leptin resistance in the liver could play a role in the development of diabetic symptoms. To investigate this hypothesis, three complementary mouse models were used to help identify the role of leptin signalling specifically in the liver. It was found that in lean mice, hepatic leptin resistance results in increased insulin sensitivity in the liver, leading to reduced hepatic glucose output but also increased lipid accumulation and secretion of larger, more triglyceride-rich very low density lipoprotein (VLDL) particles without an increase in total plasma triglycerides. In obese, hyperinsulinemic mice lacking hepatic leptin signalling, the effects of lost leptin signalling on triglyceride metabolism were exacerbated, resulting in decreased triglyceride clearance and elevated plasma triglycerides compared to controls. These effects on plasma triglycerides were reversed when hepatic leptin signalling was restored in a mouse model of total leptin resistance. Collectively, these data reveal a possible role for hepatic leptin resistance in the development of diabetic symptoms during obesity.  ii  Preface Studies from Chapter 3 are published in the following article: Huynh FK*, Levi J*, Denroche HC, Gray SL, Voshol PJ, Neumann UH, Speck M, Chua SC, Covey SD, and Kieffer TJ. (2010) Disruption of hepatic leptin signaling protects mice from age- and diet-related glucose intolerance. Diabetes 59(12): 3032-3040. Studies from Chapter 7 are published in the following article: Levi J*, Huynh FK*, Denroche HC, Neumann UH, Glavas MM, Covey SD, and Kieffer TJ. (2012) Hepatic leptin signalling and subdiaphragmatic vagal efferents are not required for leptin-induced increases of plasma IGF binding protein-2 (IGFBP-2) in ob/ob mice. Diabetologia 55(3): 752-762. *These authors contributed equally to this work. Contribution of co-authors: All studies were conceived and designed by FK Huynh, J Levi, SD Covey, and TJ Kieffer. Experiments were performed by FK Huynh with assistance from J Levi, SD Covey, HC Denroche, UH Neumann, and MM Glavas. SL Gray, PJ Voshol, and M Speck performed the hyperinsulinemic-euglycemic clamps. SC Chua provided the Leprflox/flox mice. Publications were written by FK Huynh with assistance from J Levi and editing by SD Covey and TJ Kieffer. All studies described in Chapters 3, 4, 5, 6, and 7 were performed by FK Huynh with assistance from J Levi, HC Denroche, and UH Neumann. Figure 5 was contributed by SD Covey. Figure 14 was contributed by SL Gray, PJ Voshol, and M Speck. Figure 16 was partially contributed by J Levi and is published in her Master’s thesis. For this figure, J Levi performed the PCR analysis and FK Huynh generated the mice, collected tissues, and processed the samples. Figures 40A-B were also partially contributed by J Levi and are also published in her Master’s thesis. For these figures, the mice were generated by FK Huynh and the in vivo experiments were performed by J Levi. This thesis was written by FK Huynh with editing provided by WT Gibson, CH McIntosh, SD Covey, and TJ Kieffer.  Animal Care Certificates Animal studies described in this thesis were approved by the University of British Columbia Animal Care Committee (Certificate #A06-0105, A10-0275, and A10-0059).  iii  Table of Contents Abstract ........................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Abbreviations .................................................................................................................... xii Acknowledgements...................................................................................................................... xvi Dedication ................................................................................................................................... xvii CHAPTER 1 – INTRODUCTION ................................................................................................. 1 1.1 Type 2 Diabetes ................................................................................................................... 1 Type 2 diabetes and obesity .................................................................................................... 2 1.2 The Hormone Leptin ............................................................................................................ 3 Discovery................................................................................................................................. 3 Leptin signalling ...................................................................................................................... 5 Central effects of leptin on body weight regulation ................................................................ 7 Metabolic effects of leptin ....................................................................................................... 8 Leptin action in the periphery ................................................................................................. 9 1.3 The Role of the Liver in Glucose and Lipid Metabolism .................................................. 11 Leptin action in the liver ....................................................................................................... 13 1.4 Thesis Investigation ........................................................................................................... 16 CHAPTER 2 – METHODS AND MATERIALS ........................................................................ 18 2.1 Animals .............................................................................................................................. 18 Leprflox/flox AlbCre mice ......................................................................................................... 19 Leprflox/flox AlbCre ob/ob mice................................................................................................ 20 Diabetic db/db mice expressing Lepr-b................................................................................. 22 Verification of Cre expression and Cre-mediated recombination ......................................... 22 Verification of Ad-Lepr-b expression in db/db mice ............................................................ 23 2.2 Plasma Analytes ................................................................................................................. 24 Metabolites ............................................................................................................................ 24 Hormones .............................................................................................................................. 24 Plasma leptin binding proteins .............................................................................................. 25 iv  Lipoprotein profiles ............................................................................................................... 25 Plasma apolipoprotein B levels ............................................................................................. 25 2.3 Body Composition Measurements ..................................................................................... 26 Lean to lipid mass ratio ......................................................................................................... 26 Hepatic lipid content ............................................................................................................. 26 2.4 In Vivo Assays .................................................................................................................... 27 Oral glucose tolerance test .................................................................................................... 27 Glucose-stimulated insulin secretion test .............................................................................. 27 Insulin tolerance test .............................................................................................................. 27 Hyperinsulinemic-euglycemic clamp .................................................................................... 27 Pyruvate tolerance test........................................................................................................... 28 Oral lipid tolerance test ......................................................................................................... 28 Triglyceride secretion assay .................................................................................................. 29 2.5 Analysis of Mouse Tissues ................................................................................................ 29 Measurements of β-Cell mass ............................................................................................... 29 Quantitative PCR................................................................................................................... 29 Gene arrays ............................................................................................................................ 30 Western blot analyses ............................................................................................................ 31 Measurements of lipase activity in the liver .......................................................................... 32 Liver histology ...................................................................................................................... 33 2.6 Data Analysis ..................................................................................................................... 33 CHAPTER 3 – EFFECTS OF A LIFE-LONG LOSS OF HEPATIC LEPTIN SIGNALLING ON GLUCOSE METABOLISM ........................................................................................................ 34 3.1 Introduction ........................................................................................................................ 34 3.2 Results ................................................................................................................................ 34 Generation of mice with hepatocyte-specific disruption of leptin receptor signalling ......... 34 Loss of hepatic leptin signalling does not substantially alter body weight or body composition ........................................................................................................................... 36 Loss of hepatic leptin signalling does not have a major influence on basal metabolic parameters ............................................................................................................................. 39 Loss of hepatic leptin signalling protects against age- and diet-related glucose intolerance 41 Increased glucose-stimulated plasma insulin levels and increased insulin sensitivity contribute to improved glucose tolerance in mice lacking hepatic leptin signalling ............ 43 3.3 Discussion .......................................................................................................................... 48 v  CHAPTER 4 – ACUTE EFFECTS OF HEPATIC LEPTIN SIGNALLING ON GLUCOSE METABOLISM IN OBESE HYPERINSULINEMIC MICE ...................................................... 52 4.1 Introduction ........................................................................................................................ 52 4.2 Results ................................................................................................................................ 53 Leprflox/flox AlbCre+ ob/ob mice have truncated hepatic leptin receptor transcripts .............. 53 The effects of low dose intraperitoneal injections of leptin on fasting blood glucose and insulin in Leprflox/flox AlbCre ob/ob mice ............................................................................... 54 The effects of leptin delivered via mini-osmotic pump on glucose metabolism in Leprflox/flox AlbCre ob/ob mice................................................................................................................. 58 Intravenous delivery of Ad-Lepr-b effectively restores hepatic leptin signalling in db/db mice ....................................................................................................................................... 63 The effects of restored hepatic leptin signalling on glucose metabolism in db/db mice....... 65 4.3 Discussion .......................................................................................................................... 67 CHAPTER 5 – EFFECTS OF A LIFE-LONG LOSS OF HEPATIC LEPTIN SIGNALLING ON LIPID METABOLISM ................................................................................................................. 72 5.1 Introduction ........................................................................................................................ 72 5.2 Results ................................................................................................................................ 73 Loss of hepatic leptin signalling alters lipid metabolism genes in the liver .......................... 73 Loss of hepatic leptin signalling does not affect total plasma lipid levels in the basal state 74 Mice lacking hepatic leptin signalling have larger, more triglyceride-rich VLDL particles 77 Loss of hepatic leptin signalling results in decreased apoB levels in the liver and plasma .. 78 Mice lacking liver leptin signalling have decreased hepatic lipase and increased lipoprotein lipase activity in the liver ...................................................................................................... 80 Lean mice lacking leptin signalling in the liver have normal postprandial triglyceride clearance ................................................................................................................................ 83 5.3 Discussion .......................................................................................................................... 83 CHAPTER 6 – EFFECTS OF HEPATIC LEPTIN SIGNALLING ON LIPID METABOLISM IN OBESE HYPERINSULINEMIC MICE ................................................................................. 89 6.1 Introduction ........................................................................................................................ 89 6.2 Results ................................................................................................................................ 89 Hepatic leptin signalling regulates plasma triglyceride and hepatic apoB levels in obese, hyperinsulinemic mice .......................................................................................................... 89 Hepatic leptin signalling in obese, hyperinsulinemic mice regulates lipase activity in the liver........................................................................................................................................ 93 Hepatic leptin signalling regulates triglyceride clearance in obese, hyperinsulinemic mice 94 vi  6.3 Discussion .......................................................................................................................... 96 CHAPTER 7 – THE ROLE OF HEPATIC LEPTIN SIGNALLING IN THE REGULATION OF PLASMA IGFBP-2 ...................................................................................................................... 99 7.1 Introduction ........................................................................................................................ 99 7.2 Results .............................................................................................................................. 101 Hepatic leptin signalling is not required for leptin to increase plasma IGFBP-2 levels in ob/ob mice ........................................................................................................................... 101 Lean mice lacking hepatic leptin signalling have normal plasma IGFBP-2 ....................... 102 Re-expression of functional leptin receptors in the liver db/db mice is not sufficient for restoring normal IGFBP-2 levels ........................................................................................ 103 7.3 Discussion ........................................................................................................................ 105 CHAPTER 8 - CONCLUSIONS ................................................................................................ 109 Future studies ...................................................................................................................... 114 REFERENCES ........................................................................................................................... 118 APPENDIX A – GENE ARRAY DATA ................................................................................... 139  vii  List of Tables Table 1. Composition of high fat diet #D12330 from Research Diets. ....................................... 18 Table 2. Sequences of primers used in quantitative PCR ............................................................ 30 Table 3. Metabolic parameters of mice with and without hepatic leptin signalling .................... 38 Table 4. Fasting plasma lipids in mice with and without hepatic leptin signalling ..................... 76 Table A1. Complete list of genes differentially expressed in overnight fasted Leprflox/flox AlbCre+ male mice compared to Leprflox/flox AlbCre- littermate controls .................................................. 139 Table A2. Complete list of genes differentially expressed in overnight fasted/2-hour re-fed Leprflox/flox AlbCre+ male mice compared to Leprflox/flox AlbCre- littermate controls.................. 146 Table A3. Complete list of downregulated genes in livers of random-fed leptin-treated Leprflox/flox AlbCre+ ob/ob mice compared to Leprflox/flox AlbCre- ob/ob littermate controls ...... 151  viii  List of Figures Figure 1. Leptin signalling pathway. ............................................................................................. 6 Figure 2. Hepatic insulin signalling pathway .............................................................................. 13 Figure 3. Schematic of the Leprflox and Lepr∆17 gene and protein stuctures ................................ 20 Figure 4. Breeding scheme for the generation of Leprflox/flox AlbCre ob/ob mice ........................ 21 Figure 5. Leprflox/flox AlbCre+ mice have a liver-specific loss of the leptin receptor signalling domain .......................................................................................................................................... 36 Figure 6. Attenuation of hepatic leptin signalling does not alter body composition ................... 37 Figure 7. Levels of plasma leptin binding proteins are not altered in male mice lacking hepatic leptin signalling ............................................................................................................................ 40 Figure 8. Loss of hepatic leptin signalling does not affect glucose metabolism in the fasted state ...................................................................................................................................................... 40 Figure 9. Loss of hepatic leptin signalling prevents age- and diet-related glucose intolerance .. 42 Figure 10. Attenuation of hepatic leptin signalling does not alter glucose tolerance in female mice............................................................................................................................................... 43 Figure 11. Attenuation of hepatic leptin signalling increases glucose-stimulated insulin levels 44 Figure 12. Effect of attenuated hepatic leptin signalling on glucose-stimulated insulin levels during high fat feeding.................................................................................................................. 45 Figure 13. Ablation of hepatic leptin signalling increases insulin sensitivity ............................. 46 Figure 14. Loss of hepatic leptin signalling enhances liver insulin sensitivity ........................... 47 Figure 15. Attenuation of hepatic leptin signalling results in increased insulin-stimulated phosphorylation of Akt in the liver ............................................................................................... 48 Figure 16. Leprflox/flox AlbCre+ ob/ob mice have truncated leptin receptor transcripts in the liver ...................................................................................................................................................... 54 Figure 17. Intraperitoneal injections of 0.1 ug/g leptin has minimal effect on body weight and food intake in Leprflox/flox AlbCre ob/ob mice ............................................................................... 55 Figure 18. Intraperitoneal injections of 0.1 µg/g leptin modestly lowers blood glucose in ob/ob mice but not in ob/ob mice lacking liver leptin signalling ........................................................... 56 Figure 19. Intraperitoneal injections of 0.8 µg/g leptin has minimal effect on body weight and food intake in Leprflox/flox AlbCre ob/ob mice ............................................................................... 57 Figure 20. Intraperitoneal injections of 0.8 µg/g leptin lowers blood glucose more in ob/ob mice than in ob/ob mice lacking liver leptin signalling ........................................................................ 58 Figure 21. 5 µg/day leptin via mini-osmotic pump normalizes food intake and blood glucose levels independent of hepatic leptin signalling in Leprflox/flox AlbCre ob/ob mice ........................ 59  ix  Figure 22. 0.6 µg/day leptin treatment has minimal effect on body weight and food intake in Leprflox/flox AlbCre ob/ob mice ....................................................................................................... 60 Figure 23. 0.6 µg/day leptin via mini-osmotic pump lowers blood glucose more in ob/ob mice than in ob/ob mice lacking liver leptin signalling ........................................................................ 61 Figure 24. Obese, hyperinsulinemic mice lacking hepatic leptin signalling have impaired glucose tolerance .......................................................................................................................... 61 Figure 25. The effects of ablated hepatic leptin signalling on insulin sensitvity in obese, hyperinsulinemic mice .................................................................................................................. 63 Figure 26. Restoration of hepatic leptin signalling in db/db mice after infection with Ad-Lepr-b ...................................................................................................................................................... 65 Figure 27. Restoration of hepatic leptin signalling in db/db mice does not affect body weight, plasma leptin, blood glucose, or plasma insulin levels ................................................................. 66 Figure 28. Restoration of hepatic leptin signalling in db/db mice results in impaired glucose tolerance compared to db/db controls ........................................................................................... 67 Figure 29. The effects of insulin on hepatic triglyceride secretion in mice lacking hepatic leptin signalling....................................................................................................................................... 75 Figure 30. Attenuation of hepatic leptin signalling results in increased VLDL triglycerides and larger apoB-containing lipoprotein particles ................................................................................ 78 Figure 31. Attenuation of hepatic leptin signalling decreases plasma and hepatic apoB levels . 79 Figure 32. Hepatic leptin signalling regulates lipase activity levels in the liver ......................... 81 Figure 33. Loss of hepatic leptin signalling results in hepatic lipid accumulation ..................... 82 Figure 34. Attenuation of hepatic leptin signalling results in increased hepatic lipid accumulation ................................................................................................................................. 82 Figure 35. Attentuation of hepatic leptin signalling does not affect lipid tolerance in lean mice 83 Figure 36. Hepatic leptin signalling has a subtle but consistent effect on plasma triglycerides in obese, hyperinsulinemic mice ....................................................................................................... 92 Figure 37. Restoration of hepatic leptin signalling in db/db mice restores normal hepatic apoB levels ............................................................................................................................................. 93 Figure 38. Hepatic leptin signalling regulates lipase activity levels in db/db mice .................... 94 Figure 39. Hepatic leptin signalling acutely improves lipid tolerance in ob/ob and db/db mice 96 Figure 40. Leptin increases plasma IGFBP-2 levels in ob/ob mice independent of hepatic leptin signalling..................................................................................................................................... 102 Figure 41. Lean mice lacking hepatic leptin signalling have normal plasma IGFBP-2 levels .. 103 Figure 42. Hepatic leptin signalling is not sufficient for restoring normal IGFBP-2 levels in db/db mice .................................................................................................................................. 105 Figure 43. Hepatic leptin signalling in lean mice ...................................................................... 110 x  Figure 44. Hepatic leptin resistance in obesity and type 2 diabetes .......................................... 113 Figure A1. Plasma lipocalin-2 levels are unaffected by a loss of hepatic leptin signalling ...... 162 Figure A2. Pathways affected by genes differentially expressed in Leprflox/flox AlbCre mice after an overnight fast ......................................................................................................................... 163 Figure A3. Biological processes affected by genes differentially expressed in Leprflox/flox AlbCre ob/ob mice after an overnight fast .............................................................................................. 164  xi  List of Abbreviations Acadm  acyl-CoA dehydrogenase, medium chain  ACO  acyl-CoA oxidase  Ad-Lepr-b  adenovirus expressing the long signalling isoform of the mouse leptin receptor  Ad-β-gal  adenovirus expressing β-galactosidase  AMPK  5'-adenosine monophosphate-activated protein kinase  ANOVA  analysis of variance  Ap2  adipose protein-2  ApoA4  apolipoprotein A-IV  ApoB  apolipoprotein B  ApoCIII  apolipoprotein C-III  ApoE  apolipoprotein E  AUC  area under the curve  BGU  whole body glucose utilization  BMI  body mass index  BSA  bovine serum albumin  cDNA  complementary deoxyribonucleic acid  CMV  cytomegalovirus  CPT1  carnitine palmitoyl transferase-1  CSF  cerebral spinal fluid  DNA  deoxyribonucleic acid  ECL  enhanced chemiluminescence  EDTA  ethylene diamine tetraacetic acid  EGP  endogenous glucose production xii  ELISA  enzyme-linked immunosorbent assay  ERK  extracellular signal-regulated kinase  Fasn  fatty acid synthase  FoxO1  forkhead box O1  FPLC  fast protein liquid chromatography  G6Pase  glucose-6-phosphatase  Gck  glucokinase  GIR  glucose infusion rate  GRB2  growth factor receptor bound protein-2  GS  glycogen synthase  GSK3  glycogen synthase kinase-3  HFD  high fat diet  HL  hepatic lipase  HRP  horseradish peroxidase  ICV  intracerebroventricular  IGF  insulin-like growth factor  IGFBP-2  insulin-like growth factor binding protein-2  IRS  insulin receptor substrate  ITT  insulin tolerance test  JAK  Janus kinase  KATP channels  ATP-sensitive potassium channels  LDL  low density lipoprotein  Lepr  leptin receptor  LIRKO  liver-specific insulin receptor knockout  LPL  lipoprotein lipase xiii  MRI  magnetic resonance imaging  mRNA  messenger ribonucleic acid  Mttp  microsomal triglyceride transfer protein  NMR  nuclear magnetic resonance  NP-40  nonidet P-40  OGTT  oral glucose tolerance test  P-407  poloxamer-407  PCR  polymerase chain reaction  PDE3B  phosphodiesterase-3B  PDK1  phosphoinositide-dependent kinase-1  Pdx-1  pancreatic and duodenal homeobox-1  Pepck  phosphoenolpyruvate carboxykinase  PGC-1  peroxisome proliferator-activated receptor gamma coactivator-1  PI3K  phosphatidylinositol-3-kinase  PIP3  phosphatidylinositol-3,4,5-trisphosphate  PKC  protein kinase C  POMC  proopiomelanocortin  PPARα  peroxisome proliferator-activated receptor alpha  PPARγ  peroxisome proliferator-activated receptor gamma  PTP1B  protein tyrosine phosphatase-1B  PVDF  polyvinylidene fluoride  RF  radiofrequency  RIP  rat insulin promoter  RNA  ribonucleic acid xiv  RT-PCR  reverse transcriptase polymerase chain reaction  Scd1  stearoyl-CoA desaturase-1  SDS-PAGE  sodium dodecyl sulphate polyacrylamide gel electrophoresis  SEM  standard error of the mean  SHP2  Src homology-containing tyrosine phosphatase-2  SOCS3  suppressor of cytokine signalling-3  Srebp-1c  sterol regulatory binding protein-1c  STAT  signal transducers and activators of transcription  TCA  trichloroacetic acid  Tyr  tyrosine  UBC  University of British Columbia  UCP2  uncoupling protein-2  VLDL  very low density lipoprotein  WebGestalt  Web-based Gene Set Analysis Toolkit  xv  Acknowledgements The road to a PhD is a long and arduous one that cannot be travelled alone and I have been so fortunate to have had tremendous support throughout my PhD. Tim, thanks for taking a chance on me after I bombed my first interview with you for a co-op position. Scott, thanks for being so encouraging during my co-op work term even when all my data was negative or when my data showed the opposite of what our hypothesis predicted. If it wasn’t for the encouraging atmosphere during those first few months of research, I definitely would not have gone to grad school. You guys definitely helped me discover what I think I was meant to do. Of course, I must also mention the grad students, past and present: Anna, Blair, Cathy, Gary, Heather, Irene, Jasna, Mike, Rhonda, and Ursula. I can’t think of a better group of people to have shared the highs and lows of science with. Thanks for putting up with my unique brand of sarcasm in the office. I can only hope that the people I work with in the future are as awesome and supportive as you guys have been both in and out of the lab. To my parents, I don’t know how to even begin to express my gratitude. You came to Canada with nothing and worked so hard to give us what we have. I complain and complain about all the work I have put into this thesis, but I know it pales in comparison to how hard you have worked to start a new life in Canada and to give me the opportunities that I have had. Thank you for all your love and support. Lastly, to my loving wife Crystal, you have been with me every step of the way throughout grad school. No one knows the ups and downs I have had quite like you do. Thank you for putting up with me and promising to do so for the rest of our lives. I am so excited to be starting a new life with you and I can’t wait to see what the future holds for us.  xvi  Dedication  To my supportive parents and my loving wife  xvii  CHAPTER 1 – INTRODUCTION 1.1 Type 2 Diabetes Currently, over 366 million adults worldwide are estimated to have type 2 diabetes and this number is expected to grow to 552 million by 2030 [1]. The Canadian Diabetes Association estimates that about 20 people every hour of every day are diagnosed with type 2 diabetes and by 2020, 1 in 10 Canadians will be afflicted with the disease [2]. The human cost of diabetes is clear: 14% of all North American deaths of individuals aged 20-79 in 2011 were projected to be caused by diabetes [1]. Further, a pertinent consideration in light of the current economic climate is the economic cost of diabetes. It is estimated that diabetes will cost the Canadian economy $12.2 billion dollars in 2010 and the burden is expected to rise to $16.9 billion by 2020 [2]. Worldwide, diabetes expenditures exceeded $465 billion in 2011, accounting for 11% of total healthcare expenditures in adults [1]. In 2006, a staggering $274 million of public funds and $81 million of private funds were spent on blood glucose monitoring strips alone in Canada [3] and these costs are estimated to exceed $1 billion by 2015 [4]. The costs of diabetes are unsustainable and will keep rising unless something is done to prevent or cure type 2 diabetes. To this end, it is essential that effort is invested into understanding the underlying causes of type 2 diabetes. Type 2 diabetes is characterized by resistance to insulin action and a relative insufficiency in insulin secretion. These defects in insulin secretion and action result in serious metabolic abnormalities. Normally, insulin acts on receptors to mediate glucose uptake in a variety of tissues in the body, predominantly muscle and adipose [5]. Furthermore, insulin has potent effects on the liver to suppress glycogenolysis and gluconeogenesis, thus decreasing hepatic glucose production [5]. It follows then that in type 2 diabetes, where there is resistance to the  1  actions of insulin, hyperglycemia ensues, largely due to decreased insulin-mediated glucose uptake and uncontrolled glucose production from the liver. Type 2 diabetes is also characterized by aberrant lipid metabolism, but the effects of insulin on lipid metabolism in type 2 diabetics are more complex. Insulin inhibits lipolysis of triglyceride stores in adipose tissue, leading to decreased plasma free fatty acid levels [5]. Further, insulin very potently downregulates triglyceride secretion in the form of very low density lipoproteins (VLDL) from the liver [6-8]. Thus, with insulin resistance in type 2 diabetes, there are elevated free fatty acids [9] and triglycerides [10] in the blood. However, insulin also has powerful effects on promoting fatty acid synthesis and lipid storage in adipose tissue and liver [5, 11] and these effects are actually enhanced in obesity and type 2 diabetes. Thus, it appears that while the glucometabolic effects of insulin are largely downregulated in type 2 diabetes, some of the lipometabolic effects are in fact enhanced [12-14]. Collectively, these symptoms seen in type 2 diabetes can culminate in serious complications, including cardiovascular disease, kidney failure, and nerve damage. These complications can greatly reduce quality of life and can reduce life expectancy by as much as 10 years [15]. Type 2 diabetes and obesity The causes of type 2 diabetes are currently unknown. It is a disease with a complex etiology that likely involves both genetic and environmental factors. One factor that is highly correlated with the development of type 2 diabetes is obesity, with 90% of type 2 diabetics being overweight (body mass index (BMI) of 25-29.9) or obese (BMI of 30 or above) [16]. Concurrent with the rapid rise in incidence rates of type 2 diabetes has been a similar rise in obesity rates. An aging population, increase in sedentary lifestyles, and unhealthy food choices have led to increased obesity rates around the world. Currently, over 50% of adult Canadians are overweight [17]. Further, the International Obesity Taskforce states that 1.7 billion people are 2  now at risk for weight-related diseases [18], including type 2 diabetes. Obesity has been identified as one of the main risk factors for developing type 2 diabetes [19, 20], but even with all the evidence demonstrating that obesity is a major risk factor for type 2 diabetes, a clear mechanistic link between diabetes and obesity has still not been shown.  1.2 The Hormone Leptin Discovery One factor that may help explain the relationship between obesity and diabetes is the hormone leptin. The discovery of leptin has its roots in the initial characterizations of the ob/ob and db/db mice at the Jackson Laboratory by Douglas L. Coleman [21] beginning in the 1960s. Both strains of mice were first described by the Jackson Laboratory with the ob/ob mouse having a mutation in the obese (ob) gene and the db/db mouse having a mutation in the diabetes (db) gene [22, 23]. These strains have an identical obese, diabetic phenotype when put on the same genetic background yet the ob and db mutations were mapped to chromosome 6 and chromosome 4 respectively. Thus, it was hypothesized that while the ob/ob and db/db mice had mutations in different genes, the defects arising from these mutations likely occur in the same metabolic pathway [24]. Coleman hypothesized that a circulating factor in the ob/ob or db/db mouse may be responsible for the obese phenotype. He tested this hypothesis by using parabiosis, a surgical technique by which the circulations of two mice are joined together such that circulating factors may freely cross between the two mice. When Coleman joined a db/db mouse to a wildtype mouse, the wildtype mouse consistently died of hypoglycemia and starvation [25]. Similarly, when a db/db mouse was joined to an ob/ob mouse, the ob/ob mouse had a marked decrease in food intake, weight loss, and a drop in blood glucose levels. The ob/ob mouse eventually died of  3  starvation while the db/db mouse continued to gain weight [26]. Next, when Coleman joined an ob/ob mouse to a wildtype mouse, the ob/ob mouse lost weight, had normal food intake and blood glucose levels, and this pair of mice survived for months [26]. The results of these experiments suggested that the db/db mouse produced a large amount of a satiety factor that it could not respond to while the ob/ob mouse could respond to this factor but could not produce it. Coleman’s team and others around the world raced to identify this circulating satiety factor, but it was not positively identified until 1994 when Jeffrey M. Friedman successfully used positional cloning to identify this circulating factor. This factor was subsequently named leptin, from the Greek leptos, meaning thin. Leptin is the adipose-derived gene product of the ob gene [27]. It is secreted in proportion to the amount of body fat stores [28] and thus it is believed that leptin acts as a peripheral signal to the brain to indicate a sufficient amount of energy stores [29]. Soon after its discovery, leptin was shown to be able to normalize body weight by reducing food intake and increasing energy expenditure in obese, hyperphagic leptin-deficient ob/ob mice [30-32] and these weight-reducing effects appeared to be mediated through actions on the brain [32]. Furthermore, leptin has been proposed to have a protective role against the effects of starvation. During food deprivation in mice, plasma leptin levels fall disproportionately to fat mass loss [33]. This leads to neuroendocrine alterations resulting in decreased energy expenditure and infertility, which allows for the conservation of energy until which time food becomes available again [33]. Humans with mutations in the leptin gene have also been identified. Similar to ob/ob mice, these patients present with hyperphagia, extreme obesity, and hyperinsulinemia [34-36] and these symptoms are normalized with leptin treatment [37, 38]. Many of these patients also have baseline plasma triglyceride levels that are either above normal or at the upper end of the 4  normal range [35, 36, 38-40] and these levels are decreased upon leptin treatment [36, 38]. Patients with mutations in the leptin receptor gene have also been identified and they also display marked obesity and hyperleptinemia, similar to db/db mice [41]. The discovery of leptin generated considerable excitement that the cure for obesity had been found, but it was soon discovered that most obese humans were hyperleptinemic and resistant to the weight-reducing effects of leptin [42]. Nonetheless, studies on the mechanisms of leptin resistance and studies on the metabolic effects of leptin have proven to be invaluable in the fight against obesity and diabetes. Leptin signalling Shortly after the discovery of leptin, the leptin receptor was identified as the gene product of the db gene [43, 44] and several isoforms of the leptin receptor (Lepr-a to Lepr-f) have been identified in the brain [45]. In particular, Lepr-b, the isoform with the longest intracellular domain, contains all the consensus motifs needed for maximal activation of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signalling pathway [46] (Figure 1). Of the six different receptor isoforms that have been identified, the Lepr-b isoform is believed to be responsible for the majority of leptin signalling. This is supported by the fact that db/db mice, which lack only Lepr-b [45], have the same obese, diabetic phenotype as leptindeficient ob/ob mice [24]. Lepr-b is present on the cell membrane in dimeric form, and upon leptin binding, a conformational change occurs [47]. When this conformational change occurs, JAK2, which is constitutively associated with the leptin receptor, undergoes autophosphorylation (Figure 1). Phosphorylated JAK2 (p-JAK2) then catalyzes the phosphorylation of three critical tyrosine residues on Lepr-b: Tyr-985, Tyr-1077, and Tyr-1138 [29]. Phosphorylated Tyr-985 activates Src homology-containing tyrosine phosphatase-2 (SHP2), which then binds to growth factor  5  receptor bound protein 2 (GRB2) and stimulates extracellular signal-regulated kinase (ERK) signalling [48]. Phosphorylated Tyr-1077 mediates phosphorylation of STAT5 [49], which is a transcription factor and can modulate gene expression. Similarly, phosphorylated Tyr-1138 stimulates phosphorylation of STAT3, which can also regulate gene expression. One gene that phosphorylated STAT3 (p-STAT3) can upregulate in most tissues is suppressor of cytokine signalling-3 (SOCS3), which through a negative feedback loop, can mediate dephosphorylation of both Tyr-985 and JAK2 [48, 50].  Figure 1. Leptin signalling pathway. The signalling isoform of the leptin receptor exists as a homodimer of two Lepr-b subunits. Upon binding of leptin, an intracellular signalling cascade is activated culminating in the phosphorylation and activation of ERK, STAT5, and STAT3 signalling. In a negative feedback loop, p-STAT3 promotes transcription of SOCS3, which can downregulate leptin signalling. JAK2, Janus kinase-2; SHP2, Src homology-2 domaincontaining phosphatase-2; GRB2, growth factor receptor-bound protein-2; ERK, extracellular signal-regulated kinase; STAT5, signal transducer and activator of transcription-5; STAT3, signal transducer and activator of transcription-3; SOCS3, suppressor of cytokine signalling-3.  6  Diet-induced obesity has been shown to be associated with defects in leptin signalling, resulting in leptin resistance. In rodent models, decreased activation of the JAK-STAT pathway in response to a high fat diet has been reported in several brain regions [51-54] as well as in the periphery [55-57]. It is hypothesized that an upregulation of the negative regulator SOCS3 can result in leptin resistance seen during diet-induced obesity [51, 54, 55, 58]. In fact, mice with a mutation in Tyr-985 of the leptin receptor, which is a binding site for SOCS3 [59], have increased leptin sensitivity and are resistant to diet-induced obesity [60]. Thus, maintaining normal leptin signalling may be crucial in the fight against obesity. Central effects of leptin on body weight regulation Leptin can act on several regions in the brain, including the hypothalamus, ventral tegmental area, and nucleus of the solitary tract [61]. The hypothalamus in particular has been shown to play an important role in mediating the effects of leptin on body weight regulation. The arcuate nucleus of the hypothalamus contains neurons that express proopiomelanocortin (POMC), which can be post-translationally processed into anorexigenic peptides, including αmelanocyte-stimulating hormone (α-MSH) and β-melanocyte-stimulating hormone (β-MSH). αMSH acts on neurons that express melanocortin 3 (MC3) and melanocortin 4 (MC4) receptors to decrease food intake [62]. β-MSH, which is present in humans but not rodents, exerts its antiobesity effects by acting on MC4 receptors [63, 64]. Some POMC neurons also express Lepr-b and stimulation of these neurons by leptin results in increased expression of POMC and its related peptides through a p-STAT3 dependent mechanism [29, 65]. The arcuate nucleus also contains Lepr-b-expressing neurons that express the orexigenic agouti-related peptide (AgRP) and neuropeptide Y (NPY). AgRP acts on MC3 and MC4 receptor-expressing neurons to increase food intake. Stimulation of these AgRP/NPY neurons by leptin via p-STAT3 signalling [66] results in suppression of AgRP and NPY expression [29, 67]. Interestingly, however,  7  knockout of leptin receptors in either POMC neurons or AgRP/NPY neurons results in only mild obesity [68, 69] and while deletion of leptin receptors from both POMC and AgRP/NPY neurons exacerbates the obese phenotype, leptin administration is still able to decrease food intake in these mice to some extent [69]. This suggests that the central effects of leptin on body weight regulation are highly redundant and may involve pathways outside of the well-described pathways in the arcuate nucleus. In the ventromedial hypothalamus, leptin can act on leptin receptors on steroidogenic factor 1 (SF1) neurons, resulting in their depolarization. Deletion of leptin receptors specifically in SF1 neurons results in decreased energy expenditure and mild obesity [67, 70]. When leptin receptors were deleted from both SF1 neurons and POMC neurons, the obese phenotype was more pronounced [70]. These observations again demonstrate the redundant nature of central leptin signalling on body weight regulation. Metabolic effects of leptin In addition to obesity and hyperphagia, leptin-deficient ob/ob mice and leptin receptordeficient db/db mice both display aberrant glucose and lipid metabolism, similar to metabolic derangements seen in type 2 diabetes. These symptoms are well-documented and include hyperglycemia, hyperinsulinemia, glucose intolerance, insulin resistance, hypertriglyceridemia, and hypercholesterolemia [24, 30, 71-76]. While it can be hypothesized that these symptoms are secondary to the hyperphagia and obesity seen in ob/ob and db/db mice, several observations suggest that leptin may have metabolic effects independent of its effects on body weight. First, hyperinsulinemia precedes obesity in both ob/ob and db/db mice [77, 78] and hypercholesterolemia precedes obesity in db/db mice [75]. Also, pair-feeding vehicle-treated ob/ob mice to the same levels as leptin-treated ob/ob mice is not able to reduce serum glucose and insulin levels to the same extent as leptin treatment [79], which indicates that leptin has  8  additional effects on metabolism independent of its effects on food intake. Further, leptin is also able to ameliorate hyperglycemia and hyperinsulinemia in ob/ob mice, even at low doses that have minimal effect on body weight [30, 71, 80]. Interestingly, even a single moderate dose of leptin administered to ob/ob mice reverses hyperglycemia and hyperinsulinemia within a few hours while the mice remain obese [81]. To further highlight the fact that the metabolic effects of leptin are independent of its effects on obesity, lipodystrophic mice and humans, which have little to no adipose tissue and are hypoleptinemic, also display hyperglycemia, hyperinsulinemia, insulin resistance, hypertriglyceridemia, and hepatic steatosis and these symptoms are ameliorated by leptin [82-85]. Collectively, these observations suggest that leptin has effects on metabolism independent of its effects on body weight. In fact, since leptin can affect metabolism at such low doses and short timeframes that do not affect body weight, the major effect of leptin may actually be on glucose and lipid metabolism and not on body weight. Leptin action in the periphery The manner by which leptin directly affects metabolism is not fully understood. It has been hypothesized that similar to the effects of leptin on food intake, leptin may affect metabolism by acting on the brain. Several studies have demonstrated that central intracerebroventricular (ICV) administration of leptin can affect gene expression in many organs that play a profound role in both glucose and lipid metabolism, including the liver [82, 86-88], pancreas [89], heart [90], and skeletal muscle [91]. However, many studies have demonstrated hypothalamic-independent effects of leptin [90, 92-95]. Perhaps the most compelling evidence that leptin has direct effects on the periphery is the fact that leptin receptors, including Lepr-b, are found directly in a number of tissues such as the lungs, skeletal muscle, kidneys, pancreas, heart, adipose, and liver [43, 46]. Among peripheral tissues, the highest amount of Lepr-b appears to be expressed in stomach, pancreas, and heart tissue [46].  9  Leptin has been shown to have profound effects on tissues that play an important role in glucose and lipid metabolism. In skeletal muscle, it has been shown that leptin can activate 5'adenosine monophosphate-activated protein kinase (AMPK), which leads to numerous effects, including promotion of fatty acid oxidation, decreasing triglyceride storage, and increasing insulin sensitivity [96-99]. The effects of leptin on glucose metabolism in skeletal muscle are less clear with studies showing leptin can increase glucose uptake [100-103], have no effect on glucose uptake [104, 105], or decrease glucose uptake [106]. These differential effects may depend on acute versus chronic leptin stimulation of myocytes [101, 107]. In the pancreas, leptin has a potent effect on β-cells, decreasing insulin secretion through inhibition of insulin gene transcription [81], activation of ATP-sensitive potassium (KATP) channels [108] and phosphodiesterase-3B (PDE3B) [109], or inhibition of protein kinase C (PKC) [110]. Our lab investigated the effects of leptin on β-cells in vivo, using the Cre-lox system with the rat insulin promoter (RIP)-Cre transgene to generate mice with a β-cell specific loss of leptin signalling [93]. These mice had fasting hyperinsulinemia, impaired glucosestimulated insulin secretion, glucose intolerance, insulin resistance, and mild obesity. However, they also had a partial loss of leptin receptors in the hypothalamus due to some hypothalamic RIP promoter activity. Morioka et al. [94] generated a similar mouse with a β-cell specific loss of leptin signalling using the pancreatic and duodenal homeobox-1 (Pdx-1)-Cre transgene and these mice also showed hyperinsulinemia, but they had improved glucose tolerance due to elevated glucose-stimulated insulin secretion. Interestingly, when the mice were given a high-fat diet to induce obesity, they also showed impaired glucose-stimulated insulin secretion and impaired glucose tolerance. Thus, in both models of β-cell specific loss of leptin signalling, it appears that the added stress of obesity is required for impaired glucose tolerance.  10  In adipose tissue, leptin has powerful effects on lipolysis and reducing lipid storage. Leptin can upregulate expression of acyl-CoA oxidase (ACO), carnitine palmitoyl transferase 1 (CPT1), peroxisome proliferator-activated receptor-α (PPARα), uncoupling protein-2 (UCP2), and PPAR gamma coactivator-1 (PGC-1) in adipocytes [96]. In wildtype C57BL/6 mice treated with human leptin [111], transgenic skinny mice overexpressing mouse leptin [112], or rats treated with an adenovirus expressing rat leptin under the cytomegalovirus (CMV) promoter [113], the resulting hyperleptinemia leads to a nearly complete loss of fat mass. While these effects on fat loss could be due to central effects of leptin on food intake and energy expenditure, Chen et al. showed that fat loss was independent of the effects of AdCMV-Leptin on food intake [113]. Furthermore, when Lepr-b was overexpressed specifically in adipose tissue of db/db mice using an adipose protein-2 (ap2)-Lepr-b transgene, there was a considerable loss in fat mass, resistance to obesity, and a reduction in lipogenic gene expression in adipose tissue [114]. When Lepr-b was selectively knocked down in adipose tissue of lean mice, these mice had a gain in adiposity, reiterating the lipolytic role of direct leptin action on adipocytes [115]. In terms of glucose metabolism, leptin action on isolated adipocytes results in impaired insulin signalling and reduced insulin-mediated glucose uptake and glycogen synthesis [116-118]. Thus, in terms of both glucose and lipid metabolism, the overall effect of leptin on adipocytes is antagonistic to the effects of insulin.  1.3 The Role of the Liver in Glucose and Lipid Metabolism One peripheral organ that we were particularly interested in is the liver since it can profoundly affect both glucose and lipid metabolism. In times of fasting, the liver can generate glucose via gluconeogenesis and the breakdown of hepatic glycogen stores and export this glucose to the rest of the body. Further, the liver can also export triglycerides in VLDL particles  11  to provide energy for the body. During fed states, the liver can store glucose as glycogen and triglycerides in the form of lipid droplets. The actions of the liver on controlling glucose and lipid flux during times of fasting and feeding are tightly regulated by insulin action on the liver (Figure 2). The insulin receptor is a tetramer of two alpha and two beta subunits and upon binding of an insulin molecule, the receptor undergoes autophosphorylation. The phosphorylated receptor recruits and phosphorylates a variety of insulin receptor substrates (IRSs), which in turn phosphorylate the regulatory subunit of phosphatidylinositol-3-kinase (PI3K). PI3K catalyzes the formation of the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane and this recruits phosphoinositide-dependent kinase-1 (PDK1) and Akt to the plasma membrane. PDK1 then phosphorylates Akt. Phosphorylated Akt (p-Akt) has a number of effects on hepatic function. First, p-Akt can phosphorylate and inactivate glycogen synthase kinase-3 (GSK3), resulting in decreased phosphorylation and increased activity of glycogen synthase (GS). This leads to increased glycogen synthesis. Further, p-Akt can phosphorylate the transcription factor forkhead box O1 (FoxO1), which leads to the sequestration of FoxO1 in the cytoplasm and its degradation. Without FoxO1 in the nucleus, there is decreased transcription of genes involved with hepatic glucose production and lipid export, and increased transcription of lipid synthesis genes (Figure 2) [5, 119, 120]. Therefore, insulin signalling is a potent mediator of glucose and lipid metabolism in the liver. As a result, the dysregulation of hepatic insulin action seen in type 2 diabetes is a major contributor to hyperglycemia and hyperlipidemia.  12  Figure 2. Hepatic insulin signalling pathway. After binding of insulin to the insulin receptor, a cascade of signalling events occurs, eventually leading to increased glycogen synthesis, decreased hepatic glucose production, decreased lipid export, and increased lipid storage. IRS, insulin receptor substrate; PI3K, phosphatidylinositol-3-kinase; PDK1, phosphoinositidedependent kinase-1; GSK3, glycogen synthase kinase-3; GS, glycogen synthase; FoxO1, forkhead box O1; Pepck, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; Mttp, microsomal triglyceride transfer protein; ApoCIII, apolipoprotein C-III; ApoB, apolipoprotein B; Srebp-1c, sterol regulatory binding protein-1c; PPARα, peroxisome proliferator-activated receptor alpha, PPARγ, peroxisome proliferator-activated receptor gamma.  Leptin action in the liver We hypothesize that some of the anti-diabetic effects of leptin may be exerted through direct effects on the liver. Leptin receptors have been shown by various groups to be expressed in liver cells [43, 45, 46, 72, 121-126], including Lepr-b, the isoform with JAK/STAT signalling  13  capability [46, 122, 123, 126]. Further, many studies have shown that leptin has direct effects on hepatocytes, but there is no consensus on how this affects glucose and lipid metabolism [127]. It has been shown that leptin treatment of hepatocytes in vitro can differentially regulate insulin signalling and this can have a major impact on hepatic glucose and lipid metabolism. Cohen et al. [121] demonstrated that after a 10 minute leptin pre-treatment, human hepatoma cells showed decreased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in response to insulin, suggesting that leptin may negatively regulate hepatic insulin signalling. On the other hand, Anderwald et al. [128] showed that perfusion of isolated rat liver with leptin for 90 minutes increased insulin-stimulated phosphorylation of the insulin receptor and reduced phosphoenolpyruvate carboxykinase (PEPCK) levels, suggesting that leptin can increase hepatic insulin sensitivity. Similarly, Lam et al. found that 3 hours of leptin treatment directly improved insulin-stimulated phosphorylation of the insulin receptor in HepG2 hepatocytes [129]. Leptin has also been shown to upregulate one part of the insulin signalling cascade while at the same time down regulating another [123]. Interestingly, Szanto and Kahn [123] showed that the differential effects of leptin on insulin-induced phosphorylation of IRS-1 and IRS-2 in rat hepatocytes were dependent on the length of leptin pre-treatment. Taken together, these data clearly show that leptin has direct effects on insulin signalling in hepatocytes, but the ultimate effect on metabolism may depend on a number of factors, including whether the exposure of hepatocytes to leptin is acute or chronic. Studies involving in vivo leptin treatment have demonstrated a number of effects on the liver. Several studies have shown an increase in hepatic insulin sensitivity after systemic leptin treatment in normal and leptin-deficient rodents [129-131]. Importantly, this results in an enhancement of insulin’s effects on suppressing hepatic glucose production, which is typically upregulated in type 2 diabetes. In addition, leptin has major effects on promoting lipolysis in the 14  liver. In several models of leptin deficiency, including ob/ob mice and lipodystrophic mice, hepatic steatosis is very prominent and leptin treatment is able to greatly reduce the amount of lipid stored in the liver [82, 132]. In addition to these findings, leptin treatment has also been found to modulate a number of key genes in the liver that are involved in glucose and lipid metabolism, including Pepck, glucokinase (Gck), stearoyl-CoA desaturase-1 (Scd1), sterol regulatory element binding protein (Srebp)-1 and -2, and fatty acid synthase (Fasn) [12, 87, 133]. However, a major confounding factor in all the above-mentioned in vivo leptin studies is that these effects on the liver may be mediated through central leptin action since ICV administration of leptin is also able to affect gene expression in the liver [82, 86-88]. Thus, many studies showing effects of peripheral leptin administration on the liver are unable to provide clear evidence for direct leptin action at the level of liver. There are a handful of in vivo studies that do more specifically show effects of leptin directly on the liver. Lee et al. [134] used an adenoviral approach to re-express functional leptin receptors in livers of fa/fa rats, which have a mutation in the leptin receptor gene resulting in leptin receptors with a mutated extracellular domain that are defective in JAK-STAT signalling [135]. The authors were able to successfully re-express functional leptin receptors in the liver while avoiding the introduction of leptin receptors to the hypothalamus. Using this method, it was seen that re-establishing leptin signalling specifically in the liver was able to ameliorate hepatic steatosis and reduce plasma triglycerides [134]. In another study, Cohen et al. used the Cre-lox approach to generate mice with a hepatocyte-specific ablation of the complete leptin receptor [125]. This study found few metabolic differences. While the authors showed that females lacking hepatic leptin receptors had higher plasma leptin levels than controls, they found no differences in body composition, hepatic triglycerides, or random-fed plasma glucose and insulin levels between mice homozygous for the floxed allele compared to heterozygote controls.  15  However, the characterization of these mice in terms of glucose and lipid metabolism was incomplete since many parameters were only measured at one age and other aspects of glucose and lipid metabolism such as glucose tolerance and plasma lipid levels were not investigated.  1.4 Thesis Investigation Given the high correlation of obesity and type 2 diabetes, it is essential that the molecular mechanism underlying this correlation is elucidated. Aberrant leptin action during obesity may play a role in this correlation. Since leptin receptors are expressed in the liver, it was hypothesized that direct leptin action in the liver plays an important role in controlling glucose and lipid metabolism and that dysregulation of leptin signalling in the liver contributes to the milieu of metabolic defects seen in type 2 diabetes. To address this hypothesis, mice with a hepatocyte-specific loss of leptin signalling were generated. As described in Chapters 3 and 4 of this thesis, glucose and lipid metabolism were thoroughly characterized in these mice lacking hepatic leptin signalling. Furthermore, these mice lacking leptin signalling in the liver were crossed onto a leptin-deficient ob/ob background. By treating these mice with leptin, it was possible to investigate the acute effects of leptin on the liver. Complementary to this, db/db mice were also treated with an adenovirus expressing functional leptin receptors to selectively restore leptin signalling to the liver of these mice. Chapter 5 of this thesis includes studies aimed at characterizing glucose metabolism in these unique ob/ob and db/db mouse models. Similarly, Chapter 6 contains extensive studies on lipid metabolism in these mice. Chapter 7 includes an investigation into a possible role for hepatic leptin signalling in regulating levels of insulin-like growth factor binding protein-2 (IGFBP-2), a leptin-regulated, liver-derived protein recently shown to have potent anti-diabetic effects. Collectively, the results of these studies will provide new insights into the role of leptin in  16  regulating glucose and lipid metabolism and will shed further light on a possible molecular link between obesity and type 2 diabetes.  17  CHAPTER 2 – METHODS AND MATERIALS 2.1 Animals All procedures with animals were approved by the University of British Columbia (UBC) Animal Care Committee and carried out in accordance with the Canadian Council on Animal Care guidelines. Mice were housed in a 12 h light-12 h dark cycle. Most in vivo studies involving Leprflox/flox AlbCre and Leprflox/flox AlbCre ob/ob mice were performed at the D.H. Copp Building at UBC with the exception of the triglyceride secretion assays, which were performed at the Conventional Rodent Unit of the UBC Animal Care Centre. At these facilities, mice had ad libitum access to water and a chow diet (LabDiet, #5015, Richmond, USA). For high fat diet studies, mice were given a diet where 58% of the calories were from fat (Table 1) (Research Diets, #D12330, New Brunswick, USA). All studies involving adenovirus-treated db/db mice were performed at the UBC Centre for Disease Modelling, where mice were fed with a different chow diet (Harlan Laboratories, #2918, Indianapolis, USA). Table 1. Composition of high fat diet #D12330 from Research Diets. Composition Protein Carbohydrate Fat Ingredients Casein, 80 Mesh DL-Methionine Maltodextrin 10 Corn Starch Soybean Oil Coconut Oil, hydrogenated Mineral Mix S10001 Sodium Bicarbonate Potassium Citrate Vitamin Mix V10001 Choline Bitartrate FD&C Blue Dye #1 FD&C Red Dye #40  gm% 23.0 35.5 35.8 gm 228 2 170 175 25 333.5 40 10.5 4 10 2 0.05 0.05  kcal% 16.4 25.5 58.0 kcal 912 0 680 700 225 3001.5 0 0 0 40 0 0 0  18  Leprflox/flox AlbCre mice Mice harbouring the AlbCre transgene (AlbCre+ mice; #003574, Jackson Laboratory, Bar Harbor, USA) on a C57BL/6J background were mated with Leprflox/flox mice (129 X FVB N6 background; kindly provided by Dr. Streamson C. Chua, Albert Einstein College of Medicine, Bronx, USA) to generate Leprflox/+ mice either with or without the AlbCre transgene. These offspring (~50% C57BL6/J, 50% FVB, and less than 1% 129) that were Leprflox/+ AlbCre+ and Leprflox/+ AlbCre- were mated to produce Leprflox/flox AlbCre+ mice and Leprflox/flox AlbCrelittermate controls for experiments. For all experiments, Leprflox/flox AlbCre- littermate controls were compared with Leprflox/flox AlbCre+ mice in order to minimize differences in genetic background, which can affect the phenotype of leptin deficiency [136]. The Leprflox allele contains loxP sites flanking exon 17 of the leptin receptor gene. Upon Cre-mediated recombination, a premature stop codon is introduced, resulting in the LeprΔ17 allele. This allele generates a leptin receptor protein that has an ablated signalling domain (Figure 3). The hepatocyte-specific expression of the AlbCre transgene results in Cre-mediated recombination and thus disruption of leptin signalling only in hepatocytes of Leprflox/flox AlbCre+ mice.  19  Figure 3. Schematic of the Leprflox and Lepr∆17 gene and protein structures. (A) Genomic structure of the Leprflox gene, where in Leprflox/flox AlbCre+ 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 wildtype isoform b of the leptin receptor containing the Box 1 and 2 motifs required for signal transduction. The Lepr∆17 protein lacks the Box 1 and 2 motifs and is devoid of signalling function. Primers MLepr101 and MLepr102 were used for identification of Cre-mediated recombination in genomic DNA. Primers G and 60 were used to identify Cre-mediated recombination in cDNA by RT-PCR.  Leprflox/flox AlbCre ob/ob mice To cross Leprflox/flox AlbCre mice onto an ob/ob background, Leprflox/flox AlbCre+ mice were mated with ob/+ mice (C57BL/6J background, Jackson Laboratory, Bar Harbor, USA) according to the breeding scheme outlined in Figure 4. After four rounds of breeding, Leprflox/flox AlbCre+ ob/ob mice and their Leprflox/flox AlbCre- ob/ob littermate controls were generated for experiments. To assess the role of hepatic leptin signalling in these mice, they received intraperitoneal injections of mouse recombinant leptin (0.1 µg/g or 0.8 µg/g per day for 2 days, National Hormone and Peptide Program, Torrance, USA). For each day of leptin treatment, mice were injected with half the dose at 9 am and the other half at 6 pm. Alternatively, for longer term leptin treatment, Leprflox/flox AlbCre ob/ob mice were treated with 5 µg/day, 2 µg/day, or 0.6 µg/day mouse recombinant leptin via mini-osmotic pump (Alzet, Palo Alto, USA).  20  Figure 4. Breeding scheme for the generation of Leprflox/flox AlbCre ob/ob mice. Leprflox/flox AlbCre mice were bred with ob/+ mice and after four rounds of breeding, Leprflox/flox AlbCre ob/ob mice were generated. Mendelian ratios of all possible genotypes are presented. 21  Diabetic db/db mice expressing Lepr-b Male db/db mice on a C57BL/6J background were obtained from the Jackson Laboratory (#000697, Jackson Laboratory) and injected intravenously via the tail vein with 1x109 pfu of an adenovirus (serotype 5) expressing either the long signalling isoform of the mouse leptin receptor (Ad-Lepr-b, kindly provided by Dr. Martin G. Myers and Dr. Christopher J. Rhodes [137]) or β-galactosidase (Ad-β-gal) as a control. Verification of Cre expression and Cre-mediated recombination To verify Cre expression, DNA was extracted from ear tissue using Chelex 100 resin (Bio-Rad, Mississauga, Canada). Ear biopsies were incubated in 112.5 mg/mL Chelex 100, 0.1 mg/mL proteinase K (Fisher Scientific, Ottawa, Canada), and 0.1% Tween-20 at 55°C for 45 minutes and then 95°C for 15 minutes. Samples were then quickly centrifuged and the supernatant containing DNA was used as a template for PCR. Cre expression was verified using a forward primer with the sequence AGG TGT AGA GAA GGC ACT CAG C and a reverse primer with the sequence CTA ATC GCC ATC TTC CAG CAG G. Primers for interleukin-2, which should be expressed in all mice, were also included in the PCR reaction mixture as a control to ensure that mice determined to be negative for Cre expression were not identified as negative due to a failed PCR reaction. The sequence of the interleukin-2 forward primer was CTA GGC CAC AGA ATT GAA AGA TCT and that of the reverse primer was GTA GGT GGA AAT TCT AGC ATC ATC C. To verify Cre-mediated recombination in Leprflox/flox AlbCre mice and Leprflox/flox AlbCre ob/ob mice, genomic DNA was prepared with DNeasy kits (Qiagen, Mississauga, Canada). Genomic DNA was extracted from liver tissue of Leprflox/flox AlbCre mice and Leprflox/flox AlbCre ob/ob mice and used as a template for PCR. Using primers MLepr101 and MLepr102 (Figure 3), the Leprflox allele has an expected product size of 1369 bp and the Lepr∆17 allele is predicted 22  to produce a 952 bp product. The forward primer was MLepr101 with sequence ATG CTA TCG ACA AGC AGC AGA ATG ACG and the reverse primer was primer mLepr102 with sequence CAG GCT TGA GAA CAT GAA CAC AAC AAC (Integrated DNA Technologies Inc., Coralville, USA). For RNA extractions, liver was excised and immediately placed in RNAlater (Qiagen, Mississauga, Canada). Tissues were homogenized with an Ultra Turrax and purified using an RNeasy kit (Qiagen, Mississauga, Canada). Reverse transcription 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 using primers specific for Leprflox cDNA and not genomic DNA. RT-PCR from liver cDNA was performed with primer G (TAT TCC CAT CGA GAA ATA TCA) and primer 60 (AGG CTC CAA AAG AAG AGG ACC) (Integrated DNA Technologies Inc., Coralville, USA) (Figure 3). The predicted product sizes are 343 bp for Leprflox and 267 bp for Lepr∆17. Verification of Ad-Lepr-b expression in db/db mice Liver tissue was collected 26 days post-infection from db/db mice treated with an adenovirus expressing either β-galactosidase or the long signalling isoform of the leptin receptor, Lepr-b. RT-PCR was performed to confirm the hepatic expression of wildtype Lepr-b. Primers flanking the db insertion mutation were used. The sequence of the forward primer was TGT CCT ACT GCT CGG AAC ACT G and that of the reverse primer was GGT TCA GGC TCC AGA AGA AGA GG. The predicted product sizes are 298 bp for the mutant db allele and 192 bp for wildtype Lepr-b.  23  2.2 Plasma Analytes Metabolites All blood samples were collected via the saphenous vein from restrained, unanesthetized mice. Plasma was separated by centrifuging the samples at 7,000 RPM (4,600 RCF) in a microcentrifuge at 4°C for 9 minutes. Blood glucose levels were measured using a One Touch Ultra glucometer (LifeScan Canada, Burnaby, Canada) via the saphenous vein. In the event that blood glucose levels exceeded 33.3 mM (upper limit of detection of the glucometer), plasma samples at all time points in the particular assay were obtained and glucose levels measured using a Trinder assay (Genzyme, Charlottetown, Canada). The measurement of either blood glucose or plasma glucose is specified in figure legends. Plasma cholesterol levels were measured by Cholesterol E kit (Wako Chemicals USA, Richmond, USA), plasma free fatty acids by HR Series NEFA-HR(2) kit (Wako Chemicals USA, Richmond, USA), and plasma triglycerides by Serum Triglyceride Determination kit (Sigma-Aldrich, Oakville, Canada). All assays were scaled down to fit a 96-well plate format. If samples were above the linear range of the assays, they were diluted in order to fit into the linear range. Hormones Plasma insulin levels were measured by an Ultrasensitive Mouse Insulin ELISA (ALPCO Diagnostics, Salem, USA) and plasma leptin levels were determined using a Mouse Leptin ELISA (Crystal Chem, Downers Grove, USA). Plasma IGFBP-2 levels were measured by a Mouse/Rat IGFBP-2 ELISA (ALPCO, Salem, USA). Plasma lipocalin-2 levels were measured using a NGAL (Mouse) ELISA (ALPCO, Salem, USA). All assays were performed according to the manufacturer’s protocol.  24  Plasma leptin binding proteins Levels of plasma leptin binding proteins were assessed based on the method of Guo et al. [138]. Plasma was incubated overnight with 125I-leptin (NEX340, PerkinElmer, Wellesley, MA) and then subjected to size-exclusion chromatography on a G-75 Sephadex column with an approximate bed volume of 17 mL. The buffer consisted of 0.05 M Na3PO4 and 0.15 M NaCl at pH 7.4. The incubation and chromatography were performed at 4°C. Fractions were collected and analyzed by scintillation counting on a Beckman LS6000IC scintillation counter. Lipoprotein profiles Plasma samples were fractionated by fast protein liquid chromatography (FPLC). Equal volumes of plasma from 4-hour fasted mice of the same genotype were pooled and then 200 µl of the pooled plasma were applied to a Superose 6L HR 10/30 column (GE Healthcare, Baie d’Urfe, Canada) in 154 mM NaCl and 1 mM EDTA at pH 8. Following elution, fractions were assayed for cholesterol and triglycerides using modified protocols of the Cholesterol E kit (Wako Chemicals USA, Richmond, USA) and Serum Triglyceride Determination kit (Sigma-Aldrich, Oakville, Canada) respectively. The modified assays were scaled down for a 96-well plate format and utilized equal volumes of eluted fractions with working reagents prepared as a 2X stock. Plasma apolipoprotein B levels Plasma samples were collected following a 4 hour fast. SDS-PAGE was performed on 1 μL of plasma or 15 μL of FPLC eluate using a 4-15% Mini-PROTEAN TGX Precast Gel (BioRad, Mississauga, Canada) and proteins were transferred onto PVDF membranes. Membranes were then blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline. Membranes were probed with a rabbit anti-mouse apoB antibody which detects both apoB48 and apoB100 (Meridian Life Science, K23300R, Saco, USA). The secondary antibody used was donkey-anti25  rabbit HRP (GE Life Sciences, #NA934, Little Chalfont, United Kingdom). Bands were detected using the Amersham ECL Detection Reagent (GE Life Sciences, #RPN3004, Little Chalfont, United Kingdom) and exposure to film. Densitometry analysis was performed using Adobe Illustrator software (San Jose, USA).  2.3 Body Composition Measurements Lean to lipid mass ratio Measurements were performed with a Bruker Biospec 70/30 7 Tesla MRI scanner (Bruker Biospin, Ettlingen, Germany). The 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, e.g. urine and CSF, was typically less than 5% of the total signal. The ratio of lean/fat tissue is expressed as weight/weight and was calculated from the NMR data as described [139]. Hepatic lipid content Hepatic triglycerides and cholesterol were measured by a modified protocol of Briaud et al. [140]. Liver tissue was homogenized in 3 mL of chloroform:methanol (2:1) and extracted twice with water. Five hundred microlitres of the organic layer were dried down under N2(g) and 10 μL of Thesit (Sigma-Aldrich, St Louis, USA) was added and mixed under N2(g). Similarly, standards containing both cholesterol (Wako Chemicals USA, Richmond, USA) and triolein (Sigma-Aldrich, St. Louis, USA) were also dried down under N2(g) and mixed with Thesit. Water (100 μL) was added and incubated at 37°C for 1 hour with intermittent vortexing. Triglycerides were then quantified using the Serum Triglyceride Determination kit (SigmaAldrich, St Louis, USA) and cholesterol was assayed using the Cholesterol E kit (Wako Chemicals USA, Richmond, USA).  26  2.4 In Vivo Assays Oral glucose tolerance test Mice were fasted for 4 hours and then given an oral glucose gavage (1.5 mg of glucose per g body weight from a 30% glucose solution unless otherwise specified). Glucose measurements were performed on blood samples taken from the saphenous vein at specified time points over 2 hours using a One Touch Ultra glucometer (LifeScan Canada, Burnaby, Canada). Glucose-stimulated insulin secretion test Mice were fasted for 4 hours and then administered an oral glucose gavage of 1.5 mg glucose per gram body weight. Plasma samples were collected for insulin measurements at 0, 7, 15, and 60 minutes post-gavage. Plasma insulin levels were measured as described above. Glucose measurements were also performed on blood samples taken from the saphenous vein at specified time points over 2 hours using a One Touch Ultra glucometer (LifeScan Canada, Burnaby, Canada). Insulin tolerance test Mice were fasted for 4 hours and then given an intraperitoneal injection of synthetic human insulin (Novolin® ge Toronto, Novo Nordisk, Mississauga, Canada). Doses of insulin are as specified in figure legends. Blood glucose measurements were performed on samples taken from the saphenous vein at specified time points over 2 hours using a One Touch Ultra glucometer (LifeScan Canada, Burnaby, Canada). Hyperinsulinemic-euglycemic clamp Hyperinsulinemic-euglycemic clamps were performed as previously described [141]. Mice were fasted overnight for 16 hours and anaesthetized with acepromazine (5 mg/kg), midazolam (5 mg/kg), and fentanyl (0.25 mg/kg). The tail vein was then cannulated and a 1 hour basal infusion of 3H-D-glucose (1.2 Ci/h) was initiated. Duplicate blood samples were 27  obtained 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/h). Euglycemia (~5.0 mM) was maintained by variable infusion of 12.5% Dglucose for 45 minutes. Once glucose clamping was achieved, triplicate blood samples (50 L) were obtained from the tail vein at 10 min intervals. Animals were sacrificed by cervical dislocation and tissues were dissected and frozen in liquid N2. Plasma samples were counted using a Beckman LS6000IC scintillation counter after extraction by TCA precipitation. Whole body glucose utilization (mol/kg∙min) was determined as the ratio of the specific activity of glucose to the rate of 3H-D-glucose appearance. Endogenous glucose production (mol/kg∙min) was calculated as the difference between whole body glucose utilization and exogenous glucose infusion. All data pertaining to hyperinsulinemic-euglycemic clamps included in this thesis were obtained by Dr. Sarah L. Gray (University of Northern British Columbia, Prince George, Canada), Dr. Peter J. Voshol (University of Cambridge, Cambridge, United Kingdom), and Madeleine Speck (University of British Columbia, Vancouver, Canada). Pyruvate tolerance test Mice were fasted overnight for 16 hours and then given an intraperitoneal injection of 2 g/kg body weight pyruvate (Sigma-Aldrich, St Louis, MO) in saline. Blood glucose levels were monitored for the next 2 hours with a One Touch Ultra Glucometer (Life Scan Inc., Burnaby, Canada) in samples collected from the saphenous vein. Oral lipid tolerance test Mice were fasted for 4 hours and then given 5 μL/g extra virgin olive oil via oral gavage. Blood samples were taken over the next 4-5 hours, plasma prepared and assayed for triglycerides as described above.  28  Triglyceride secretion assay A 15% stock solution of poloxamer-407 (P-407) was prepared by stirring P-407 (SigmaAldrich, Oakville, Canada) in sterile PBS overnight at 4°C. Following a 4 hour fast, mice were injected intraperitoneally with 1 g/kg of P-407 from the 15% stock solution. This was followed by an intraperitoneal injection of 0.6 U/kg or 0.725 U/kg insulin (Novolin®, Novo Nordisk, Mississauga, Canada). Blood samples were taken throughout the next 4 hours, plasma prepared and assayed for triglycerides as described above.  2.5 Analysis of Mouse Tissues Measurements of β-Cell mass Pancreata were removed, fixed in 4% paraformaldehyde overnight at 4°C, and stored in 70% ethanol prior to embedding in paraffin and sectioning. For each mouse, insulin positive area was assessed on three pancreas sections separated by 35 μm. Sections were incubated with 3% hydrogen peroxide (Fisher Scientific, Ottawa, Canada), blocked in serum free protein block (DakoCytomation, Inc., Carpinteria, USA), and stained with guinea pig anti-insulin antibody (LINCO Research Inc., St. Charles, USA) overnight at 4°C. The sections were then incubated for 1 hour at room temperature in biotinylated goat anti-guinea pig IgG and then 30 minutes in Vectastain R.T.U. Elite ABC reagent. They were then stained with Peroxidase Substrate Kit DAB (all from Vector Laboratories, Burlingame, USA) and mounted. Whole sections were scanned with a ScanScope CS System, and insulin positive area was measured by V9 Positive Pixel Count Algorithm on ImageScope software (all from Aperio, Vista, USA). Quantitative PCR Tissues were extracted and immediately placed in RNAlater (Qiagen, Mississauga, Canada) overnight at 4°C after which the RNAlater was removed and tissue stored at -80°C.  29  RNA was prepared using TRI Reagent (Applied Biosciences, Carlsbad, USA). Genomic DNA was removed from the RNA samples by treatment with DNase I (RNase-free) (New England Biolabs, Pickering, Canada) according to the manufacturer instructions. One µg of RNA was used for cDNA synthesis using a polyT primer with the iScript Select cDNA synthesis kit (BioRad, Mississauga, Canada). For each sample, 2 L of 100-fold diluted cDNA were used with the desired primers and the SsoFast EvaGreen Supermix with Low ROX master mix (Bio-Rad, Mississauga, Canada) as specified by the manufacturer instructions. Reactions were monitored in a StepOne Plus thermocycler (Applied Biosystems, Carlsbad, USA). Ppia was used as a reference gene and was selected from a panel of 8 candidates based on transcript stability by geNorm analysis [142]. Transcript abundance was calculated by the Pfaffl method using a single control sample as the calibrator point [143]. Primer sequences are presented in Table 1. Table 2. Sequences of primers used in quantitative PCR. Official gene names for phosphenolpyruvate carboxykinase (Pepck), glucose-6-phosphatase (G6pc), apolipoprotein B (Apob), hepatic lipase (Lipc), lipoprotein lipase (Lpl), and insulin-like growth factor binding protein-2 (Igfbp2) are shown. Forward Primer 5'-GCAGAACACAAGGGCAAGATCATC-3' 5'-CGTCACAGTTTTCTCCTCCTCAGC-3' 5'-TGGTTGAGCTGATCAAGACC-3' 5'-TCACACTGGACAAAGACATCG-3' 5'-GTGACCGATTTCATCAAGTTTGGAG-3' 5'-ACCCCTTGCCAGCAGGAGTTGGA-3'  Pepck G6pc Apob Lipc Lpl Igfbp2  Reverse Primer 5'-GATGTAGCCGATGGGCGTG-3' 5'-CTCTGCAAATCAGCCGAGGCAG-3' 5'-AAACACGTGGTAGTTTTGAATGG-3' 5'-CTTCACTTCACAGTTCACAAAGAC-3' 5'-GACGGACACAAAGTTAGCACCAC-3' 5'-TCCCTGGATGGGCTTCCCGGT-3'  Gene arrays Liver tissues were harvested and immediately placed in RNAlater (Qiagen, Mississauga, Canada) overnight at 4°C after which the RNAlater was removed and tissue was stored at -80°C. RNA was prepared using an RNeasy Kit (Qiagen, Mississauga, Canada) and genomic DNA was removed from the RNA samples by treatment with DNase I (RNase-free) (New England Biolabs, Pickering, Canada) according to the manufacturer instructions. One µg of RNA was used for 30  cDNA synthesis using a polyT primer with the iScript Select cDNA synthesis kit (Bio-Rad, Mississauga, Canada). cDNA samples were given to the Centre for Molecular Medicine and Therapeutics Genotyping and Gene Expression Core Facility (Vancouver, Canada) for assaying on the Illumina MouseWG-6 v2 Expression BeadChip or the MouseRef-8 v2 Expression BeadChip (Illumina Inc., San Diego, USA). Data were analyzed using the GenomeStudio® data analysis software (Illumina Inc., San Diego, USA). Functional enrichment analysis was performed using the Web-based Gene Set Analysis Toolkit (WebGestalt) [144, 145]. Western blot analyses Liver samples were harvested and immediately frozen in liquid nitrogen. Liver lysates were prepared by homogenization in ice cold lysis buffer containing 50 mM Tris-HCl at pH 8, 120 mM NaCl, 30 mM NaF, 5 mM EDTA, 1% (w/v) NP-40 (Sigma-Aldrich, St Louis, USA), protease inhibitor cocktail (Sigma-Aldrich, St Louis, USA), and phosphatase inhibitor cocktail (Sigma-Aldrich, St Louis, USA). SDS-PAGE was performed on an 8% acrylamide gel and proteins transferred onto a PVDF membrane. Membranes were then blocked with either 5% skim milk or 5% bovine serum albumin (BSA Fraction V, Fisher Scientific, Ottawa, Canada) in Tris-buffered saline. Western blots were performed using antibodies against phosphorylated Akt at Ser-473 (Cell Signalling Technologies, #4060, Danvers, USA), total Akt (Cell Signalling Technologies, #9272, Danvers, USA), apolipoprotein B (Meridian Life Science, #K23300R, Saco, USA), phosphorylated STAT3 (Cell Signalling Technologies, #9145, Danvers, USA) and total STAT3 (Cell Signalling Technologies, #9139, Danvers, USA). Loading controls were assessed by stripping the membrane with 2% SDS, 50 mM Tris-HCl, and 0.1 M βmercaptoethanol at pH 6.7 for 45 minutes at 60°C. Membranes were then blocked and re-probed for the appropriate loading control. Secondary antibodies used were donkey-anti-rabbit HRP (GE Life Sciences, #NA934, Little Chalfont, United Kingdom) or sheep-anti-mouse HRP (GE  31  Life Sciences, #NA931, Little Chalfont, United Kingdom). Bands were detected using the Amersham ECL Detection Reagent (GE Life Sciences, #RPN3004, Little Chalfont, United Kingdom) and exposure to film. Densitometry analysis was performed using AlphaView analysis software (ProteinSimple, Santa Clara, USA). Measurements of lipase activity in the liver Liver lysates were prepared by homogenization in ice cold lysis buffer consisting of 50 mM Tris-HCl at pH 8, 120 mM NaCl, 5 mM EDTA, 30 mM NaF, 1% (w/v) NP-40 (SigmaAldrich, St. Louis, USA), phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, USA), and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, USA). Lysates were then assessed for hepatic lipase (HL) and lipoprotein lipase (LPL) activity as described [146]. To measure total lipase activity, lysates were incubated in 0.6 mM glycerol tri[9,10-3H]oleate (1 mCi/mmol), 25 mM piperazine-N/N-bis(2-ethanesulfonic acid) at pH 7.5, 0.05% (wt/vol) albumin, 50 mM MgCl2, and 2% (v/v) heat-inactivated chicken serum (containing the LPL co-activator apolipoprotein CII). After an incubation of 30 minutes at 30°C, the amount of [3H]-oleate released was determined by addition of a fatty acid extraction solution (38.5% methanol, 34.2% chloroform, 27.3% heptane) and 2.86 mM NaOH. After vortexing and centrifugation, the upper phase containing radioactive sodium [3H]-oleate was assessed for radioactivity by liquid scintillation counting. To measure non-LPL lipase activity, lysates were first incubated in 1 M NaCl at room temperature for 10 minutes to inhibit LPL activity. Lysates were then subject to the same reaction conditions as above except that heat-inactivated chicken serum (containing the LPL co-activator apolipoprotein CII) was omitted. The resulting activity measurements were indicative of non-LPL activity and were assumed to be predominantly due to HL activity. LPL activity was determined by subtracting the non-LPL activity measurements from the total  32  activity measurements. All measurements of lipase activity were performed by Ying Wang in the laboratory of Dr. Brian Rodrigues (University of British Columbia, Vancouver, Canada). Liver histology Liver tissues were harvested, fixed in 4% paraformaldehyde overnight, and stored in 70% ethanol. Samples were sectioned, processed, and stained by Wax-it Histology Services (Vancouver, Canada). Sections of 5 µm thickness were stained with hematoxylin and eosin and visualized with light microscopy under oil immersion.  2.6 Data Analysis Data were analyzed using SigmaPlot 12.0 (Systat, San Jose, USA) with the exception of the gene array data, which were analyzed using the GenomeStudio® data analysis software (Illumina Inc., San Diego, USA). Data are presented as mean ± standard error of the mean (SEM) and were analyzed using a Student’s t-test or two-way analysis of variance (ANOVA) with a Holm-Sidak post-hoc test as appropriate.  33  CHAPTER 3 – EFFECTS OF A LIFE-LONG LOSS OF HEPATIC LEPTIN SIGNALLING ON GLUCOSE METABOLISM 3.1 Introduction The weight-independent effects of leptin on glucose metabolism may be mediated through direct effects of leptin on the liver. Many in vitro studies have been performed to attempt to elucidate the effect of leptin on glucose metabolism in hepatocytes, but these results are mixed and have not established a clear role for leptin in the liver. Few in vivo studies have been performed to assess the specific role of leptin on hepatic glucose metabolism. Most notably, Cohen et al. [125] used a Cre-lox approach to generate a mouse with a hepatocytespecific loss of the entire leptin receptor. These authors found that a hepatocyte-specific ablation of leptin receptors did not alter body weight, body composition, or free-feeding levels of glucose or insulin. However, other aspects of glucose metabolism were not explored. The studies in this chapter expanded on the work by Cohen et al. [125] by investigating glucose metabolism at several different ages, on a high fat diet, and under the setting of a glucose challenge. For these studies, a Cre-lox approach was also used. However, distinct from the Cohen study, we generated mice with a hepatocyte-specific ablation of leptin signalling domains as opposed to ablation of the complete receptor. By thoroughly examining glucose metabolism in these mice, it was possible to specifically elucidate the role of hepatic leptin signalling in vivo. This work has been published in the journal Diabetes [72].  3.2 Results Generation of mice with hepatocyte-specific disruption of leptin receptor signalling To generate mice with hepatocyte-specific disruption of leptin signalling, Leprflox/flox mice, which have loxP sites flanking exon 17 of the leptin receptor gene (Lepr)(Figure 3) were  34  used [68, 147]. Exon 17 of Lepr is present in the long, signalling isoform of the leptin receptor (Lepr-b) and encodes the JAK binding site. Upon Cre-mediated recombination at the loxP sites, exon 17 is excised (herein referred to as Lepr17), and a resulting frame shift mutation generates an altered 3 terminus [147] that no longer encodes tyrosine 985 and tyrosine 1138, which are sites of JAK-mediated tyrosine phosphorylation [48]. Mice homozygous for the Lepr17 allele are deficient in leptin-stimulated STAT phosphorylation [70, 148]. The Leprflox/flox mice were crossed with AlbCre+ mice, which have the cre transgene under the control of the albumin promoter, conferring hepatocyte-specific expression of Cre [149]. The resulting Leprflox/+ mice with and without the AlbCre transgene were bred to generate Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- mice. Following generation of Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- littermate controls, the extent and specificity of Cre-mediated excision of exon 17 of the leptin receptor was investigated. In all tissues tested, there was a PCR product of ~1370 bp, which corresponds to the expected product size of the Leprflox allele (Figure 5A). Although present, there was very little of this amplicon generated from liver DNA of Leprflox/flox AlbCre+ mice, likely contributed by non-hepatocytes. Instead, the major product amplified from liver DNA of Leprflox/flox AlbCre+ mice was ~950 bp in size, which corresponds to the expected size of the Leprflox allele following Cre-mediated excision (resulting in Lepr17). Thus, it appears that in the Leprflox/flox AlbCre+ mice, excision at the Leprflox allele among the tissues tested was restricted to the liver and that the majority of hepatocytes carry the Lepr17 rather than Leprflox allele. Consistent with this, an analysis of Lepr-b mRNA transcripts by RT-PCR revealed that while the only amplified product detected from the liver of Leprflox/flox AlbCre- mice corresponded to the anticipated 343 bp product from the wildtype Lepr-b transcript, in liver of the Leprflox/flox AlbCre+ mice, only an amplicon of the predicted size of the Lepr17 transcript was detected (Figure 5B). The Lepr17 35  transcript has been previously shown to result in impaired leptin-stimulated JAK/STAT signalling in mouse lines derived from the same Leprflox/flox mice used in our study [68, 70]. Thus, our mice, which we have shown to predominantly express the Lepr17 allele specifically in the liver, likely have impaired leptin-stimulated hepatic JAK/STAT activation.  Figure 5. Leprflox/flox AlbCre+ mice have a liver-specific loss of the leptin receptor signalling domain. (A) Genomic DNA from tissues of Leprflox/flox mice with and without the AlbCre transgene was used as template for PCR of the Leprflox allele. The predicted product sizes are 1369 bp for Leprflox and 952 bp for LeprΔ17. (B) RNA was extracted from the livers of Leprflox/flox mice and used as template for RT-PCR with primers flanking exon 17. The predicted product sizes are 343 bp for Leprflox and 267 bp for LeprΔ17 transcripts. Arrows to the left mark the migration of molecular weight markers in bp. These data were contributed by SD Covey.  Loss of hepatic leptin signalling does not substantially alter body weight or body composition To characterize the effects of ablated hepatic leptin signalling, body weight and body composition were assessed. Unlike mice with a complete loss of leptin signalling (db/db mice), which are much heavier than control mice (C57BL/6), the loss of hepatic leptin signalling did not substantially alter body weight relative to controls in either gender at the various ages tested (Table 2). To assess body composition, the total lean to lipid mass ratios of Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- littermate controls were assessed at 6 and 16 weeks of age by NMR. At both ages and in each gender, there was a similar ratio of lean to lipid mass in mice with and 36  without hepatic leptin signalling (Figures 6B and 6D). Thus, a loss of hepatic leptin signalling does not alter body weight or adiposity.  Figure 6. Attenuation of hepatic leptin signalling does not alter body composition. (A and C) Leprflox/flox AlbCre+ mice and littermate controls were assessed for body weight as well as (B and D) body composition as measured by NMR at 6 and 16 weeks old. At 6 weeks old, n≥7 and at 16 weeks old, n≥5 for Leprflox/flox AlbCre+ and littermate controls. Data are expressed as meanSEM. P values were determined by Student’s t-test.  37  Table 3. Metabolic parameters of mice with and without hepatic leptin signalling. The genetic background of Leprflox/flox AlbCre mice is mixed and is described in detail in Methods and Materials. Wildtype controls and db/db mice were on a pure C57BL/6 genetic background. Metabolic Parameter  6 wks  ♂  ♀  12 wks  ♂  ♀  16 wks  ♂  ♀  a  b  a  Body Weight (g)  Fasting Glucose (mM)  Random Fed Glucose (mM)  Fasting Insulin (ng/ml)  Lepr AlbCreflox/flox Lepr AlbCre+  25.70.5 (22) 25.60.5 (24)  9.70.3 (22) 9.30.3 (24)  8.30.4 (7) 7.60.3 (8)  1.170.13 (5) 1.090.11 (5)  C57BL/6 db/db  20.50.5 (8) 30.00.7 (8)*  11.10.3 (16) 15.11.8 (8)*  9.00.4 (8) 28.11.6 (8)*  0.860.06 (8) 7.890.91 (8)*  Lepr AlbCreflox/flox Lepr AlbCre+  20.50.3 (18) 19.70.3 (17)  8.30.3 (18) 8.50.3 (16)  7.30.3 (8) 7.20.2 (5)  0.530.15 (6) 0.340.05 (3)  C57BL/6 db/db  16.00.4 (8) 31.60.5 (8)*  8.00.3 (8) 22.11.6 (8)*  8.10.4 (8) 28.90.5 (8)*  0.530.09 (4) 10.181.38 (7)*  Lepr AlbCreflox/flox Lepr AlbCre+  34.00.9 (29) 32.60.9 (35)  10.10.3 (29) 9.90.3 (35)  8.30.4 (6) 8.00.3 (7)  1.380.30 (6) 1.320.29 (7)  C57BL/6 db/db  28.81.0 (14) 50.50.9 (8)*  11.10.4 (14) 28.01.0 (8)*  9.00.2 (8) 28.11.1 (8)*  2.610.76 (8) 11.22.2 (8)*  Lepr AlbCreflox/flox Lepr AlbCre+  22.20.9 (27) 23.10.5 (22)  7.90.3 (27) 7.70.2 (22)  8.00.3 (5) 8.00.2 (5)  0.540.10 (5) 0.480.17 (5)  C57BL/6 db/db  18.70.6 (8) 51.70.6 (8)*  7.20.4 (8) 28.12.2 (8)*  7.00.1 (8) 24.82.4 (8)*  0.490.06 (8) 7.460.76 (8)*  Lepr AlbCreflox/flox Lepr AlbCre+  41.61.1 (24) 39.11.1 (25)  10.90.5 (24) 10.60.3 (26)  7.80.4 (5) 8.60.5 (7)  2.000.41 (18) 1.700.25 (23)  C57BL/6 db/db  31.71.0 (14) 56.70.9 (8)*  9.70.3 (14) 25.62.8 (8)*  8.20.5 (8) 25.11.8 (8)*  2.320.77 (8) 12.02.10 (8)*  Lepr AlbCreflox/flox Lepr AlbCre+  26.91.2 (12) 25.41.0 (8)  7.50.4 (12) 7.20.2 (8)  7.50.3 (5) 7.30.3 (5)  0.820.20 (4) 0.410.01 (4)*  C57BL/6 db/db  20.40.7 (8) 57.90.6 (8)*  8.40.5 (8) 21.72.5 (8)*  7.50.2 (8) 21.02.8 (8)*  0.370.06 (8) 9.671.42 (6)*  flox/flox  flox/flox  flox/flox  flox/flox  flox/flox  flox/flox  a  Mice were fasted for 4 hours during the light cycle Samples were collected between 11pm – 1am flox/flox * P≤0.05 versus control (Lepr AlbCre- or C57BL/6) by Student’s t-test b  38  Loss of hepatic leptin signalling does not have a major influence on basal metabolic parameters Next, the effects of a loss of hepatic leptin signalling on basal metabolic parameters were investigated. Plasma leptin levels were not significantly different between groups in either gender at 18 weeks of age (5.66±1.11 ng/mL vs. 4.36±2.63 ng/mL for male Leprflox/flox AlbCrevs. Leprflox/flox AlbCre+, P=0.308, n≥4 and 4.11±0.78 ng/mL vs. 6.80±1.64 ng/mL for female Leprflox/flox AlbCre- vs. Leprflox/flox AlbCre+, P=0.123, n≥3). Similarly, plasma leptin binding protein levels also appeared to be unchanged between Leprflox/flox AlbCre+ and littermate controls (Figure 7). Insulin and blood glucose levels were also measured in Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- littermate controls in both the 4 hour fasted state as well the fed state during the light and dark phases, respectively. For comparison, these parameters were also measured in mice lacking functional leptin receptors in all tissues (db/db) and the relevant wildtype controls (C57BL/6). The complete loss of leptin action in the db/db mice resulted in increased body mass, hyperglycemia (in both the fasted and fed state) and fasting hyperinsulinemia in both genders at 6, 12 and 16 weeks of age (Table 2). In contrast, a liver-specific loss of leptin signalling did not significantly alter blood glucose levels (in either fasted or random fed state) of either gender at any of the ages investigated. Consistent with this, no alterations in hepatic PEPCK and G6Pase transcript levels between male Leprflox/flox AlbCre+ and littermate controls after a 4 hour fast were found (Figure 8A). Further, pyruvate-induced gluconeogenesis after an overnight fast was similar in male Leprflox/flox AlbCre+ mice and their littermate controls (Figure 8B). Fasting plasma insulin levels in males with hepatocyte-specific ablation of leptin signalling were also similar to levels in littermate controls. However, the Leprflox/flox AlbCre+ females had lower fasting plasma insulin levels compared to their littermate controls at all ages measured and this difference was statistically significant at 16 weeks of age (Table 2).  39  Figure 7. Levels of plasma leptin binding proteins are not altered in male mice lacking hepatic leptin signalling. Twenty-one week old male mice were fasted for four hours and then a cardiac puncture was performed to collect blood. Plasma was prepared and incubated overnight with 125I-leptin at 4°C and then subjected to size-exclusion chromatography. Fractions were collected and analyzed by scintillation counting, n=3 in each group. Data are mean±SEM.  Figure 8. Loss of hepatic leptin signalling does not affect glucose metabolism in the fasted state. (A) Levels of PEPCK and G6Pase transcripts were measured in the livers of 22 week old male mice following a 4 hour fast, n≥8. (B) Thirty week old male Leprflox/flox AlbCre- and Leprflox/flox AlbCre+ mice were fasted overnight and then given an injection of 2 g/kg body weight pyruvate. Blood glucose measurements were then monitored for 2 hours, n=8 in each group. Data are expressed as meanSEM. P values were determined by Student’s t-test.  40  Loss of hepatic leptin signalling protects against age- and diet-related glucose intolerance To assess if the response to a nutritional challenge may be altered by a loss of hepatic leptin signalling, oral glucose tolerance tests (OGTTs) were performed on Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- littermate controls at several different ages, along with db/db and C57BL/6 wildtype controls. As expected, the complete absence of leptin signalling in the male db/db mice resulted in striking glucose intolerance in young 6 week old mice (Figure 9F). Surprisingly, in contrast to the db/db mice, the 6 week old male mice lacking hepatic leptin signalling had a trend towards slightly improved glucose tolerance (Figure 9A). This trend became more prominent as the mice aged and the differences in the peak blood glucose values were statistically significant in 16 week old mice and beyond (Figures 9B-D). At 16 and 42 weeks old, the Leprflox/flox AlbCre+ mice had an 18% improvement in glucose excursion relative to the littermate controls as measured by area under the curve (AUC) analysis. Interestingly, there was no difference in glucose tolerance in females when comparing the Leprflox/flox AlbCre+ females and their littermate controls at the same ages as the males (Figure 10). Since it appeared that male mice lacking hepatic leptin signalling were protected from an age-related deterioration of glucose tolerance, it was interesting to see if this protection would persist when they were fed a high fat diet. Leprflox/flox AlbCre+ and their littermate controls were fed a high fat diet for 20 weeks starting at 4 weeks of age. It was evident that the high fat diet caused a metabolic stress since there was a two-fold increase in fasting insulin levels between chow-fed mice and their high fat fed counterparts (Figures 11A and 12). Similar to the chow fed mice, the high fat fed male mice lacking hepatic leptin signalling had improved glucose tolerance relative to littermate controls (Figure 9E) despite no differences in body weight (55.9±4.0 g for Leprflox/flox AlbCre+ and 54.3±2.6 g for Leprflox/flox AlbCre-, P=0.365, n≥6). The magnitude of this improvement in glucose tolerance was greater in high fat fed mice than the chow fed mice, as there was a 40% improved glucose excursion (by AUC analysis) in mice lacking hepatic leptin signalling. Thus, 41  the loss of hepatic leptin signalling allows mice to retain normal glucose tolerance as they age even when on a high fat diet.  Figure 9. Loss of hepatic leptin signalling prevents age- and diet-related glucose intolerance. Oral glucose tolerance tests were performed on male (A-E) Leprflox/flox AlbCre+ mice and littermate controls or (F) db/db mice and C57BL/6 controls at the indicated ages. (E) Mice were fed a high fat diet (HFD) for 20 weeks, fasted for 4 h, and gavaged with 1.22 mg/g glucose. Data are expressed as meanSEM and n 6. *Denotes P≤0.05 versus wildtype control by Student’s t-test.  42  Figure 10. Attenuation of hepatic leptin signalling does not alter glucose tolerance in female mice. Oral glucose tolerance tests were performed on female Leprflox/flox AlbCre+ mice and littermate controls at 6 (A), 12 (B), and 16 (C) weeks of age. Mice were fasted for 4 hours and gavaged with 1.5 mg/g glucose. For Leprflox/flox AlbCre+, n=3 and for Leprflox/flox AlbCre-, n≥7. Data are expressed as meanSEM.  Since it has been reported that a Cre transgene, the RIPCre transgene, itself can cause glucose intolerance [150], it is possible that the differences in glucose tolerance in our studies were a direct result of excision of Lepr exon 17 or possibly the presence of the AlbCre transgene. To test this, we performed OGTTs in male Leprflox/+ AlbCre+ and their Leprflox/+ AlbCrelittermate controls at 16 weeks of age. Glucose excursion was very similar between the two groups of mice when analyzed by AUC analysis (Leprflox/+ AlbCre- 1570140 versus Leprflox/+ AlbCre+ 1556220 and P=0.480, n≥7). This reveals that the presence of the Cre transgene itself is not the cause of the altered glucose tolerance (Figure 9) but rather the loss of leptin signalling in hepatocytes, and that the phenotype is only present when both copies of the leptin receptor allele are disrupted. Increased glucose-stimulated plasma insulin levels and increased insulin sensitivity contribute to improved glucose tolerance in mice lacking hepatic leptin signalling To explore the potential mechanism of improved glucose tolerance in the Leprflox/flox AlbCre+ mice, aspects of insulin secretion in mice with and without hepatic leptin signalling were next examined. As seen in Figure 11A, following a glucose gavage, the Leprflox/flox 43  AlbCre+ mice had 1.35 fold increase in plasma insulin relative to the Leprflox/flox AlbCrelittermate controls (AUC of 25233 and 18631 respectively, P=0.053, n≥13). This increase was not associated with an increase in β-cell mass (Figures 11B-C). Similarly, a trend for increased glucose-stimulated insulin levels was also seen in Leprflox/flox AlbCre+ mice on a high fat diet (Figure 12).  Figure 11. Attenuation of hepatic leptin signalling increases glucose-stimulated insulin levels. (A) Plasma insulin levels were monitored following a gavage of 1.5 mg/g body weight glucose to assess steady state levels of glucose-stimulated insulin secretion in 16-20 week old Leprflox/flox AlbCre+ mice and their littermate controls, n13 in each group. (B) Insulin positive area as a function of total pancreas area and (C) total pancreatic β-cell mass, n≥5 mice per genotype and 3 sections measured per pancreas from 22 week old male mice. All data are expressed as meanSEM. P values were determined by Student’s t-test.  44  Figure 12. Effect of attenuated hepatic leptin signalling on glucose-stimulated insulin levels during high fat feeding. Plasma insulin levels were monitored following a gavage of 1.22 mg/g body weight glucose to assess steady state levels of glucose-stimulated insulin secretion in 18 week old Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- mice fed a high fat diet for 14 weeks, n5 in each group. Data are expressed as meanSEM.  While the increased plasma insulin following a glucose challenge likely contributes to the enhanced glucose tolerance in mice lacking hepatic leptin signalling, it is possible that altered insulin sensitivity may also contribute to this phenotype. To assess whole body insulin sensitivity, insulin tolerance tests (ITTs) were performed in male and female mice at 19 weeks of age. The Leprflox/flox AlbCre+ females displayed increased insulin sensitivity compared to the Leprflox/flox AlbCre- controls (Figure 13A), while in male mice there was no difference in whole body insulin sensitivity (Figure 13B).  45  Figure 13. Ablation of hepatic leptin signalling increases insulin sensitivity. Insulin tolerance tests were performed on (A) 19-week old females (n4) and (B) 21-week old males (n6) with and without the AlbCre transgene. All data expressed as meanSEM. *Denotes P≤0.05 versus littermate controls by Student’s t-test.  To more specifically explore insulin sensitivity at the level of the liver in male mice, hyperinsulinemic-euglycemic clamp studies were performed. Figures 14A and 14B show that hyperinsulinemia was achieved while maintaining euglycemia during the clamp and that no differences in blood glucose or plasma insulin levels were observed between Leprflox/flox AlbCre+ and their Leprflox/flox AlbCre- littermate controls. In the basal (non-hyperinsulinemic) state, whole body glucose utilization and endogenous glucose production were similar for the Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- littermate controls (Figure 14C). As expected, in both Leprflox/flox AlbCre+ and Leprflox/flox AlbCre- mice, glucose utilization increased and endogenous glucose production decreased in the hyperinsulinemic phase. While whole body glucose utilization was similar between the mice with and without hepatic leptin signalling, there was a significant difference in insulin-induced suppression of endogenous glucose production (P=0.03). In the littermate controls, insulin suppressed glucose production by 39%, while in the Leprflox/flox AlbCre+ mice, insulin suppressed glucose production by 68%. Since the majority of endogenously produced glucose comes from hepatocytes, these data provide evidence that a loss 46  of hepatic leptin signalling mediates enhanced insulin sensitivity in the liver. This notion is supported by the fact that levels of phosphorylated Akt, a key mediator of insulin signalling, were increased in mice lacking hepatic leptin signalling when compared to littermate controls (Figure 15). Taken together, these data suggest that a loss of hepatic leptin signalling leads to increased insulin sensitivity in the liver.  Figure 14. Loss of hepatic leptin signalling enhances liver insulin sensitivity. Male Leprflox/flox AlbCre+ mice and littermate controls (16-20 weeks old) were used in a hyperinsulinemic-euglycemic clamp as described in Materials and Methods, n≥6. (A-B) Plasma insulin and blood glucose levels during basal and hyperinsulinemic states. (C) Whole body glucose utilization (BGU), endogenous glucose production (EGP), and glucose infusion rate (GIR). Data are expressed as meanSEM. P values were determined by Student’s t-test. These data were contributed by SL Gray, PJ Voshol, and M Speck.  47  Figure 15. Attenuation of hepatic leptin signalling results in increased insulin-stimulated phosphorylation of Akt in the liver. Following the hyperinsulinemic-euglycemic clamp, liver tissues were harvested and flash frozen. Liver lysates were prepared and Western blots performed for phosphorylated and total Akt levels. The antibody for phosphorylated Akt was directed against Ser-473. Representative blots from two Leprflox/flox AlbCre- and two Leprflox/flox AlbCre+ mice are shown in (A). Quantification of all samples by densitometry is shown in (B), n≥8. Data are expressed as meanSEM. P values were determined by Student’s t-test.  3.3 Discussion Similar to the work of Cohen et al. [125], the data in this chapter show that the loss of hepatic leptin signalling does not have a major influence on glucose metabolism in the mildly fasting or random fed state. However, these data extend the work of Cohen et al. to reveal that leptin action on the liver does influence glucose metabolism under certain metabolic stressors. Specifically, as male mice aged, those lacking leptin signalling in the liver performed substantially better during a glucose challenge, remarkably even when fed a high fat diet. This improved glucose tolerance was likely related to increased hepatic insulin sensitivity in 16-20 week old mice as well as a trend towards increased glucose-stimulated plasma insulin levels, perhaps caused by changes in interorgan communication between the liver and pancreas, which has been demonstrated by several studies [80, 151, 152]. Taken together, the data show that aged mice lacking hepatic leptin signalling appear to benefit from the combined effects of  48  increased hepatic insulin sensitivity and increased glucose-stimulated insulin levels, resulting in markedly improved glucose tolerance. Although there was an improvement in insulin sensitivity as well as reduced fasting insulin levels in female mice lacking hepatic leptin signalling compared to controls, this did not manifest in differences in glucose clearance during an oral glucose challenge. It is possible that this relates to the better insulin sensitivity and glucose tolerance in general of female mice relative to their male counterparts [153, 154]. Since the female controls did not develop glucose intolerance during the time frame examined, there was no observed improvement in glucose tolerance in female mice lacking hepatic leptin signalling despite the improved insulin sensitivity. Many studies have investigated the effect of leptin on insulin sensitivity, but there is no clear consensus in the literature if leptin has pro- or anti-insulin sensitizing effects on the liver [127]. In agreement with the data presented in this chapter, several studies have found that leptin can attenuate insulin-induced activities in the liver [121, 155]. Other studies, however, revealed that leptin can increase insulin-mediated actions in the liver [128, 129, 156]. Consistent with leptin mediating both pro- and anti-insulin sensitizing effects, work from our laboratory showed that leptin could increase insulin receptor phosphorylation while at the same time increasing expression of protein tyrosine phosphatase-1B (PTP1B) [129], a negative regulator of insulin receptor signalling [157, 158]. Reports regarding the effects of leptin on components of the insulin signalling pathway in hepatocytes also differ considerably. For example, leptin has been reported to alter insulin signalling by increasing association of p85 with IRS-1 and decreasing IRS-2 tyrosine phosphorylation [121], and alternatively by increasing IRS-2 tyrosine phosphorylation and association with p85 and decreasing IRS-1 tyrosine phosphorylation [128]. Other studies have found leptin to reduce insulin dependent IRS-1/IRS-2 association with p85 49  [155] while still other studies found that leptin was unable to modify the effects of insulin on IRS-1/IRS-2 phosphorylation [158]. Thus, it appears there is a complex relationship between leptin and its effects on hepatic insulin sensitivity and signalling. Chronic versus acute actions of leptin may be one factor dictating if leptin exerts pro- or anti-insulin sensitizing effects on the liver. From the current work, the functional significance of a chronic loss of leptin activity was assessed in vivo and this ultimately leads to increased hepatic insulin sensitivity. It is interesting that a complete loss of leptin receptor signalling (db/db mice) is associated with a worsening of glucose homeostasis, whereas the loss of leptin action on the liver appears to function in the opposite manner. This observation suggests that leptin has divergent effects on different tissues. While it has been shown that leptin can act directly on the brain to cause a secondary increase in insulin sensitivity in the liver [88, 137], our study shows that losing functional leptin receptors in the liver can also increase hepatic insulin sensitivity. Thus, the present study provides evidence that under certain conditions, leptin action on hepatocytes may function to curtail and control the extent of insulin action on the liver. This is consistent with the action of leptin to limit insulin effects in the periphery by directly suppressing insulin secretion from pancreatic β-cells [93, 94, 108] and decreasing lipid storage in adipose tissue [114, 118]. It is possible that leptin plays an important role in keeping insulin effects in check so as to protect against hypoglycemia after post-prandial insulin release. This may explain why the most striking effects on glucose metabolism that we saw as a result of ablated hepatic leptin signalling occurred during the post-prandial state (Figure 9) or during a state of hyperinsulinemia (Figures 13, 14, and 15) and not when insulin levels are low (Figure 8). Therefore, leptin may differentially regulate glucose metabolism by acting either on the brain or the periphery and the overall effect of leptin may depend on the current metabolic state. Clearly, a complex relationship exists between the effects of leptin on the brain and the periphery and a disruption of  50  this relationship may result in metabolic abnormalities such as diabetes and obesity. Nonetheless, given the remarkable ability of a loss of hepatic leptin signalling to protect against glucose intolerance during aging and a high fat diet, two of the most prevalent risk factors for type 2 diabetes, hepatic leptin signalling is a candidate therapeutic target and further studies are warranted in this area.  51  CHAPTER 4 – ACUTE EFFECTS OF HEPATIC LEPTIN SIGNALLING ON GLUCOSE METABOLISM IN OBESE HYPERINSULINEMIC MICE 4.1 Introduction The results from Chapter 3 show the effects of hepatic leptin signalling in lean mice that have a life-long loss of leptin signalling in the liver. However, leptin may have temporal effects on glucose metabolism since chronic versus acute leptin treatment has been shown to have differential effects on glucose metabolism [107, 123]. Thus, in Leprflox/flox AlbCre+ mice, which have a chronic, life-long loss of hepatic leptin signalling, it is possible that only the chronic effects of leptin action on the liver are observed. Furthermore, since Leprflox/flox AlbCre+ mice lack hepatic leptin signalling from the prenatal stage, there is also the possibility that Leprflox/flox AlbCre+ mice develop compensation to the effects of lost liver leptin signalling. To address these temporal issues that may affect the phenotype of Leprflox/flox AlbCre+ mice, hepatic leptin signalling in other complementary mouse models in which there is temporal control over the loss or gain of hepatic leptin signalling was investigated. Leprflox/flox AlbCre mice were crossed with leptin-deficient ob/ob mice to generate Leprflox/flox AlbCre- ob/ob controls and Leprflox/flox AlbCre+ ob/ob mice, which are leptin-deficient and lack functional leptin receptors in the liver. By treating Leprflox/flox AlbCre- ob/ob mice with leptin, it was possible to observe the acute effects of restored leptin signalling in all tissues while leptin treatment in Leprflox/flox AlbCre+ ob/ob mice allowed us to acutely restore leptin signalling in all tissues except the liver. Unlike Leprflox/flox AlbCre+ mice, which could have developmental differences compared to their littermate controls due to a life-long loss of hepatic leptin signalling, Leprflox/flox AlbCre+ ob/ob mice have no such differences since these mice are equivalent to their littermate controls until treated with exogenous leptin. Furthermore, before leptin treatment, these mice are obese and hyperinsulinemic. Thus, by choosing the dose of leptin carefully, we 52  were able to maintain obesity and hyperinsulinemia and observe the effects of hepatic leptin signalling under more metabolically stressed conditions. To complement the Leprflox/flox AlbCre ob/ob mouse model, we also obtained db/db mice, which have a total loss of functional leptin receptors, and treated them with an adenovirus to transiently re-express leptin signalling selectively in the liver. Since db/db mice are obese and hyperinsulinemic, the effects of restored hepatic leptin signalling under the setting of metabolic stress were readily observable. This chapter contains the results of the investigation into glucose metabolism in these mice.  4.2 Results Leprflox/flox AlbCre+ ob/ob mice have truncated hepatic leptin receptor transcripts Leptin-deficient ob/ob mice with a hepatocyte-specific loss of leptin signalling were generated using a Cre-lox approach. To do this, Leprflox/flox mice with loxP sites flanking exon 17 were used. When crossed with mice expressing a Cre transgene, Leprflox/flox mice undergo Cremediated recombination, resulting in the Lepr∆17 allele, which generates truncated leptin receptors that lack the signalling domain and are deficient in leptin-mediated STAT3 phosphorylation [148]. Further, when Leprflox/flox mice were crossed with Heat-shock-Cre line 1 mice, which ubiquitously express Cre, the resulting Lepr∆17/∆17 mice were obese, hyperphagic, hyperglycemic, hyperinsulinemic, and sterile, a phenotype reminiscent of leptin-receptor deficient db/db mice [159]. Thus, Leprflox/flox mice were crossed with AlbCre+ mice and ob/+ mice to generate mice with a hepatocyte-specific loss of leptin signalling on a leptin-deficient ob/ob background (Figure 4). Cre-mediated recombination in the Leprflox/flox AlbCre+ ob/ob mice resulted in the generation of the Lepr∆17 allele (Figures 16A-B), which is restricted to hepatocytes (Figure 5). Figures 16A and 16B show that Cre-mediated recombination in  53  hepatocytes was achieved as expected on the ob/ob background. The faint amplicon seen in Figure 16B at ~343 bp suggests that Cre-mediated recombination was not complete in the liver of Leprflox/flox AlbCre+ ob/ob mice, but this amplicon is likely contributed by non-hepatocytes in the liver, which do not express the AlbCre transgene.  Figure 16. Leprflox/flox AlbCre+ ob/ob mice have truncated leptin receptor transcripts in the liver. (A) Genomic DNA was extracted from liver tissue of Leprflox/flox AlbCre- ob/ob and Leprflox/flox AlbCre+ ob/ob mice and used as a template for PCR. Using primers MLepr101 and MLepr102, the Leprflox allele has an expected product size of 1369 bp and the Lepr∆17 allele is predicted to produce a 952 bp product. (B) RNA was extracted from liver tissue of Leprflox/flox AlbCre ob/ob mice and used as a template for RT-PCR using primers G and 60, which flank exon 17 of the leptin receptor gene. The predicted product sizes are 343 bp for Leprflox and 267 bp for Lepr∆17. These data were partially contributed by J Levi and are published in her Master’s thesis.  The effects of low dose intraperitoneal injections of leptin on fasting blood glucose and insulin in Leprflox/flox AlbCre ob/ob mice Leprflox/flox AlbCre ob/ob mice were first treated with a low dose of 0.1 µg/g leptin via intraperitoneal injection for two days. This dose of leptin has previously been shown to have minimal effects on body weight and food intake but have a marked effect on lowering blood glucose levels [30]. Figure 17 shows that indeed in Leprflox/flox AlbCre ob/ob mice, intraperitoneal injections of 0.1 µg/g leptin had no effect on body weight or food intake and thus obesity was maintained in these mice.  54  Figure 17. Intraperitoneal injections of 0.1 µg/g leptin has minimal effect on body weight and food intake in Leprflox/flox AlbCre ob/ob mice. (A) Body weight and (B) overnight food intake were measured in male Leprflox/flox AlbCre ob/ob mice treated with 0.1 µg/g leptin for 2 days. Mice were given half the dose at 9 am and the other half at 6 pm on each day of treatment. Data are mean±SEM, n≥5.  Next, the effect that this dose of leptin had on blood glucose and plasma insulin levels was examined. Upon treatment with 0.1 µg/g leptin, both Leprflox/flox AlbCre+ ob/ob mice and their littermate controls had a very minor response in terms of blood glucose levels. However, between days 2 and 10 post-leptin injection, ob/ob mice with hepatic leptin signalling tended to have a slight decrease in blood glucose levels while ob/ob mice lacking hepatic leptin signalling had a slight increase. When plotted as percent of baseline, it was apparent that blood glucose levels decreased by as much as 12% in Leprflox/flox AlbCre- ob/ob mice (Figure 18A-B). However, in ob/ob mice lacking hepatic leptin signalling, blood glucose levels actually increased by as much as 26% (Figure 18A-B). Interestingly, it appears that 2 days of leptin treatment had an effect on blood glucose levels for up to 2 weeks, well past the half-life of leptin, which has been reported to be less than 6 hours when leptin is delivered by intraperitoneal injection [33]. This suggests that leptin may have long-term effects on blood glucose. Furthermore, at this dose of leptin, plasma insulin levels were unchanged and both groups of mice remained hyperinsulinemic. This indicates that low-dose leptin treatment to ob/ob mice may increase 55  insulin sensitivity, resulting in lower blood glucose levels without changing plasma insulin levels. This effect of leptin on increasing insulin sensitivity seems to be lost in ob/ob mice lacking hepatic leptin signalling.  Figure 18. Intraperitoneal injections of 0.1 µg/g leptin modestly lowers blood glucose in ob/ob mice but not in ob/ob mice lacking liver leptin signalling. Leprflox/flox AlbCre ob/ob males were treated with 0.1 µg/g leptin for 2 days. Mice were given half the dose at 9 am and the other half at 6 pm on each day of treatment. Four hour fasted blood glucose levels were followed before, during, and after leptin treatment. Raw blood glucose values are shown in (A) and blood glucose values as a percent of baseline are shown in (B). Plasma insulin levels after a 4 hour fast are shown in (C). P values were determined by two-way ANOVA with a HolmSidak post-hoc test.  Leprflox/flox AlbCre ob/ob mice were next treated with a higher dose of leptin in order to see if this could produce a more prominent effect on blood glucose levels. The dose of leptin was increased to 0.8 µg/g for 2 days. This dose was enough to very modestly decrease food intake and attenuate body weight gain, but these mice clearly remained obese (Figure 19). When blood glucose levels were measured in these mice, it was apparent that this higher dose of leptin was able to augment the differences seen at the lower dose of 0.1 µg/g leptin between Leprflox/flox AlbCre+ ob/ob mice and their littermate controls (Figure 20A-B). Fasting blood glucose levels in Leprflox/flox AlbCre- ob/ob controls dropped by 34% after leptin treatment while those of Leprflox/flox AlbCre+ ob/ob mice only decreased by 19% (Figure 20B). This effect on blood glucose levels lasted up to 19 days after leptin injections in Leprflox/flox AlbCre- ob/ob mice 56  whereas in ob/ob mice lacking hepatic leptin signalling, blood glucose levels returned to baseline in about 10 days. Plasma insulin levels again remained unaffected by this dose of leptin in both groups of mice (Figure 20C). These data again suggest that acute leptin signalling in the liver may have effects on increasing insulin sensitivity in metabolically stressed obese mice.  Figure 19. Intraperitoneal injections of 0.8 µg/g leptin have minimal effect on body weight and food intake in Leprflox/flox AlbCre ob/ob mice. (A) Body weight and (B) overnight food intake were measured in male Leprflox/flox AlbCre ob/ob mice treated with 0.1 µg/g mouse recombinant leptin for 2 days. Mice were given half the dose at 9 am and the other half at 6 pm on each day of treatment. Data are mean±SEM, n≥5.  57  Figure 20. Intraperitoneal injections of 0.8 µg/g leptin lowers blood glucose more in ob/ob mice than in ob/ob mice lacking liver leptin signalling. Leprflox/flox AlbCre ob/ob males were treated with 0.8 µg/g mouse recombinant leptin for 2 days. Mice were given half the dose at 9 am and the other half at 6 pm on each day of treatment. Four hour fasted blood glucose levels were followed before, during, and after leptin treatment. Raw blood glucose values are shown in (A) and blood glucose values as a percent of baseline are shown in (B). Plasma insulin levels after a 4 hour fast are shown in (C). P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test.  The effects of leptin delivered via mini-osmotic pump on glucose metabolism in Leprflox/flox AlbCre ob/ob mice Since leptin administered via intraperitoneal injection is quickly cleared, Leprflox/flox AlbCre ob/ob mice were next treated with a higher continuous dose of leptin via subcutaneous mini-osmotic pump in order to maintain leptin levels in the bloodstream. This higher continuous dose allowed for the determination of whether an even larger difference in fasting blood glucose levels between Leprflox/flox AlbCre+ ob/ob mice and their littermate controls could be observed and whether this difference could be maintained over the length of leptin treatment. Further, by continuously delivering leptin, we were able to perform more in vivo assays during leptin treatment. Leprflox/flox AlbCre ob/ob mice were first treated with a high dose of 5 µg/day for 7 days by subcutaneously implanting a mini-osmotic pump. This high dose of leptin led to a profound reduction in food intake (Figure 21B), which decreased to levels below those previously  58  measured in Leprflox/flox mice [93]. This decrease in food intake resulted in an over 10% decrease in body weight in both groups of mice during just 7 days of leptin treatment (Figure 21A). Interestingly, this high dose of leptin also resulted in normalized blood glucose levels in both Leprflox/flox AlbCre+ ob/ob mice and their littermate controls (Figure 21C). Thus, it seems that at a high dose of leptin, the effects of hepatic leptin signalling on fasting blood glucose levels are masked by the effects of the marked reduction in food intake and body weight.  Figure 21. 5 µg/day leptin via mini-osmotic pump normalizes food intake and blood glucose levels independent of hepatic leptin signalling in Leprflox/flox AlbCre ob/ob mice. (A) Body weight and (B) overnight food intake, and (C) 4 hour fasted blood glucose were measured in male Leprflox/flox AlbCre ob/ob mice treated with 5 µg/day mouse recombinant leptin for 7 days. Data are mean±SEM, n≥5.  Next, Leprflox/flox AlbCre ob/ob mice were treated with a lower dose of leptin over a longer treatment period in order to maintain obesity and observe the effects of hepatic leptin signalling under metabolically stressed conditions. To do this, a low dose of 0.6 µg/day was delivered via mini-osmotic pump. Figure 22A shows that this dose of leptin increased plasma leptin levels by only a very small amount, just above the detection limit of the leptin ELISA. This resulted in the maintenance of obesity with a steady decrease in food intake over the 28 day treatment period (Figure 22B-C). Interestingly, at this low dose of leptin, it was again observed that ob/ob mice lacking hepatic leptin signalling had elevated blood glucose levels compared to 59  ob/ob mice with leptin signalling in the liver and this elevation in blood glucose levels persisted long after the leptin pump was removed (Figure 23A). Furthermore, similar to the mice treated with low dose intraperitoneal injections of leptin, fasting plasma insulin levels in Leprflox/flox AlbCre ob/ob mice treated with 0.6 µg/day via mini-osmotic pump were not different (Figure 23C). Although glucose tolerance tests revealed that Leprflox/flox AlbCre+ ob/ob mice had impaired glucose tolerance, glucose-stimulated insulin secretion did not differ from Leprflox/flox AlbCre- ob/ob littermate controls (Figure 24). These data suggest that in obese, hyperinsulinemic mice, the lack of hepatic leptin signalling leads to insulin resistance both in the fasted and postprandial states.  Figure 22. 0.6 µg/day leptin treatment has minimal effect on body weight and food intake in Leprflox/flox AlbCre ob/ob mice. Male Leprflox/flox AlbCre ob/ob mice were treated with 0.6 µg/day mouse recombinant leptin for 28 days via mini-osmotic pump starting on day 1. Mice were monitored for 4 hour fasted plasma leptin levels (A), body weight (B), and overnight food intake. The dotted line indicates the limit of detection of the leptin ELISA. Data are shown as mean±SEM, n≥5 for both groups.  60  Figure 23. 0.6 µg/day leptin via mini-osmotic pump lowers blood glucose more in ob/ob mice than in ob/ob mice lacking liver leptin signalling. Male Leprflox/flox AlbCre ob/ob mice were treated with 0.6 µg/day mouse recombinant leptin for 28 days. (A) Four hour fasted blood glucose levels were monitored before, during, and after leptin treatment. (B) Four hour fasted plasma insulin levels were measured before and during leptin treatment. Data are mean±SEM, n≥5. P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test.  Figure 24. Obese, hyperinsulinemic mice lacking hepatic leptin signalling have impaired glucose tolerance. Male Leprflox/flox AlbCre ob/ob mice were treated with 0.6 µg/day mouse recombinant leptin for 28 days. On day 22 of leptin treatment, mice were subjected to an oral glucose tolerance test. (A) Plasma glucose measurements and (B) plasma insulin measurements were taken after an oral gavage of 1.5 mg/g glucose at t=0. Data are mean±SEM, n≥5. P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test. Insulin tolerance tests were next performed to see if Leprflox/flox AlbCre+ ob/ob mice treated with 0.6 µg/day leptin were indeed more resistant to insulin than their littermate controls. 61  As seen in Figure 23 and again in Figure 25A, low dose leptin-treated Leprflox/flox AlbCre+ ob/ob mice had higher fasting blood glucose levels than Leprflox/flox AlbCre- ob/ob controls. In response to insulin, blood glucose levels remained elevated in Leprflox/flox AlbCre+ ob/ob mice compared to controls (Figure 25A), but when expressed as a percent of baseline, it is apparent that the response to insulin was the same in both groups of mice after taking into account the differences at t=0 (Figure 25B). When this experiment was repeated in another cohort of mice, it was observed that before leptin treatment, ob/ob mice with and without hepatic leptin signalling were equally insulin resistant (Figure 25C), but after 15 days of low dose leptin treatment, the ob/ob mice lacking hepatic leptin signalling had improved insulin sensitivity compared to ob/ob mice with leptin signalling in the liver (Figure 25D). Thus, two different cohorts of low dose leptintreated Leprflox/flox AlbCre ob/ob mice showed different responses to an insulin tolerance test and so the effect of hepatic leptin signalling in obese, hyperinsulinemic mice is still unclear.  62  Figure 25. The effects of ablated hepatic leptin signalling on insulin sensitivity in obese, hyperinsulinemic mice. Male Leprflox/flox AlbCre ob/ob mice were treated with 0.6 µg/day mouse recombinant leptin for 28 days via mini-osmotic pump. On day 15 of leptin treatment, mice were subjected to an insulin tolerance test by administering a dose of 1.25 U/kg human insulin intraperitoneally following a 4-hour fast. Blood glucose levels were monitored over the next two hours. Raw blood glucose measurements are shown in (A) and values are expressed as percent of baseline in (B). In another cohort, mice were subjected to an insulin tolerance test on day -18 (C) and day 15 (D) of leptin treatment. Data are shown as mean±SEM, n≥4. P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test.  Intravenous delivery of Ad-Lepr-b effectively restores hepatic leptin signalling in db/db mice To attempt to clarify the role of hepatic leptin signalling on glucose metabolism in obese, hyperinsulinemic mice, glucose metabolism was investigated in another mouse model that is complementary to Leprflox/flox AlbCre ob/ob mice. We obtained db/db mice, which have a whole body loss of leptin signalling, and treated them with an adenovirus (serotype 5) expressing the 63  long, signalling isoform of the leptin receptor (Ad-Lepr-b) via the tail vein, which confers liverselective expression of the adenoviral construct [160]. Although as much as 90% of adenovirus serotype 5 is taken up by Kupffer cells in the liver, our results likely do not reflect the effects of Ad-Lepr-b on Kupffer cells since Kupffer cells die upon exposure to adenoviruses [161]. This would happen in both control mice infected with an adenovirus expressing β-galactosidase (Adβ-gal) as well as Ad-Lepr-b treated mice. The adenoviruses that manage to evade Kupffer cells have a high propensity to infect hepaotcytes [162]. Thus, our data reflect the effects of restoration of Lepr-b specifically in hepatocytes. Using this adenoviral approach, it was possible to observe whether restoring leptin signalling in hepatocytes of db/db mice would show the opposite effects on glucose metabolism that were seen in low dose leptin-treated Leprflox/flox AlbCre+ ob/ob mice. To confirm expression of Lepr-b in db/db mice after infection with Ad-Lepr-b, RT-PCR was performed on liver tissue using primers that flank the insertion mutation present in the Lepr gene of db/db mice. Figure 26A shows that when RT-PCR was performed on liver from db/db mice infected with Ad-β-gal, a PCR product was obtained at the expected size of ~298 bp, which contains the db insertion mutation. In db/db mice infected with Ad-Lepr-b, the predominant PCR product was ~192 bp, which is the expected product size for wildtype Lepr-b. This reexpression of Lepr-b resulted in functional leptin receptors since db/db mice treated with AdLepr-b had significantly more hepatic phosphorylation of STAT3 than controls treated with Adβ-gal (Figures 26B-C).  64  Figure 26. Restoration of hepatic leptin signalling in db/db mice after infection with AdLepr-b. Liver tissue was collected 26 days post-infection from db/db mice treated with an adenovirus expressing either β-galactosidase or the long signalling isoform of the leptin receptor, Lepr-b. (A) RT-PCR was performed to confirm the hepatic expression of wild-type Lepr-b. The predicted product sizes are 298 bp for the mutant db allele and 192 bp for wild-type Lepr-b. (B) Representative Western blot showing phosphorylated and total STAT3 levels in livers of db/dbAd-β-gal and db/db-Ad-Lepr-b mice. Quantification by densitometric analysis is shown in (C), n=8. P value was determined by a Student’s t-test.  The effects of restored hepatic leptin signalling on glucose metabolism in db/db mice The effects of restoring hepatic leptin signalling in db/db mice on body weight, blood glucose, and plasma insulin levels were measured first. It was clear that restored hepatic leptin signalling in db/db mice had no effect on body weight in these mice as both groups of mice remained obese (Figure 27A). Furthermore, both groups of db/db mice remained extremely hyperleptinemic with plasma leptin levels of approximately 100 ng/mL at all time points tested (Figure 27B). Interestingly, fasting blood glucose and plasma insulin levels both exhibited a downward trend after virus treatment, but this decrease happened in both db/db mice treated with Ad-Lepr-b and those treated with Ad-β-gal (Figure 27B-C). In fact, it has been shown that adenoviral infections can result in an acute phase immune response [163, 164] and this can have a direct effect on glucose metabolism in the liver [164]. Thus, it seems that while hepatic leptin  65  signalling has no effect on fasting blood glucose and plasma insulin levels in these mice, the viral infection itself may have effects on glucose metabolism.  Figure 27. Restoration of hepatic leptin signalling in db/db mice does not affect body weight, plasma leptin, blood glucose, or plasma insulin levels. Male db/db mice were treated with Ad-Lepr-b or Ad-β-gal virus on day 0 as indicated. (A) Body weight, (B) 4 hour fasted plasma insulin, (C) blood glucose, and (D) plasma insulin levels were measured before and after virus treatment. Data are shown as mean±SEM, n≥5 for both groups.  Since low dose leptin-treated ob/ob mice lacking hepatic leptin signalling had impaired glucose tolerance compared to their controls (Figure 24A), glucose tolerance in db/db mice with restored hepatic leptin signalling was also measured to identify any improvements in glucose 66  tolerance. Surprisingly, db/db mice with restored hepatic leptin signalling also had impaired glucose tolerance compared to Ad-β-gal treated controls (Figure 28A) with no differences in glucose-stimulated insulin levels (Figure 28B). Therefore, it seems that in obese, hyperinsulinemic mice, the effect of hepatic leptin signalling on glucose tolerance is complex with our data suggesting that both a loss and overexpression of leptin receptors in the liver can lead to glucose intolerance.  Figure 28. Restoration of hepatic leptin signalling in db/db mice results in impaired glucose tolerance compared to db/db controls. Male db/db mice were treated with Ad-Lepr-b or Ad-βgal virus on day 0. Fifteen days post virus infection, an oral glucose tolerance test was performed by administering an oral gavage of 1.5 mg/g glucose. (A) Plasma glucose levels and (B) plasma insulin levels were measured over the next hour. Data are shown as mean±SEM, n≥5 for both groups. P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test.  4.3 Discussion The literature clearly shows that the effects of hepatic leptin signalling on glucose metabolism are complex. Many attempts have been made to elucidate the role of liver leptin signalling, but there is no agreement on the exact role of leptin in regulating glucose metabolism in the liver. Perhaps epitomizing the complexity of liver leptin signalling on glucose metabolism in the liver is the fact that some groups have shown in the same study that leptin can 67  simultaneously upregulate one aspect of insulin signalling while downregulating another [57, 121, 123, 128] and this may depend on the length of time of leptin treatment [57, 123]. Indeed, our laboratory showed that when hepatocytes were treated with leptin in vitro, there was an increase in insulin receptor phosphorylation but a concurrent increase in PTP1B, a negative regulator of insulin signalling [129]. This was also demonstrated in vivo since ob/ob mice treated with leptin also had an increase in hepatic insulin receptor phosphorylation as well as an increase in PTP1B [129]. To attempt to determine the role of leptin signalling on glucose metabolism specifically in the liver in vivo, glucose metabolism in mice with a chronic, life-long liver-specific loss of leptin signalling was examined [72]. These data, shown in Chapter 3, demonstrate that lean mice with a chronic, life-long loss of hepatic leptin signalling had a protection from age- and dietrelated glucose intolerance and had increased insulin sensitivity. This suggests that in lean mice, leptin is a negative regulator of hepatic insulin signalling. Notably, these mice were largely normal in the basal fasted state and the effects of lost liver leptin signalling only seemed to be observable under hyperinsulinemic conditions. Furthermore, since these mice have a chronic loss of leptin signalling in the liver, these effects likely only reveal the chronic effects of leptin on hepatic glucose metabolism. To see whether there are differences in acute versus chronic leptin signalling in the liver in vivo, ob/ob mice lacking functional hepatic leptin receptors were generated and treated acutely with leptin injections. By using low doses of leptin, it was possible to observe the effects of hepatic leptin signalling in metabolically stressed obese mice that were hyperinsulinemic even in the fasted state. Furthermore, obese, hyperinsulinemic db/db mice were treated with an adenovirus to transiently restore functional leptin receptors in the liver.  68  The results showed that when Leprflox/flox AlbCre+ ob/ob mice were given acute injections of low dose leptin, they had elevated fasting blood glucose levels compared to the Leprflox/flox AlbCre- ob/ob controls. This was also seen when these mice were given a low dose of leptin over a longer time period via an osmotic pump. Therefore, unlike Leprflox/flox AlbCre+ mice, which are lean and have normal fasting blood glucose levels, metabolically stressed Leprflox/flox AlbCre+ ob/ob mice have elevated fasted blood glucose when treated with low dose leptin. However, when Leprflox/flox AlbCre+ ob/ob mice were given a high dose of leptin, there was no effect of liver leptin signalling on fasting blood glucose levels. Similarly, db/db mice with restored hepatic leptin signalling also had extremely high levels of plasma leptin and the same fasting blood glucose levels as their controls. Interestingly, at low doses of leptin, the effects on fasting blood glucose lasted well after leptin treatment was ceased while at high doses, the effects on fasting blood glucose wore off quickly after the cessation of leptin treatment. It is speculated that at low doses, the small amount of peripherally administered leptin is cleared before it reaches the brain and thus the effects observed are more likely due to direct peripheral effects. At high doses, the chance of leptin reaching the brain before being cleared is higher and therefore at high doses, the possibility of brain leptin actions overriding peripheral effects is greater. Overall, it appears that the glucometabolic effects of leptin on the liver may be dependent on the dose of leptin. Intriguingly, this suggests that one explanation for the discrepancies seen in the literature regarding the effects of leptin on hepatic glucose metabolism could be the different doses of leptin used from study to study. The mechanism by which acute low dose leptin affects hepatic glucose metabolism is not clear. The data show that ob/ob mice lacking hepatic leptin signalling have elevated fasting blood glucose levels compared to ob/ob controls with no changes in fasting plasma insulin levels, which suggests that ob/ob mice lacking hepatic leptin signalling may be insulin resistant  69  compared to controls. However, the results on insulin sensitivity in these mice were inconclusive and, if anything, the data suggested that low dose leptin-treated Leprflox/flox AlbCre+ ob/ob mice were more insulin sensitive compared to controls. Alternatively, the elevated fasting blood glucose levels in low dose leptin-treated Leprflox/flox AlbCre+ ob/ob mice may have been due to effects independent of insulin. Leptin has been shown to be able to inhibit the effects of glucagon in rat hepatocytes [165, 166] and this was specifically shown to be a result of acute but not chronic leptin exposure [166]. Therefore, in low dose leptin-treated Leprflox/flox AlbCre+ ob/ob mice, elevated fasting blood glucose levels may be due to a loss of leptin-mediated inhibition of glucagon action and further studies should be done to investigate this possibility. In the post-prandial state, ob/ob mice lacking hepatic leptin signalling treated with low dose leptin had impaired glucose tolerance compared to their ob/ob controls. Unexpectedly, db/db mice with restored hepatic leptin signalling also had impaired glucose tolerance. It is possible that both a loss of hepatic leptin signalling and excessive leptin signalling in the liver of obese, hyperinsulinemic mice results in impaired glucose tolerance. However, this is unlikely since lean mice lacking leptin signalling in the liver had improved glucose tolerance under the metabolic stressors of aging and a high fat diet. Thus, the studies on glucose tolerance in Leprflox/flox AlbCre ob/ob mice and db/db mice with restored hepatic leptin signalling should be repeated before any firm conclusions are made. Our studies attempting to clarify the role of hepatic leptin signalling in vivo reflect the data currently in the literature and demonstrate the complex nature of leptin action on hepatic glucose metabolism. Collectively, our data show that the effects of leptin on glucose metabolism in the liver in vivo may depend on a number of factors including the dose of leptin used, acute versus chronic exposure to leptin, and also the metabolic state of the animal. Therefore, future  70  studies should control for these factors and hopefully, a clearer picture of the complicated relationship between hepatic leptin signalling and glucose metabolism can be achieved.  71  CHAPTER 5 – EFFECTS OF A LIFE-LONG LOSS OF HEPATIC LEPTIN SIGNALLING ON LIPID METABOLISM 5.1 Introduction Leptin has been shown to have many effects on lipid metabolism. Mice with a deficiency in leptin signalling, including ob/ob mice and lipoatrophic mice, display severe hepatic steatosis and systemic leptin treatment is able to greatly reduce the amount of lipid stored in the liver [71, 82, 132]. Further, it has been shown that peripheral leptin treatment can reduce hypercholesterolemia in ob/ob mice [167], and leptin can ameliorate hyperlipidemia in lipoatrophic mice [168]. Importantly, restricting food intake in ob/ob mice cannot improve lipid metabolism as effectively as leptin treatment [169, 170]. Thus, it is clear that leptin has potent effects on lipid metabolism independent of its effects on body weight, but whether direct leptin action on the liver in vivo is involved in these effects on lipid metabolism has not been fully investigated. When Cohen et al. generated the mouse with a liver-specific knockout of the complete leptin receptor, they showed that these mice had no alterations in hepatic lipid triglyceride levels [125]. However, this was the only aspect of lipid metabolism measured in these mice. Therefore, a rigorous investigation was performed on lipid metabolism in Leprflox/flox AlbCre mice, Leprflox/flox AlbCre ob/ob mice, and db/db mice with restored hepatic leptin signalling. The effects of ablated hepatic leptin signalling on lipid metabolism in Leprflox/flox AlbCre mice are presented in this chapter. Results from a thorough study on lipid metabolism in leptin-treated Leprflox/flox AlbCre ob/ob mice and db/db mice with a replacement of leptin signalling in the liver are available in Chapter 6.  72  5.2 Results Loss of hepatic leptin signalling alters lipid metabolism genes in the liver To obtain an unbiased overview of liver genes that may be regulated by hepatic leptin signalling, gene array analyses were performed on liver tissue from Leprflox/flox AlbCre+ mice and their littermate controls that were either fasted overnight or fasted overnight and then re-fed for 2 hours (Tables A1-A2). An array was also performed on leptin-treated Leprflox/flox AlbCre ob/ob mice (Table A3). The gene that had the highest fold change between Leprflox/flox AlbCre+ mice and their littermate controls both after an overnight fast and after a 2-hour re-feed was lipocalin-2. This was especially interesting since lipocalin-2 has been shown to play a role in glucose and lipid metabolism, with one laboratory showing that lipocalin-2 knockout mice are protected from age and obesity-induced insulin resistance [171] and another laboratory showing lipocalin-2 deficiency potentiates diet-induced insulin resistance [172]. Since hepatic lipocalin-2 levels are positively correlated with lipocalin-2 levels in the plasma [173], plasma lipocalin-2 levels were measured in Leprflox/flox AlbCre mice in order to validate our gene array results. Unfortunately, plasma lipocalin-2 levels were not different in Leprflox/flox AlbCre+ mice and their littermate controls (Figure A1). A functional enrichment analysis on the gene array data was next performed and this revealed that lipid metabolic pathways were greatly affected by a loss of hepatic leptin signalling in both lean and obese mice (Figures A2-A3). Previous studies have also shown that leptin treatment of ob/ob mice has major effects on lipid metabolism genes in the liver [87, 133, 169]. Furthermore, similar to our findings presented in Chapter 4, Liang and Tall showed that the duration of leptin exposure also had an impact on which genes were altered by leptin in the liver [133]. Interestingly, the study by Liang and Tall found that several apolipoprotein genes were altered when they treated ob/ob mice with leptin. The results in Appendix A also show several  73  alterations in apolipoprotein gene expression, including apolipoprotein A-IV, apolipoprotein M, and apolipoprotein B (Tables A1-A3). This suggests that apolipoprotein levels may be directly regulated by hepatic leptin signalling. In fact, one of the most highly downregulated genes in leptin-treated Leprflox/flox AlbCre+ ob/ob mice was Apob, a gene in which polymorphisms are associated with hypertriglyceridemia and familial combined hyperlipidemia in humans [174, 175]. Indeed, apoB levels have even been used as part of the criteria for diagnosing familial combined hyperlipidemia [176]. Therefore, lipid metabolism was thoroughly examined in Leprflox/flox AlbCre mice, Leprflox/flox AlbCre ob/ob mice, and db/db mice with restored hepatic leptin signalling with a focus on apoB and apoB-related effects on triglyceride metabolism. Loss of hepatic leptin signalling does not affect total plasma lipid levels in the basal state To investigate the effects of ablated hepatic leptin signalling on lipid metabolism, plasma lipid levels were first measured in fasted Leprflox/flox AlbCre mice. Plasma cholesterol, triglycerides, and free fatty acids levels after a 4 hour fast were unchanged between Leprflox/flox AlbCre+ mice and their littermate controls at 6, 12, and 16 weeks of age (Table 3). To more thoroughly assess lipid metabolism, triglyceride secretion from the liver was evaluated in the fasting state. To measure VLDL-TG secretion from the liver, fasted mice were injected with P407, a compound shown to inhibit triglyceride uptake with fewer side effects than Triton WR1339 [177]. P-407 was a potent inhibitor of triglyceride uptake as plasma triglyceride levels increased by 10-fold 5 hours after injection (Figure 29A). However, the accumulation in plasma triglycerides occurred at the same rate in both Leprflox/flox AlbCre+ mice and their littermate controls. Next, since insulin can suppress VLDL-TG secretion [6-8] and it was seen previously that the livers of Leprflox/flox AlbCre+ mice are more sensitive to the glucometabolic effects of insulin (Chapter 3) [72], it was examined whether a bolus of insulin would also differentially affect VLDL-TG secretion in mice with and without hepatic leptin signalling. As expected, in  74  response to an insulin bolus, there was a decrease in the rate of triglyceride accumulation in both Leprflox/flox AlbCre+ mice and their littermate controls (Figures 29B and 29C). Surprisingly, in the mice lacking hepatic leptin signalling, there was a trend towards increased levels of plasma triglycerides after insulin injection compared to controls (Figure 29B). Indeed, at a higher dose of insulin (Figure 29C), this trend was even more pronounced. While not statistically significant, these surprising observations suggest that the effects of insulin on suppressing triglyceride secretion from the liver may be muted in mice lacking hepatic leptin signalling.  Figure 29. The effects of insulin on hepatic triglyceride secretion in mice lacking hepatic leptin signalling. Mice were fasted for four hours, injected with P-407 at t=0, and then were either not injected (A), injected with 0.6 U/kg insulin (B), or 0.725 U/kg insulin (C) at t=2 hours. Data from (A) were obtained from one cohort of mice while data from panels (B) and (C) were obtained from another cohort. Plasma triglyceride levels were monitored throughout the experiment. Data are expressed as mean±SEM, n≥4. P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test.  75  Table 4. Fasting plasma lipids in mice with and without hepatic leptin signalling. The genetic background of Leprflox/flox AlbCre mice is mixed and is described in detail in Methods and Materials. Wildtype controls and db/db mice were on a pure C57BL/6 genetic background.  6 wks  ♂  ♀  Cholesterol (mg/dL) Lepr AlbCreflox/flox Lepr AlbCre+  15610 (7) 1666 (12)  0.570.05 (18) 0.650.05 (23)  0.700.08 (8) 0.880.08 (8)  C57BL/6 db/db  771 (8) 1194 (8)*  0.520.03 (8) 0.540.02 (8)  0.730.06 (8) 1.030.16 (8)*  13715 (7) 13410 (10)  0.570.10 (16) 0.480.06 (23)  0.590.11 (4) 0.780.08 (4)  807 (4) 1859 (8)*  0.400.04 (4) 0.560.06 (8)  0.960.09 (4) 1.660.06 (8)*  Lepr AlbCreflox/flox Lepr AlbCre+  18510 (10) 1999 (16)  0.420.03 (15) 0.340.04 (21)  0.930.07 (5) 0.700.06 (5)*  C57BL/6 db/db  1004.5 (8) 1257.1 (8)*  0.460.02 (8) 0.480.07 (8)  1.140.08 (8) 1.260.14 (8)  Lepr AlbCreflox/flox Lepr AlbCre+  1549 (8) 1428 (12)  0.220.04 (14) 0.190.03 (14)  0.840.11 (6) 0.700.15 (2)  C57BL/6 db/db  1084 (8) 22515 (8)*  0.300.03 (8) 0.420.07 (8)  0.900.07 (8) 1.980.12 (8)*  17620 (6) 18311 (7)  0.560.05 (6) 0.500.05 (7)  0.880.09 (6) 0.960.07 (7)  1294.6 (8) 1615.2 (7)*  0.370.04 (8) 0.470.09 (8)  1.310.10 (8) 1.330.10 (8)  Lepr AlbCreflox/flox Lepr AlbCre+  16714 (6) 14113 (6)  0.450.06 (5) 0.360.07 (5)  1.010.09 (5) 0.950.15 (5)  C57BL/6 db/db  963 (7) 23710 (8)*  0.210.03 (7) 0.380.08 (8)*  0.970.04 (5) 1.680.12 (8)*  flox/flox  flox/flox  Lepr AlbCreflox/flox Lepr AlbCre+ C57BL/6 db/db  12 wks  ♂  ♀  16 wks  ♂  flox/flox  flox/flox  flox/flox  Lepr AlbCreflox/flox Lepr AlbCre+ C57BL/6 db/db  ♀  a  Fasting Lipids Triglycerides (mM)  flox/flox  Free Fatty Acids (mM)  a  Mice were fasted for 4 hours during the light cycle flox/flox * P≤0.05 versus control (Lepr AlbCre- or C57BL/6) by Student’s t-test.  76  Mice lacking hepatic leptin signalling have larger, more triglyceride-rich VLDL particles Given these subtle effects of hepatic leptin signalling on plasma triglyceride, it was hypothesized that perhaps the effect of liver leptin signalling in lean mice was too subtle to observe when we measured plasma triglyceride levels in whole plasma. It is possible that there may be slight but important changes in the composition of lipoproteins in mice lacking hepatic leptin signalling. Leptin has been implicated in regulating the amount of triglyceride incorporation into VLDL [7], so the amount of lipid present on different sizes of lipoproteins was more closely assessed in mice with and without hepatic leptin signalling. To do this, plasma was fractionated by size via fast protein liquid chromatography (FPLC) to evaluate lipoprotein profiles in Leprflox/flox AlbCre+ mice and their Leprflox/flox AlbCre- littermate controls. Fractionation of plasma showed that mice with and without hepatic leptin signalling had no differences in the distribution or amount of cholesterol (Figure 30A). Interestingly, when triglyceride levels were measured, it was found that Leprflox/flox AlbCre+ mice had elevated triglycerides in fractions consistent in size with VLDL particles (Figure 30B). Furthermore, it appeared that the VLDL-TG in mice lacking hepatic leptin signalling eluted from the column slightly earlier than triglycerides in the control mice, indicating larger particles. Next, western blots were performed for apoB since each VLDL particle is associated with one apoB molecule [178]. Interestingly, the western blots confirmed that particles containing apoB in the plasma of Leprflox/flox AlbCre+ mice began to elute one fraction earlier than in Leprflox/flox AlbCre- mice (Figure 30C). In fact, when apoB content was measured across all fractions, it was noticed that in general, apoB-containing particles in mice lacking hepatic leptin signalling eluted earlier than those in control mice (Figures 30C-F). These data reveal that mice lacking hepatic leptin signalling have larger apoB-containing lipoprotein particles and have VLDL-sized particles with increased amounts of triglycerides. 77  Figure 30. Attenuation of hepatic leptin signalling results in increased VLDL triglycerides and larger apoB-containing lipoprotein particles. Pooled 4-hour fasted plasma samples from 17-30 week old male Leprflox/flox AlbCre+ mice and littermate controls (n≥3 each) were subjected to FPLC and the fractions were assayed for cholesterol (A) and triglycerides (B). Data represent the average of two (A) and three (B) experiments with different cohorts of mice. (C) Four-hour fasted plasma samples (n≥3) were pooled from 18-27 week old Leprflox/flox AlbCre+ mice and their littermate controls. The pooled samples were fractionated and then western blots for apoB were performed on fractions 6-37. Mean pixel intensity of the apoB100 band (D), apoB48 band (E), and combined apoB100 and apoB48 bands (F) are shown for each fraction. Data are mean±SEM, *P≤0.05 by Student’s t-test.  Loss of hepatic leptin signalling results in decreased apoB levels in the liver and plasma As there appeared to be a slight decrease in total apoB levels in mice lacking hepatic leptin signalling (Figure 30F), total apoB levels in whole plasma were measured from individual mice. Indeed, plasma apoB100 levels were significantly lower by 18% in Leprflox/flox AlbCre+ 78  mice compared to controls (Figures 31A-B). There was also a non-significant trend for plasma apoB48 levels to be lower in mice lacking hepatic leptin signalling (Figures 31A and 31C). Since apoB can come from the small intestine as well as the liver, hepatic apoB transcript levels were measured to see if changes in the liver could account for the decreased plasma apoB levels. As seen in Figure 31D, hepatic apoB mRNA levels were lower in Leprflox/flox AlbCre+ mice by 24%, suggesting that decreased plasma apoB in these mice can be accounted for by decreased hepatic expression of apoB.  Figure 31. Attenuation of hepatic leptin signalling decreases plasma and hepatic apoB levels. (A) Western blots for apoB were performed on four-hour fasted plasma samples from 2530 week old Leprflox/flox AlbCre mice, n≥6. Quantification of apoB100 and apoB48 for all samples by densitometry is shown in (B) and (C), n≥6. (D) ApoB transcript levels were 79  measured in the liver of 22 week old male Leprflox/flox AlbCre mice following a four hour fast, n=9. Data are mean±SEM. P values were determined by Student’s t-test. Mice lacking liver leptin signalling have decreased hepatic lipase and increased lipoprotein lipase activity in the liver The data indicate that mice lacking hepatic leptin signalling have less total plasma apoB, larger apoB-containing lipoprotein particles and increased amounts of triglycerides in VLDLsized particles. It is hypothesized that these observations can be explained by increased triglyceride incorporation per VLDL particle. However, a reduction in lipase activity could also explain some of these observations because patients with HL deficiency display abnormally large lipoprotein particles [179]. Indeed, mice lacking leptin signalling in the liver had 23% lower HL mRNA (Figure 32A) and a trend towards 30% lower HL activity levels (Figure 32C) compared to controls. Hepatic LPL levels were also measured in mice lacking liver leptin signalling and it was found that there was a substantial 4.5-fold increase in LPL activity in the liver (Figure 32D). This was surprising given that LPL is not normally expressed in adult mouse liver [180, 181]. To determine whether a loss of hepatic leptin signalling induces the liver to produce LPL, hepatic LPL mRNA levels were measured and no difference in transcript levels were found between Leprflox/flox AlbCre+ mice and their littermate controls (Figure 32B), demonstrating that the increased hepatic LPL activity in lean mice with a loss of liver leptin signalling was not transcriptionally regulated. The contribution of hepatic LPL to total triglyceride lipase activity in the liver increased from 17% in control mice to 57% in mice lacking hepatic leptin signalling (Figure 32E). Overall, these alterations to HL and LPL activity resulted in increased total triglyceride lipase activity in the liver of Leprflox/flox AlbCre+ mice (Figure 32E). Since overexpression of LPL in different tissues can cause lipid accumulation in those tissues [180, 182], a histological examination of liver from Leprflox/flox AlbCre mice was performed and this revealed that a loss of hepatic leptin signalling led to enlarged lipid droplets in the liver (Figure 33). Further, when a biochemical extraction of lipids from liver tissues was 80  performed, it was found that mice lacking leptin signalling in the liver had 16% more triglycerides and 48% more cholesterol in their livers when compared to littermate controls (Figure 34A-B). These data clearly reveal a role for hepatic leptin signalling in regulating lipase activity in the liver.  Figure 32. Hepatic leptin signalling regulates lipase activity levels in the liver. (A-B) Hepatic lipase and lipoprotein lipase transcript levels were measured in the liver of 22-week-old male Leprflox/flox AlbCre mice following a four hour fast, n≥8. (C-D) Activity levels of hepatic lipase and lipoprotein lipase were assessed in liver lysates from 22 week old male Leprflox/flox AlbCre mice, n=6 per group. Total triglyceride lipase activity in the liver (HL+LPL) is shown in (E). Data are expressed as mean±SEM. P values were determined by Student’s t-test.  81  Figure 33. Loss of hepatic leptin signalling results in hepatic lipid accumulation. Liver sections from male Leprflox/flox AlbCre mice were stained with hematoxylin and eosin. Representative images from three 22-week-old male Leprflox/flox AlbCre- littermate controls are shown in (A-C). Three representative images from 22-week-old male Leprflox/flox AlbCre+ mice are shown in (D-F). Images are magnified 100x under oil immersion.  Figure 34. Attenuation of hepatic leptin signalling results in increased hepatic lipid accumulation. Twenty-one week old male mice were fasted for four hours and then the liver was harvested. Lipids were isolated by a chloroform:methanol extraction and reconstituted into Thesit micelles. Samples were then assayed for triglycerides (A) and cholesterol (B), n≥8 in each group. Data are expressed as meanSEM. P values were determined by Student’s t-test.  82  Lean mice lacking leptin signalling in the liver have normal postprandial triglyceride clearance In light of alterations in lipase activity in the liver and since it has been suggested that leptin may act on the liver to promote postprandial triglyceride clearance [183], an oral lipid tolerance test was performed on mice with a life-long loss of leptin signalling in the liver (Figure 35). In response to an oral gavage of olive oil, plasma triglyceride levels in both Leprflox/flox AlbCre+ mice and their Leprflox/flox AlbCre- littermate controls increased at the same rate, peaking 2 hours post-gavage at levels that were 3-fold higher than basal. By 4 hours postgavage, plasma triglycerides in mice with and without hepatic leptin signalling had returned to baseline levels. Thus, a chronic loss of hepatic leptin signalling does not appear to affect postprandial triglyceride clearance.  Figure 35. Attenuation of hepatic leptin signalling does not affect lipid tolerance in lean mice. Oral lipid tolerance tests were performed on male Leprflox/flox AlbCre+ mice and littermate controls at 16 weeks of age. Mice were fasted for four hours and gavaged with 5 µL/g of olive oil. Data are expressed as mean±SEM, n=6 per group.  5.3 Discussion It has long been known that leptin has potent effects on lipid metabolism in vivo, but whether these effects of leptin are a result of direct leptin action on the liver, an organ which plays a major role in lipid metabolism, has not been fully addressed. To answer this question, 83  lipid metabolism was investigated in mice lacking hepatic leptin receptor signalling domains and this revealed a previously unreported role for hepatic leptin action in modulating lipase activity in the liver. It was found that Leprflox/flox AlbCre+ mice, with a life-long loss of leptin signalling in the liver, have decreased HL activity and a striking increase in LPL activity in the liver. While it has been previously reported that HL mRNA levels in the liver are decreased in ob/ob mice and restored with leptin treatment [133], the current data show that direct effects of leptin on the liver can positively regulate hepatic HL activity. Further, a novel finding that leptin resistance in the liver leads to a marked increase in hepatic LPL activity was also revealed. This is very interesting given that LPL is typically not expressed in adult liver [180, 181]. Since an overexpression of LPL in different tissues can cause lipids to accumulate in those tissues [180, 182], it is speculated that the elevation of LPL activity in Leprflox/flox AlbCre+ mice contributes to the enlarged lipid droplets and elevated hepatic triglycerides seen in these mice. Furthermore, with such a large increase in hepatic LPL activity levels, it was somewhat surprising that these mice did not have major changes in plasma triglycerides. However, it was previously reported that when LPL was restored specifically in the liver of hypertriglyceridemic LPL knockout mice, there was no effect on plasma triglycerides [180]. In contrast to our findings, Cohen et al. [125] reported that mice with a lack of the complete leptin receptor in the liver have normal hepatic lipid levels. While our mouse model lacked the leptin receptor signalling domain in the liver but retained the extracellular and transmembrane domains, mice studied by Cohen et al. had a knockout of the complete receptor. Thus, there may be a role for the extracellular and transmembrane domains of the leptin receptor in liver lipid metabolism. However, it should also be noted that Cohen et al. used different methods to assess hepatic lipid levels, including possibly using mice of different age than our study (age unspecified by Cohen et al.) and the use of mice heterozygous for the floxed leptin  84  receptor gene as controls. These differences in methods could potentially explain the differences in hepatic lipid content seen in our study and by Cohen et al. since mice heterozygous for the leptin receptor (db/+) or leptin (ob/+) gene have been shown to have changes in metabolism when compared to wildtype mice [184], with some changes in ob/+ mice being age-related [185]. Under the conditions in our study, mice with an ablation of the hepatic leptin receptor signalling domain have an increase in hepatic triglyceride and cholesterol levels when compared to littermate controls, suggesting that the ability of leptin to ameliorate hepatic steatosis in rodent models of type 2 diabetes [134] and lipodystrophy [186] may be mediated at least in part through leptin signalling at the level of the liver. It was previously reported that Leprflox/flox AlbCre+ mice have increased hepatic insulin sensitivity (Chapter 3) [72]. Given that insulin is a potent promoter of lipogenesis [187] and suppressor of lipid export from the liver [188], enhanced hepatic insulin sensitivity would be expected to contribute to increased hepatic lipid levels in Leprflox/flox AlbCre+ mice. Furthermore, insulin has a well-described role on decreasing plasma apoB levels [6-8]. Consistent with this, the data show that in mice lacking leptin signalling in the liver, increased hepatic insulin sensitivity was associated with decreased plasma apoB levels even in the fasting state. While it is possible that this effect on apoB is mediated directly by leptin signalling independent of insulin, it is speculated that it is actually the effect of leptin on insulin signalling that mediates changes in apoB since it has been shown that leptin itself does not affect plasma apoB levels [7, 189]. The observation that Leprflox/flox AlbCre+ mice had decreased plasma apoB levels (Figure 31) yet increased triglycerides in VLDL particles (Figure 30) suggests that Leprflox/flox AlbCre+ mice may have fewer VLDL particles in total but more triglycerides being incorporated per apoB-containing VLDL particle. It is hypothesized that the increased incorporation of 85  triglycerides is due to the elevated liver triglycerides in mice lacking hepatic leptin signalling [72], leading to increased substrate availability. This can result in more triglyceride incorporation into each VLDL particle [7, 190], leading to enlarged, more triglyceride-rich VLDL particles [190]. Interestingly, patients with HL deficiency have been shown to have triglyceride-rich lipoproteins that are also larger in size [179]. Furthermore, Bamji-Mirza et al. [191] showed that an overexpression of HL in a rat liver cell line resulted in secretion of triglyceride-poor VLDL. Therefore, decreased HL activity in Leprflox/flox AlbCre+ mice may also contribute to defects in lipid loading, leading to enlarged, triglyceride-rich VLDL particles. Our observations are largely compatible with a theory proposed by Huang et al. [7]. These authors proposed that insulin is responsible for modulating the number of VLDL particles, while hepatic leptin action can modulate the amount of triglycerides available for incorporation into each VLDL particle through the effects of leptin on increasing fatty acid oxidation. In our unique model of increased hepatic insulin sensitivity with extreme hepatic leptin resistance, there is less plasma apoB, which suggests fewer VLDL particles, and increased hepatic triglycerides, which may lead to larger, more triglyceride-rich VLDL particles. According to this model, one might expect that hepatic triglyceride secretion would be suppressed more in Leprflox/flox AlbCre+ mice because they are more insulin sensitive than controls [72]. Surprisingly, while it was observed that insulin suppressed hepatic triglyceride secretion in both groups of mice, those lacking hepatic leptin signalling actually had higher plasma triglycerides than controls after insulin (Figure 29). This may be due to enhanced lipogenic effects of insulin in the Leprflox/flox AlbCre+ mice, which together with the lack of leptin signalling can result in even more substrate availability during hyperinsulinemic conditions, allowing for more triglycerides per VLDL particle. Complementary to these data, liver insulin receptor knockout (LIRKO) mice have increased plasma apoB yet decreased plasma triglycerides and no alterations in liver triglycerides  86  [192]. Thus, insulin signalling in the liver may also have effects on triglyceride loading onto VLDL independent of its effects on substrate availability and our data suggest that leptin signalling in the liver may serve to counter this effect. Perhaps the most surprising aspect of the current data is the mild nature of the phenotype seen in these lean mice lacking hepatic leptin signalling. Despite all the data showing major changes in hepatic lipid metabolism genes upon leptin treatment in models of leptin deficiency such as ob/ob mice or lipodystrophic mice [82, 133, 169], the data suggest that many of these changes are likely due to indirect effects of leptin on the liver. Furthermore, unlike mice with a global loss of leptin signalling (db/db mice), which show hyperlipidemia and extreme hepatic steatosis, mice with a liver-specific loss of leptin signalling have normal total fasting plasma triglycerides and cholesterol levels and only have a minor increase in liver lipids. However, consistent with the literature [134, 193], the data do support the fact that direct leptin action on the liver plays an antisteatotic role in vivo; though the level of hepatic steatosis in Leprflox/flox AlbCre+ mice was not as severe as in livers of db/db mice. Furthermore, it appears that while mice with a hepatocyte-specific loss of leptin signalling have increased incorporation of triglycerides into VLDL particles, they do not develop hypertriglyceridemia due to their concurrent reduction in hepatic apoB production. It is possible that a more pronounced phenotype may be observed if mice lacking hepatic leptin signalling are crossed with mice that are more metabolically stressed or with mice containing specific lipid abnormalities such as apoE or LDL receptor-null mice, so future studies in this direction may be warranted. Nonetheless, it has been shown that slight abnormalities in lipoprotein particle size [194, 195], even when total plasma lipid levels are the same [195], are associated with increased risk of cardiovascular disease. In fact, patients with metabolic syndrome have a higher proportion of larger VLDL than healthy patients and this was true even in patients with normal plasma  87  triglyceride levels [196]. Thus, the subtle effects of hepatic leptin resistance on lipid metabolism may prove to have a major impact on health.  88  CHAPTER 6 – EFFECTS OF HEPATIC LEPTIN SIGNALLING ON LIPID METABOLISM IN OBESE HYPERINSULINEMIC MICE 6.1 Introduction As discussed in Chapters 3 and 4, leptin action on the liver may differentially affect insulin signalling and glucose metabolism depending on the dose of leptin, the length of leptin exposure, and the metabolic state of the animal. These same factors may also affect how leptin influences lipid metabolism in the liver since insulin also has a potent effect on hepatic lipid metabolism. Furthermore, even with all the changes in lipid metabolism genes, as determined by gene array (Tables A1-A2, Figure A1), it was surprising that there was only a minor perturbation in lipid metabolism in Leprflox/flox AlbCre mice. It was therefore hypothesized that hepatic leptin signalling would have a more profound effect on lipid metabolism under conditions of metabolic stress. Indeed, when a gene array was performed on liver from the more metabolically stressed Leprflox/flox AlbCre ob/ob mice, it was seen that after acute leptin treatment, lipid metabolism genes were again among those demonstrating the greatest changes in Leprflox/flox AlbCre+ ob/ob mice and their Leprflox/flox AlbCre- ob/ob controls (Table A3, Figure A2). Therefore, lipid metabolism was also investigated in low dose leptin-treated Leprflox/flox AlbCre+ ob/ob mice as well as in metabolically stressed db/db mice with restored hepatic leptin signalling.  6.2 Results Hepatic leptin signalling regulates plasma triglyceride and hepatic apoB levels in obese, hyperinsulinemic mice Previously, FPLC fractionation of plasma showed that Leprflox/flox AlbCre+ mice had elevated triglycerides in VLDL particles in the fasted state (Figure 30), but this effect was quite subtle such that we did not observe differences in fasting triglycerides when measured in whole plasma (Table 3). Furthermore, we observed that under hyperinsulinemic conditions, the loss of 89  hepatic leptin signalling resulted in increased triglyceride secretion from the liver (Figure 29). Therefore, it is possible that the effects of hepatic leptin signalling on VLDL triglycerides could be augmented under hyperinsulinemic conditions. To investigate the effects of hepatic leptin signalling on fasting plasma triglycerides under more strenuous metabolic conditions, we crossed the Leprflox/flox AlbCre mice onto an obese, hyperinsulinemic ob/ob background to generate ob/ob mice lacking functional hepatic leptin receptors (Leprflox/flox AlbCre ob/ob mice). Upon treating these mice with leptin, we were able to observe the acute effects of hepatic leptin signalling under obese, hyperinsulinemic conditions. These mice were treated with low dose leptin so as to maintain obesity and hyperinsulinemia (Figure 22). This dose of leptin lowered plasma cholesterol and triglycerides in ob/ob mice with and without hepatic leptin signalling (Figure 36A-C). However, plasma triglyceride levels in Leprflox/flox AlbCre+ ob/ob mice did not decrease as much as in their Leprflox/flox AlbCre- ob/ob littermate controls (Figure 36C). By the last day of leptin treatment, the Leprflox/flox AlbCre+ ob/ob mice had 36% higher plasma triglycerides than their littermate controls (Figure 36C). Interestingly, the effects of leptin treatment appeared to persist even after leptin therapy ceased, with plasma triglyceride levels in both groups only returning to near pre-leptin levels 50 days after the leptin pump was removed (Figure 36C). This indicates that leptin treatment in ob/ob mice has long-term effects on triglyceride metabolism. Since this effect on fasting plasma triglycerides in Leprflox/flox AlbCre ob/ob mice was subtle, it was important to see if these results could be reproduced in a complementary mouse model. Thus, leptin receptor-deficient db/db mice were treated with an adenovirus expressing Lepr-b (Ad-Lepr-b) to reconstitute hepatic leptin signalling selectively in the liver. It was previously shown by our lab that this method confers liver selective expression of the viral construct [160]. Furthermore, it is clear that this method can potently restore p-STAT3 signalling in the liver of Ad-Lepr-b treated db/db mice (Figure 26). Upon treatment with Ad-  90  Lepr-b, the db/db mice remained obese and hyperinsulinemic (Figure 27). Also, it appears that db/db mice treated with Ad-Lepr-b and control db/db mice treated with Ad-β-gal both had a response to the virus itself independent of the Lepr-b or β-gal constructs (Figure 36). This is likely due to the acute phase immune response to the virus, which has been shown to have effects on lipid metabolism [163]. Nonetheless, no differences were observed in plasma cholesterol or free fatty acids between Ad-Lepr-b and Ad-β-gal treated db/db mice (Figure 36DE). However, there was an effect on plasma triglyceride levels; db/db mice with restored hepatic leptin signalling had lower fasting plasma triglycerides than the Ad-β-gal treated controls between 1 and 3 weeks post-infection with Ad-Lepr-b treated mice reaching 31% lower plasma triglycerides 12 days post-infection (Figure 36F). Indeed, when hepatic apoB transcript levels were measured, we found that hepatic apoB mRNA levels in db/db mice were 26% lower than C57BL/6 controls and upon re-expression of functional leptin receptors in the liver of db/db mice, hepatic apoB transcript levels returned to wildtype levels (Figure 37). These data again suggest that mice lacking hepatic leptin signalling secrete fewer but more triglyceride-rich VLDL particles. Taken together, the data show that under the metabolic stressors of obesity and hyperinsulinemia hepatic leptin signalling is required for maintaining normal plasma triglyceride levels.  91  Figure 36. Hepatic leptin signalling has a subtle but consistent effect on plasma triglycerides in obese, hyperinsulinemic mice. (A-C) Seven-week-old male Leprflox/flox AlbCre ob/ob mice were treated with 0.6 µg/day mouse recombinant leptin for 28 days via mini-osmotic pump starting on day 1. Mice were tracked for 4-hour-fasted plasma cholesterol (A), free fatty acids (B), and triglycerides (C), n≥5 for both groups. (D-F) Male db/db mice were treated with Ad-β-gal or Ad-Lepr-b at 12 weeks of age and tracked for 4-hour-fasted plasma cholesterol (D), free fatty acids (E), and triglycerides (F), n≥6 for both groups. Data are shown as mean±SEM. P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test.  92  Figure 37. Restoration of hepatic leptin signalling in db/db mice restores normal hepatic apoB levels. Hepatic apoB transcript levels were measured in C57BL/6 controls and db/db mice 4 weeks post infection with either Ad-β-gal or Ad-Lepr-b, n≥5. Data are mean±SEM. P values were determined by Student’s t-test. Hepatic leptin signalling in obese, hyperinsulinemic mice regulates lipase activity in the liver Similar to lean mice that have a liver-specific loss of leptin signalling (Figure 32), Adbeta-gal treated db/db mice, which have a whole body loss of leptin signalling, also had a ~30% decrease in HL activity compared to wildtype C57BL/6 controls (Figure 38A). When functional leptin receptors were overexpressed selectively in the liver of db/db mice, HL activity levels went up even beyond levels seen in wildtype mice (Figure 38C). Furthermore, db/db mice treated with Ad-β-gal had a 2-fold increase in LPL activity levels and these activity levels returned to wildtype levels after the restoration of hepatic leptin signalling (Figure 38D). It was also observed that in the total lack of leptin signalling, hepatic LPL activity contributed to 60% of total triglyceride lipase activity in the liver and when leptin signalling was selectively restored to the liver, hepatic LPL activity contributed only 20% to total triglyceride lipase activity, which is similar to wildtype C57BL/6 levels (Figure 38F). Interestingly, in db/db mice, it seems that leptin regulation of LPL activity is transcriptionally controlled whereas regulation of HL activity 93  is not. Nonetheless, the functional end result is that with the loss of hepatic leptin signalling, hepatic HL activity is decreased and LPL activity is increased.  Figure 38. Hepatic leptin signalling regulates lipase activity levels in db/db mice. (A) Hepatic lipase and (B) lipoprotein lipase transcript levels were measured in the liver of 16-weekold C57BL/6 controls (n=5) and db/db males treated with Ad-β-gal or Ad-Lepr-b, n≥7. (C) Hepatic lipase, (D) lipoprotein lipase, and (E) total lipase activity levels (HL+LPL) were measured in liver lysates from C57BL/6 controls (n=5) and db/db mice treated with either Ad-βgal or Ad-Lepr-b, n=7. Data are expressed as mean±SEM. P values were determined by Student’s t-test. Hepatic leptin signalling regulates triglyceride clearance in obese, hyperinsulinemic mice Although lean mice lacking hepatic leptin signalling had no alterations in oral lipid tolerance (Figure 35), it is possible that leptin signalling in the liver may play a more prominent role regulating postprandial triglyceride clearance in more metabolically stressed mice. When 94  obese, hyperinsulinemic ob/ob mice lacking functional liver leptin signalling were treated with leptin, lipid tolerance was not improved to the same extent as in mice with functional hepatic leptin receptors (Figure 39A). Interestingly, the effects of leptin on lipid tolerance seemed to persist even after leptin therapy was ceased, indicating again that leptin treatment in ob/ob mice has long-term effects on lipid metabolism (Figure 39B-C). Lipid tolerance was also assessed in db/db mice treated with Ad-Lepr-b or Ad-β-gal. Although adenovirus treatment itself seemed to worsen lipid tolerance in both groups of db/db mice (Figure 39D-E), likely due to an immune response [163], lipid tolerance in the mice that received the Ad-Lepr-b virus was improved compared to those mice that received the Ad-β-gal virus (Figure 39D-E). Thus, these data suggest that lipid metabolism is differentially affected by a loss of hepatic leptin signalling in lean mice compared to hepatic leptin signalling in obese, hyperinsulinemic mice. Collectively, the data reveal that in metabolically stressed ob/ob and db/db mice, hepatic leptin signalling has a positive effect on promoting triglyceride clearance.  95  Figure 39. Hepatic leptin signalling acutely improves lipid tolerance in ob/ob and db/db mice. (A-C) Seven-week-old male Leprflox/flox AlbCre ob/ob mice were treated with 0.6 µg/day mouse recombinant leptin for 28 days via mini-osmotic pump starting on day 1. Oral lipid tolerance tests were performed by fasting the mice for 4 hours and then administering an oral gavage of 5 µl/g olive oil. Mice were subjected to an oral lipid tolerance test after 7 days of leptin treatment (A), 9 days post pump removal (B), and 75 days post pump removal (C), n≥5. (D-E) Male db/db mice were subjected to an oral lipid tolerance test (D) 9 days prior to and (E) 7 days after treatment with either Ad-β-gal or Ad-Lepr-b, n≥6. Data are expressed as mean±SEM. P values were determined by two-way ANOVA with a Holm-Sidak post-hoc test.  6.3 Discussion It was previously shown in Chapter 5 that lean mice lacking liver leptin signalling had a subtle increase in VLDL triglycerides that was only observable after FPLC fractionation of plasma. However, when hepatic leptin signalling was investigated under conditions of metabolic stress, it was found that low dose leptin-treated ob/ob mice that lacked hepatic leptin signalling did have an observable elevation in fasting triglycerides even when measured in whole plasma. 96  Accordingly, when db/db mice had leptin signalling restored to the liver, they had an improvement in fasting plasma triglycerides compared to db/db controls. In addition, postprandial triglyceride clearance in lean mice lacking hepatic leptin signalling was normal, but when lipid tolerance was measured in leptin-treated ob/ob mice lacking hepatic leptin signalling, there was a marked worsening of lipid tolerance compared to leptin-treated ob/ob controls. Similarly, in db/db mice with restored hepatic leptin signalling, there was an improvement in lipid tolerance compared to db/db controls. Interestingly, the effects of leptin on promoting triglyceride clearance may also contribute to the elevated fasting plasma triglycerides seen in leptin-treated ob/ob mice lacking liver leptin signalling. Thus, in obese, hyperinsulinemic mice, hepatic leptin signalling plays a more pronounced role in regulating plasma triglyceride levels than in lean mice. Unlike the role of hepatic leptin signalling in glucose metabolism, the control of lipid metabolism by hepatic leptin signalling seems to be more straightforward. Similar to its effects in lean mice, hepatic leptin signalling in obese, hyperinsulinemic mice was observed to be a positive regulator of hepatic apoB levels and hepatic lipase activity in the liver and a negative regulator of hepatic lipoprotein lipase activity. This was demonstrated in lean Leprflox/flox AlbCre+ mice with normal plasma leptin levels as well as obese db/db mice with a transient restoration of hepatic leptin signalling, which had extremely high leptin levels. Thus, the regulation of hepatic apoB and hepatic lipase activity by leptin was independent of leptin dose and metabolic status. It seems that the evidence overwhelmingly supports a role for hepatic leptin signalling in decreasing lipid storage. It has already been shown that leptin has potent effects on decreasing hepatic lipid storage through effects on increasing β-oxidation [7, 98, 134, 193]. Furthermore, our data in Chapter 3 show that leptin has effects on inhibiting insulin signalling in the liver (Figure 15) [72], and this may also result in decreased lipogenic gene  97  expression, which is also antisteatotic. Furthermore, since tissues with an overexpression of LPL have increased lipid accumulation [180, 182], the results in this chapter suggest that another mechanism by which leptin reduces hepatic lipid accumulation is through suppression of LPL activity. Taken together, the data reveal a possible role for hepatic leptin resistance in the development of dyslipidemia. It appears that leptin resistance in lean mice results in only minor perturbations in hepatic triglyceride and VLDL triglyceride levels. However, these perturbations become more pronounced under conditions of metabolic stress such as obesity and hyperinsulinemia. Given that many obese humans are leptin resistant [197], the data suggest that defects in lipid metabolism seen in obese humans may stem in part from resistance to leptin action in the liver. Although the effects of liver leptin signalling on lipid metabolism appear subtle, our data show that these effects are more pronounced in obesity and hyperinsulinemia and these effects may be compounded over years of obesity. Intriguingly, polymorphisms in the LEPR [198], HL [199], and LPL [200] genes have been linked with familial combined hyperlipidemia, the most common genetically linked hyperlipidemia in humans. In fact, HL and LPL are among the genes most frequently reported to be associated with familial combined hyperlipidemia [175]. Thus, alterations to HL and LPL activity in the liver due to hepatic leptin resistance may result in increased risk of dyslipidemia and perhaps contribute to the development of metabolic syndrome.  98  CHAPTER 7 – THE ROLE OF HEPATIC LEPTIN SIGNALLING IN THE REGULATION OF PLASMA IGFBP-2 7.1 Introduction There are 7 insulin-like growth factor binding proteins (IGFBPs) that circulate and bind insulin-like growth factors (IGFs) to modulate their functions [201-203]. It has been shown that IGFBPs can regulate the half-life of IGFs, sequester IGFs from IGF receptors and thereby inhibit IGF activity, or even facilitate IGF binding to its receptors to enhance activity [203]. It seems that the exact role of IGFBP binding to IGFs is tissue-specific and depends on which IGFBP is bound [203]. However, it has recently been shown that IGFBPs have functions independent of binding to IGFs and these actions may be mediated through direct binding of IGFBPs to cell surface receptors [80, 204-206]. The IGF-independent effects of IGFBP-2 in particular are gaining interest since it appears that IGFBP-2 has direct effects on modulating glucose metabolism. Interestingly, mice over-expressing IGFBP-2 under the CMV promoter had mildly reduced post-natal body weight [207] and IGFBP-2 knockout mice had slightly increased body weight and body fat mass [208]. Moreover, mice over-expressing IGFBP-2 under its native promoter were protected from high fat diet-induced obesity and glucose intolerance and had increased insulin sensitivity [209], while IGFBP-2 knockout mice on a chow diet showed no alterations in glucose tolerance [208]. Furthermore, in humans, there is an association between obesity and insulin resistance with low levels of IGFBP-2 [210]. Overall, there appears to be an inverse relationship between IGFBP-2 and obesity-related insulin resistance, but much is still unknown about the exact nature of this relationship. Using an unbiased gene array approach, in which leptin-treated ob/ob [80] or lipodystrophic [82] mice were compared to their respective saline-treated controls, it was 99  discovered that leptin, a powerful regulator of body weight and glucose metabolism, is also a potent regulator of hepatic IGFBP-2 mRNA levels [80, 82]. Leptin was shown to increase hepatic transcription of IGFBP-2 when administered either peripherally or centrally to lipodystrophic mice [82]. As well, administration of leptin to ob/ob mice at low doses that did not decrease body weight also potently upregulated hepatic IGFBP-2 transcript levels. Interestingly, it was recently demonstrated that peripheral administration of leptin to ob/ob mice potently upregulates both hepatic IGFBP-2 mRNA and circulating IGFBP-2 levels [80]. Moreover, Hedbacker et al. [80] showed that administration of an IGFBP-2 expressing adenovirus to ob/ob mice had similar anti-diabetic effects as leptin in various mouse models of insulin resistance and insulin-deficient diabetes and these effects were independent of body weight loss. Interestingly, in a small cohort of leptin-deficient people, it was observed that these patients had about 50% lower levels of plasma IGFBP-2 than controls and these levels were increased after 6 months of leptin treatment [80]. These results indicate that leptin is involved in regulating circulating IGFBP-2 levels and that IGFBP-2 may play an intricate role in mediating the effects of leptin on metabolism. However, the exact site and mechanism by which leptin regulates IGFBP-2 production is still unclear. The liver, which expresses the long, signalling isoform of the leptin receptor [46, 121, 122], may be an important site for leptin action on increasing plasma IGFBP-2. It has been shown that increased hepatic IGFBP-2 mRNA is associated with increased circulating IGFBP-2 [80, 211, 212], suggesting that the liver is a major source of plasma IGFBP-2. Further, subcutaneous administration of leptin to the periphery is able to increase both liver IGFBP-2 mRNA [80, 82] and plasma IGFBP-2 levels [80]. Thus, leptin may act directly on the liver to increase expression and secretion of IGFBP-2. It is hypothesized that leptin may increase hepatic IGFBP-2 expression by acting directly on leptin receptors in the liver. To address this  100  hypothesis, plasma IGFBP-2 levels were measured in Leprflox/flox AlbCre mice, leptin-treated Leprflox/flox AlbCre ob/ob mice, and db/db mice with a replacement of leptin signalling in the liver. The results of these studies are presented in this chapter and these results have been published as part of a larger study published in Diabetologia [213].  7.2 Results Hepatic leptin signalling is not required for leptin to increase plasma IGFBP-2 levels in ob/ob mice Ob/ob mice have about ten times lower plasma IGFBP-2 levels than wildtype controls and it has been shown that leptin treatment to ob/ob mice can increase both hepatic and plasma IGFBP-2 levels in a dose-dependent manner [80, 213]. In order to determine whether direct leptin action on hepatic leptin receptors is the mechanism by which leptin upregulates plasma IGFBP-2, Leprflox/flox AlbCre+ ob/ob mice were treated with leptin to see if leptin can still increase plasma IGFBP-2 levels in ob/ob mice lacking functional liver leptin signalling. Female Leprflox/flox AlbCre+ ob/ob and Leprflox/flox AlbCre- ob/ob littermate controls were administered leptin (5 µg/day) for 2 weeks via mini-osmotic pumps. IGFBP-2 levels started to increase 2 days into leptin treatment and steadily increased until leptin therapy was ceased (Figures 40A-B). Surprisingly, circulating leptin levels during leptin treatment were higher in the ob/ob mice lacking hepatic leptin receptor signalling domains compared to littermate ob/ob controls with full-length hepatic leptin receptors. This suggests that the leptin receptor signalling domain may play a role in leptin clearance by the liver. Despite this difference in plasma leptin levels, it is clear that leptin delivery via osmotic pump still resulted in increased plasma leptin levels above pre-treatment levels in both the Leprflox/flox AlbCre+ ob/ob and Leprflox/flox AlbCreob/ob mice and by the last day of leptin treatment, plasma IGFBP-2 levels had risen by nearly ten-fold above pre-leptin levels in both groups of mice (Figure 40B). Further, in the Leprflox/flox 101  AlbCre+ ob/ob mice, the higher leptin levels resulted in significantly higher plasma IGFBP-2 levels than Leprflox/flox AlbCre- ob/ob mice at some time points. Even at a lower dose of leptin for a shorter duration, plasma IGFBP-2 levels still rose to the same extent in ob/ob mice with and without hepatic leptin signalling (Figure 40C). Thus, functional hepatic leptin receptors are not required for leptin to increase plasma IGFBP-2 levels in ob/ob mice.  Figure 40. Leptin increases plasma IGFBP-2 levels in ob/ob mice independent of hepatic leptin signalling. Female Leprflox/flox AlbCre ob/ob mice were treated with a continuous leptin infusion (5 μg/day) via mini-osmotic pumps for 14 days. Pumps were implanted subcutaneously on day 0 following a 4 hour fast. (A) Plasma leptin and (B) plasma IGFBP-2 levels were measured following a 4 hour fast, n≥5. (C) Male Leprflox/flox AlbCre ob/ob mice were treated with a continuous leptin infusion (2 μg/day) via mini-osmotic pumps for 7 days and plasma IGFBP-2 levels were measured following a 4 hour fast, n≥3. All data are expressed as mean±SEM. *P≤0.05 compared to littermate controls by Student’s t-test. Panels A and B were partially contributed by J. Levi and are also published in her Master’s thesis.  Lean mice lacking hepatic leptin signalling have normal plasma IGFBP-2 Figure 40A shows that a continuous leptin infusion of 5 μg/day via mini-osmotic pumps for 14 days led to supraphysiological plasma leptin levels in Leprflox/flox AlbCre+ ob/ob mice. Thus, it is possible that with a lack of hepatic leptin signalling in ob/ob mice, supraphysiological levels of leptin are required to achieve normal plasma IGFBP-2 levels. To eliminate this possibility, plasma IGFBP-2 levels were measured in Leprflox/flox AlbCre+ mice, which have normal physiological leptin levels [72]. We found that at both 7 and 17 weeks of age, Leprflox/flox 102  AlbCre+ mice had the same levels of plasma IGFBP-2 as their Leprflox/flox AlbCre- littermate controls (Figure 41A). Furthermore, it is clear that the loss of hepatic leptin signalling did not affect other aspects of the growth hormone-IGF-1 axis either (Figure 41B-C). Therefore, even at physiological levels of leptin, hepatic leptin signalling is not required for maintaining normal plasma IGFBP-2 levels. Taken together, our data clearly demonstrate that regardless of how much leptin is circulating in the plasma, hepatic leptin signalling is not required for leptin to increase plasma IGFBP-2 levels.  Figure 41. Lean mice lacking hepatic leptin signalling have normal plasma IGFBP-2 levels. (A) Plasma IGFBP-2 levels were measured in 7 and 17 week old Leprflox/flox AlbCre male mice after a 4 hour fast, n≥8. (B) Levels of plasma IGF-1 were measured in 8 and 17 week old Leprflox/flox AlbCre male mice after a 4 hour fast, n≥8. (C) Plasma growth hormone levels were measured in 22 week old Leprflox/flox AlbCre male mice after a 4 hour fast, n≥5. Data are mean±SEM. P values were determined by Student’s t-test. Re-expression of functional leptin receptors in the liver db/db mice is not sufficient for restoring normal IGFBP-2 levels It is possible that in our models of mice lacking hepatic leptin signalling, the Lepr∆17 protein, which retains the extracellular and transmembrane domains of the leptin receptor, is still able to respond to leptin and mediate hepatic IGFBP-2 expression through a STAT3-independent pathway. Therefore, a different mouse model that did not depend on the Lepr∆17 allele was used to test whether hepatic leptin signalling is involved in leptin-mediated increases in plasma  103  IGFBP-2. Db/db mice, which have a whole body loss of leptin signalling, were obtained and treated with an adenovirus expressing the long, signalling isoform of the leptin receptor (AdLepr-b) via the tail vein, which confers liver-selective expression of the adenoviral construct [160]. Using this method, it was determined whether restoring leptin signalling in the liver of db/db mice could increase hepatic or plasma IGFBP-2 levels. Hepatic IGFBP-2 mRNA levels were measured in db/db mice treated with Ad-β-gal and compared to levels in C57BL/6 controls. It was found that indeed when whole body leptin signalling was absent, hepatic IGFBP-2 transcript levels were <4% of C57BL/6 controls (Figure 42A). Further, before virus treatment, both groups of db/db mice had plasma IGFBP-2 levels that were comparable to the low levels seen in ob/ob mice [80, 213] (Figure 40). Interestingly, even when hepatic leptin signalling was restored in db/db mice, neither hepatic transcript nor plasma IGFBP-2 levels rose (Figures 42A-B). These data clearly show that hepatic leptin signalling alone is not sufficient for mediating the effects of leptin in increasing hepatic or plasma IGFBP-2.  104  Figure 42. Hepatic leptin signalling is not sufficient for restoring normal IGFBP-2 levels in db/db mice. Liver tissue was collected 26 days post-infection from db/db mice treated with an adenovirus expressing either β-galactosidase or the long signalling isoform of the leptin receptor, Lepr-b. (A) Hepatic IGFBP-2 transcript levels were measured by quantitative PCR, n≥5. (B) Plasma IGFBP-2 levels were measured following virus treatment, n=6. All data are shown as mean±SEM. NS, not significantly different by Student’s t-test.  7.3 Discussion The best established effects of leptin are its effects on reducing body weight by decreasing food intake and increasing energy expenditure. However, even in one of the first studies showing the effects of leptin in ob/ob mice, it was seen that at doses too low to affect body weight, leptin could still reverse hyperglycemia [30]. Further, even in mouse models of type 1 diabetes, leptin can reverse hyperglycemia independent of its effects on body weight and food intake [105, 214, 215]. Thus, one of the most potent effects of leptin in mice is actually on lowering glucose levels rather than on reducing body weight. Because of its anti-diabetic potential, it is of great interest to uncover the mechanism by which leptin potently reverses hyperglycemia even in the absence of weight loss. It has been suggested that plasma IGFBP-2 may play a role in mediating the effects of leptin on glucose metabolism [80]. Leptin treatment has been shown to significantly increase 105  circulating IGFBP-2 levels in ob/ob mice and remarkably, IGFBP-2 administration via adenovirus was able to ameliorate diabetes in leptin-deficient ob/ob mice [80]. Work from our laboratory showed that even at the lowest dose of leptin used (0.2 µg/day), which had effects on glucose homeostasis but not body weight, leptin was able to significantly increase IGFBP-2 levels compared to PBS-treated mice [213]. The effect of leptin on circulating levels of IGFBP2 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, wildtype mice [213]. These data further establish leptin as a potent regulator of plasma IGFBP-2 levels. However, the mechanism by which leptin increases plasma IGFBP-2 levels is still unknown. It was hypothesized that the liver may be a site mediating the effects of leptin on increasing plasma IGFBP-2 levels [80, 82]. However, leptin was able to increase plasma IGFBP-2 levels in ob/ob mice lacking hepatic leptin signalling, indicating that hepatic leptin signalling is not required for leptin to increase levels of plasma IGFBP-2. Furthermore, when functional hepatic leptin receptors were re-expressed in hyperleptinemic db/db mice, which have low plasma IGFBP-2 levels, neither hepatic nor plasma IGFBP-2 levels rose towards wildtype levels. In addition, hepatic IGFBP-2 mRNA levels were not shown to be differentially regulated by leptin in any of our gene array studies comparing Leprflox/flox AlbCre or leptin-treated Leprflox/flox AlbCre ob/ob mice (Tables A1-A3). Taken together, the data indicate that direct leptin action on the liver is neither required nor sufficient for mediating the effects of leptin on plasma IGFBP-2. It is noteworthy that there were some time points where Leprflox/flox AlbCre+ ob/ob mice treated with 5 µg/day leptin had significantly higher IGFBP-2 levels compared to controls, perhaps a reflection of the higher circulating leptin levels during leptin treatment. This suggests that the hepatic leptin receptor signalling domain may be involved in leptin clearance, resulting 106  in the Leprflox/flox AlbCre+ ob/ob mice having higher leptin levels during leptin treatment. Even though the kidney is reported to be the major site of leptin clearance [216, 217], it is possible that at high levels of plasma leptin, such as those seen during continuous exogenous delivery of 5 µg/day leptin, the liver may be an important contributor to leptin clearance. Nonetheless, in both the Leprflox/flox AlbCre+ ob/ob mice and Leprflox/flox AlbCre- ob/ob controls treated with 5 µg/day leptin, leptin levels rose well above pre-treatment levels and on the last day of leptin treatment, IGFBP-2 levels were nearly ten-fold higher than pre-leptin infusion levels in both groups. In addition, Leprflox/flox AlbCre+ mice which had normal plasma leptin levels [72], also had normal plasma IGFBP-2 levels. Even hyperleptinemic db/db mice with functional hepatic leptin receptors did not have altered IGFBP-2 levels compared to control db/db mice. Overall, our data show that regardless of plasma leptin levels, direct action on the liver is not required for leptin to increase plasma IGFBP-2. The results of our study eliminate direct action of leptin on hepatocytes as the sole mediator of leptin-induced increases in plasma IGFBP-2, but other possible mechanisms remain to be tested. Intriguingly, central intracerebroventricular (ICV) doses of leptin that do not increase peripheral leptin levels are also able to increase hepatic IGFBP-2 transcription [82]. Therefore, leptin may act on the brain to indirectly increase hepatic and plasma IGFBP-2 levels. However, work from our laboratory has shown that when subdiaphragmatic parasympathetic vagal efferents were severed in ob/ob mice, leptin was still able to increase plasma IGFBP-2 levels [213] though the possibility remains that central leptin action can affect hepatic production and secretion of IGFBP-2 via sympathetic inputs to the liver which remained intact in these mice. In addition, it is important to consider that while the liver is believed to be the major source of circulating IGFBP-2, other tissues may also contribute to plasma IGFBP-2 levels and these other tissues may play a bigger role when direct or indirect hepatic stimulation by leptin is  107  absent. In fact, IGFBP-2 expression has been detected in several tissues, including kidney [218, 219], adrenal gland [220], brain [218, 221], and adipose [222, 223], all of which are known to secrete hormones and express the long, signalling isoform of the leptin receptor [46, 224, 225]. Visceral white adipose tissue in particular has recently been shown to express IGFBP-2 mRNA in levels that correlate with plasma IGFBP-2 levels and in the absence of leptin (ob/ob mice) or leptin signalling (db/db mice), IGFBP-2 mRNA expression in visceral white adipose is reduced [223]. Another possibility is that leptin may indirectly increase hepatic and plasma IGFBP-2 by modulating levels of other hormones that in turn act on the liver to increase IGFBP-2 levels. Thus, while the most obvious mechanism by which leptin might regulate hepatic and plasma IGFBP-2 has been eliminated, there are other possibilities that remain to be addressed. Considering the remarkable effects of leptin and IGFBP-2 on ameliorating diabetes, more research should be done to better understand the regulation of plasma IGFBP-2 levels by leptin.  108  CHAPTER 8 - CONCLUSIONS When leptin was first discovered in 1994, there were high hopes that it would be the cure for obesity. However, it was quickly discovered that most obese humans are not leptin deficient and actually require inordinately high amounts of exogenous leptin to cause very moderate amounts of weight loss [226, 227]. Thus, it was concluded that most obese humans are resistant to the effects of leptin on weight loss. Much focus has been on the central mechanisms of leptin resistance since this is proposed as the cause of obesity in most people. Furthermore, leptin resistance in the brain can affect glucose and lipid metabolism in the periphery, which may lead to the metabolic abnormalities that are associated with obesity [61, 88, 137, 228]. However, it is undeniable that leptin receptors, including the long signalling Lepr-b isoform, are present in the periphery and this highly suggests that leptin has direct effects on peripheral organs. Indeed, resistance to leptin action is also seen in peripheral tissues during obesity [55-57] and this may also contribute to abnormal glucose and lipid metabolism. Work from our laboratory and others has shown that one function of leptin in the periphery is to limit the potent effects of hyperinsulinemia. Leptin has a well-described role acting on pancreatic β-cells to inhibit insulin secretion [81, 93, 108-110] and thus reducing the overall influence of insulin on metabolism. Furthermore, leptin impairs the actions of insulin signalling in adipocytes [116, 117], resulting in decreased insulin-induced glucose uptake [116], decreased lipogenesis [229, 230], and increased lipolysis [229]. What this thesis now shows is that in vivo, leptin also acts on the liver to inhibit some of the actions of insulin. The proposed functions of leptin in the liver of lean mice are outlined in Figure 43. Given the powerful effects of insulin, the inhibitory function of leptin on insulin-mediated suppression of hepatic glucose production may be protective against life-threatening hypoglycemia. Importantly, however, by inhibiting insulin action, leptin can also inhibit insulin-mediated lipogenesis, leading to 109  decreased lipid storage. Furthermore, it is now shown that leptin can also weakly increase HL activity but potently suppress LPL activity in lean mice. This suppression of LPL likely also contributes to the effect of leptin on decreasing lipid accumulation in the liver since overexpression of LPL in the liver leads to increased hepatic lipid accumulation [180, 182]. In addition, it was shown that leptin also has effects on promoting apoB expression, which can lead to increased export of VLDL. This also can contribute to decreasing lipid accumulation in the liver. Thus, the data overwhelmingly suggest that under normal physiological circumstances, the major function of hepatic leptin signalling is to decrease lipid accumulation in the liver.  Figure 43. Hepatic leptin signalling in lean mice. Leptin signalling in lean mice inhibits insulin action and reduces hepatic lipid accumulation. Green and red arrows indicate a net increase or decrease respectively. LPL, lipoprotein lipase; HL, hepatic lipase; HGP, hepatic glucose production; apoB, apolipoprotein B; VLDL, very low density lipoprotein.  110  The fundamental focus of this thesis was to determine whether leptin action on the liver is able to mechanistically link obesity with insulin resistance and type 2 diabetes. The data suggest that it is very possible that hepatic leptin signalling plays a role in this process. In the early stages of obesity, when leptin levels begin to rise above normal levels, the increased leptin may act on the liver to induce hepatic insulin resistance since leptin antagonizes hepatic insulin action. From what is known about liver-specific insulin receptor knockout (LIRKO) mice, hepatic insulin resistance alone is enough to induce hyperinsulinemia [231]. As hyperinsulinemia ensues, this can potentiate the development of obesity through insulinmediated lipogenesis in adipose tissue, resulting in even higher leptin levels and eventually leptin resistance (Figure 44). As we have seen in our mice with obesity, hyperinsulinemia, and hepatic leptin resistance, this can lead to dire consequences. Hyperinsulinemia can strongly induce resistance to the glucometabolic effects of insulin, resulting in enhanced hepatic glucose production even when the insulin antagonizing effects of leptin are lost [232, 233]. Interestingly, however, in models of type 2 diabetes, there is in fact selective insulin resistance in the liver, whereby the liver is resistant to the glucometabolic effects of insulin but more sensitive to the lipometabolic effects of insulin [12-14]. This results in increased hepatic lipogenesis and decreased apoB, which can both lead to increased lipid accumulation. The data in this thesis suggest that with hepatic leptin resistance, the lipogenic actions of insulin are even further increased due to the loss of leptin inhibition of insulin (Figure 44). Of note, it is still unknown how hyperinsulinemia in obesity and type 2 diabetes confers resistance to glucometabolic actions of insulin but not lipometabolic actions. Intriguingly, our data suggest that perhaps hepatic leptin resistance contributes to this selective insulin resistance by enhancing insulin actions on lipid accumulation. Indeed, LIRKO mice, which have total hepatic insulin resistance but normal leptin sensitivity [234], do not have hepatic triglyceride accumulation nor hypertriglyceridemia  111  [192]. Thus, it is possible that hepatic leptin resistance contributes to selective insulin resistance in obese, diabetic states, resulting in increased triglycerides in the liver and in plasma. In addition to the insulin-enhancing effects of leptin resistance in the liver during obesity and hyperinsulinemia, enhanced LPL activity was also observed, which can also lead to increased lipid accumulation in the liver (Figure 44). Moreover, it was observed that in the lack of hepatic leptin signalling, there was increased triglyceride incorporation into VLDL particles and in the obese state, lack of leptin signalling in the liver led to impaired triglyceride clearance. It is speculated that these are protective mechanisms against further increases in hepatic lipid accumulation. However, the downside of this protection is the resulting hypertriglyceridemia, which is detrimental to other parts of the body. Collectively, the data show that the loss of hepatic leptin signalling due to obesity may contribute to the development of hyperinsulinemia, selective insulin resistance, and hypertriglyceridemia seen in type 2 diabetes (Figures 43-44).  112  Figure 44. Hepatic leptin resistance in obesity and type 2 diabetes. Under obese, hyperinsulinemic conditions, loss of hepatic leptin signalling potentiates the lipogenic actions of insulin. Green and red arrows indicate a net increase or decrease respectively. TG, triglyceride; LPL, lipoprotein lipase; HL, hepatic lipase; HGP, hepatic glucose production; apoB, apolipoprotein B; VLDL, very low density lipoprotein.  Current treatments for obese type 2 diabetic patients include drugs that increase insulin sensitivity or even using insulin itself to try and overcome insulin resistance. However, given the selective insulin resistance in type 2 diabetics, this may not be the best strategy since enhanced insulin action can lead to increased lipogenesis resulting in weight gain, hepatic steatosis, and hypertriglyceridemia. Perhaps the best way to treat obese diabetic patients is to restore insulin action but also restore leptin signalling so that the effects of insulin are kept in check. In the liver, restoration of insulin action alone would reduce hepatic glucose output but 113  would likely increase lipid accumulation and lipotoxicity [235]. Restoration of leptin signalling alone would improve hepatic steatosis and hypertriglyceridemia, but our data show that this does not improve glucose parameters. Thus, the current clinical use of insulin to treat severe type 2 diabetes may be one-sided and future research into the ideal treatment for obesity-associated type 2 diabetes should be focused on the restoration of both insulin and leptin action. Future studies Although leptin is being used clinically to treat conditions of leptin deficiency with good success [38, 227], the relative few patients with leptin deficiency means that these studies have been done on small sample sizes. Thus, before leptin or leptin sensitizing drugs can be used clinically to treat common obesity and type 2 diabetes, much more work needs to be done to define the role of leptin under these disease states. Furthermore, it is especially important to investigate the actions of leptin in a tissue-specific manner since leptin has advantageous effects on decreasing lipid accumulation in metabolically active organs such as skeletal muscle and liver but has potentially detrimental effects on other tissues. Leptin can promote atherosclerosis by causing accumulation of cholesterol esters in foam cells [236] and increased aggregation of platelets [237]. Also, leptin has been shown to promote development of cancer in several tissues including breast [238] and prostate [239]. Therefore, it is essential that the role of leptin on specific tissues be further investigated before leptin is used clinically to treat common obesity and type 2 diabetes. Work from this thesis has helped to define the role of leptin signalling in the liver in terms of its effects on glucose and lipid metabolism, but many questions still remain. The data generated from the gene array studies have provided several new avenues that can be pursued. For example, the gene array showed that Leprflox/flox AlbCre+ mice have much higher hepatic lipocalin-2 mRNA expression than their littermate controls. While this did not translate into 114  higher plasma lipocalin-2 levels (Figure A1), it does not rule out whether loss of hepatic leptin signalling affects lipocalin-2 levels in the liver without affecting lipocalin-2 secretion. Therefore, hepatic lipocalin-2 mRNA levels in a larger cohort of Leprflox/flox AlbCre mice should be verified by qPCR. Even with no difference in plasma lipocalin-2, it is possible that lipocalin2 levels specifically in the liver may affect liver function since treatment of cultured hepatocytes with lipocalin-2 results in insulin resistance [240]. Furthermore, hepatic lipocalin-2 expression is correlated with liver injury and inflammation [241] and thus, our preliminary data suggest that mice lacking hepatic leptin signalling may have liver damage and this should be investigated further. Previous gene array studies in which ob/ob mice were treated systemically with leptin have shown that hepatic expression of apolipoprotein A-IV (ApoA4) [133, 169], several genes encoding different glutathione S-transferases [87, 169], and acyl-Coenzyme A dehydrogenase, medium chain (Acadm, also known as Mcad) [133] is altered by leptin treatment. Our data now show that these genes may be regulated by direct action of leptin on the liver (Tables A1-A3). These genes are especially interesting since ApoA4, which was shown to be elevated in overnight fasted Leprflox/flox AlbCre+ mice compared to their littermate controls, is a circulating polypeptide produced by the intestinal mucosa and liver that has been shown to be able to inhibit food intake [242]. Glutathione S-transferases are involved in maintaining cellular redox homeostasis and it was hypothesized by Liang and Tall that regulation of glutathione S-transferases by leptin may allow for the elimination of reactive oxygen species, which can arise from increased fatty acid oxidation and oxidative phosphorylation [133]. Acadm is involved with the breakdown of fatty acids during fatty acid oxidation and loss of Acadm results in hepatic steatosis [243]. Interestingly, hepatic Acadm is increased by leptin treatment to ob/ob mice [133], possibly due to direct effects on the liver (Table A3). This suggests that leptin regulation of Acadm may be a  115  mechanism by which leptin limits lipid accumulation in the liver. Therefore, there are several interesting genes that could be investigated to further define the role of leptin action on the liver. Data from this thesis have demonstrated that the effects of leptin action on the liver depend highly on the dose of leptin, length of exposure to leptin, and metabolic state of the animal. The effects of hepatic leptin were investigated in lean mice with life-long loss of hepatic leptin signalling and in obese, hyperinsulinemic mice with a gain or loss of hepatic leptin signalling at various levels of plasma leptin. However, lean mice with a conditional loss of leptin signalling in the liver is another mouse model that could help further the understanding of hepatic leptin action. One advantage of this model that would make it distinct from the previous three models used is that these mice would develop normally with full leptin action in the liver. Therefore, compensatory mechanisms for lost hepatic leptin action that could arise through development would be absent in these mice. The tools to generate this mouse model are already available in the laboratory, namely our Leprflox/flox mice and adenovirus expressing Cre recombinase. However, our data show that the immune response to an adenovirus could affect glucose and lipid metabolism and perhaps a less immunogenic virus such as an adeno-associated virus could be used to deliver Cre recombinase to the liver. The data in this thesis regarding leptin regulation of IGFBP-2 could also be followed up on. Studies from this thesis as well as others in the laboratory have shown that neither hepatic leptin signalling nor subdiaphragmatic vagal efferents are required for leptin to increase hepatic mRNA and plasma levels of IGFBP-2 [213]. More studies could be done to investigate the exact mechanism by which leptin regulates IGFBP-2. For example, ob/ob mice could be treated with leptin and then samples harvested from several tissues that have been shown to express both leptin receptors and IGFBP-2, including kidney [218, 219], adrenal gland [220], brain [218, 221], and adipose tissue [222, 223]. Quantitative PCR could be performed on these samples to 116  determine if these tissues might also produce IGFBP-2 in response to leptin. While the liver is thought to be the main producer of plasma IGFBP-2, it is possible that these other tissues can also contribute to plasma IGFBP-2 levels. Furthermore, the importance of hepatic production of IGFBP-2 could be examined by knocking down IGFBP-2 expression in the liver of ob/ob mice and then treating them with leptin to see if leptin can still ameliorate diabetes in these mice. These studies will help reveal the mechanism and importance of leptin regulation of IGFBP-2. It is clear that many more avenues of study can be pursued as a result of findings in this thesis. Helping to define the pathways affected by leptin signalling in a tissue-specific manner will aid in the development of safer and targeted leptin-based therapeutics. It is hoped that these findings defining the role of leptin action in the liver will ultimately help in the fight against obesity and type 2 diabetes.  117  REFERENCES [1] Unwin N, Whiting D, Guariguata L, Ghyoot G, Gan D (eds) (2011) International Diabetes Federation Diabetes Atlas. 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(2007) The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance. Diabetes 56: 2533-2540 [241] Borkham-Kamphorst E, Drews F, Weiskirchen R (2011) Induction of lipocalin-2 expression in acute and chronic experimental liver injury moderated by pro-inflammatory cytokines interleukin-1beta through nuclear factor-kappaB activation. Liver international : official journal of the International Association for the Study of the Liver 31: 656-665 [242] Tso P, Liu M, Kalogeris TJ, Thomson AB (2001) The role of apolipoprotein A-IV in the regulation of food intake. Annu Rev Nutr 21: 231-254 [243] Tolwani RJ, Hamm DA, Tian L, et al. (2005) Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice. PLoS Genet 1: e23 138  APPENDIX A – GENE ARRAY DATA Table A1. Complete list of genes differentially expressed in overnight fasted Leprflox/flox AlbCre+ male mice compared to Leprflox/flox AlbCre- littermate controls.a  lipocalin 2  Differential Scoreb 338.70  1.35E-34  Fold Changed 12.35  Apoa4  apolipoprotein A-IV  338.70  1.35E-34  3.11  5033411D12Rik  RIKEN cDNA 5033411D12 gene  338.70  1.35E-34  2.62  Hpx  hemopexin  338.70  1.35E-34  1.82  Ifitm2  interferon induced transmembrane protein 2  338.70  1.35E-34  1.79  Cdk5rap3  CDK5 regulatory subunit associated protein 3  338.70  1.35E-34  1.37  Ccnd1  cyclin D1  163.50  7.82E-07  2.50  Gnat1  guanine nucleotide binding protein, alpha transducing 1  93.49  4.47E-10  2.65  Arhgef3  Rho guanine nucleotide exchange factor 3  92.92  5.11E-10  1.33  Egfr  epidermal growth factor receptor, transcript variant 1  82.31  1.10E-07  1.57  Cfi  complement component factor i  73.36  4.61E-08  1.27  Sep15  selenoprotein  71.05  7.85E-08  1.26  Slco2a1  solute carrier organic anion transporter family, member 2a1  67.60  1.74E-07  1.43  Inhbe  inhibin beta E  66.99  2.00E-07  1.34  Rfc2  replication factor C (activator 1) 2  65.96  2.54E-07  1.52  Egr1  early growth response 1  65.30  2.95E-07  1.88  Ptplad1  protein tyrosine phosphatase-like A domain containing 1  62.42  5.72E-07  1.25  Gene Symbol  Gene Name  Lcn2  P-valuec  139  Gene Symbol  Gene Name  Differential Scoreb  P-valuec  Fold Changed  Serpina3n  serine (or cysteine) peptidase inhibitor, clade A, member 3N  61.02  7.91E-07  1.58  Cpb2  carboxypeptidase B2 (plasma)  56.94  2.02E-06  1.36  Anxa2  annexin A2  56.69  2.14E-06  1.58  Qprt  quinolinate phosphoribosyltransferase  56.40  2.29E-06  1.36  Blvrb  biliverdin reductase B (flavin reductase (NADPH))  56.39  2.30E-06  1.21  Pigp  phosphatidylinositol glycan anchor biosynthesis, class P  55.83  2.61E-06  1.27  Itih3  inter-alpha trypsin inhibitor, heavy chain 3  49.81  1.04E-05  1.36  Orm2  orosomucoid 2  48.52  1.41E-05  3.28  Gdf15  growth differentiation factor 15  40.22  9.51E-05  1.77  C4bp  complement component 4 binding protein  36.71  0.000213  1.38  Arfgap2  ADP-ribosylation factor GTPase activating protein 2  36.40  0.000229  1.38  Drg1  developmentally regulated GTP binding protein 1  36.07  0.000247  1.21  Hdlbp  high density lipoprotein (HDL) binding protein  35.71  0.000268  1.34  Mt1  metallothionein 1  35.30  0.000295  2.14  Dynll1  dynein light chain LC8-type 1  31.92  0.000643  1.22  Osbpl9  oxysterol binding protein-like 9, transcript variant 2  31.92  0.000643  1.17  Morf4l2  mortality factor 4 like 2  31.36  0.000730  1.22  Zfhx3  zinc finger homeobox 3  31.29  0.000742  1.32  140  Gene Symbol  Gene Name  Differential Scoreb  P-valuec  Fold Changed  Psma7  proteasome (prosome, macropain) subunit, alpha type 7  31.22  0.000755  1.21  Use1  unconventional SNARE in the ER 1 homolog (S. cerevisiae)  31.03  0.000788  1.29  Xbp1  X-box binding protein 1  30.87  0.000819  1.31  Mcfd2  multiple coagulation factor deficiency 2  30.72  0.000848  1.22  Nup210  nucleoporin 210  27.66  0.001713  1.58  Hc  hemolytic complement  27.66  0.001713  1.49  Lasp1  LIM and SH3 protein 1  27.54  0.001763  1.38  Maged1  melanoma antigen, family D, 1  27.54  0.001763  1.37  Cxcl14  chemokine (C-X-C motif) ligand 14  27.11  0.001945  1.60  Pim3  proviral integration site 3  27.11  0.001945  1.23  Mrpl13  mitochondrial ribosomal protein L13  27.11  0.001945  1.17  Mrps33  mitochondrial ribosomal protein S33, nuclear gene encoding mitochondrial protein, transcript variant 2  26.16  0.002418  1.16  Mmd2  monocyte to macrophage differentiation-associated 2  26.13  0.002435  1.47  Wbp5  WW domain binding protein 5  24.97  0.003186  1.35  Rab18  RAB18, member RAS oncogene family  24.97  0.003186  1.28  Mrps31  mitochondrial ribosomal protein S31, nuclear gene encoding mitochondrial protein  24.91  0.003230  1.22  Yif1a  Yip1 interacting factor homolog A (S. cerevisiae)  24.51  0.003542  1.22  Acta2  actin, alpha 2, smooth muscle, aorta  23.96  0.004022  1.47  Lsr  lipolysis stimulated lipoprotein receptor  23.50  0.004463  1.37  Krtcap2  keratinocyte associated protein 2  22.87  0.005165  1.15 141  nicotinamide N-methyltransferase  Differential Scoreb 22.03  0.006268  Fold Changed 1.19  Bgn  biglycan  21.79  0.006623  1.71  Mbc2  membrane bound C2 domain containing protein  21.55  0.006996  1.57  Nap1l1  nucleosome assembly protein 1-like 1  21.49  0.007090  1.25  Psma5  proteasome (prosome, macropain) subunit, alpha type 5  21.49  0.007090  1.21  Sec16a  SEC16 homolog A (S. cerevisiae)  21.13  0.007709  1.38  Nmt1  N-myristoyltransferase 1  20.87  0.008193  1.25  Prdx3  peroxiredoxin 3  20.86  0.008199  1.27  Lum  lumican  20.76  0.008388  1.40  Cygb  cytoglobin  20.02  0.009946  1.57  Angptl3  angiopoietin-like 3  -19.58  0.011019  -1.43  Kif1b  kinesin family member 1B  -19.82  0.010414  -1.24  Ttpa  tocopherol (alpha) transfer protein  -19.98  0.010049  -1.16  Grcc10  gene rich cluster, C10 gene  -20.01  0.009985  -1.39  Ndufb9  NADH dehydrogenase (ubiquinone) 1 beta subcomplex 9  -20.57  0.008765  -1.53  Ddah1  dimethylarginine dimethylaminohydrolase 1  -20.70  0.008504  -1.58  Ndufa7  NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 7 (B14.5a)  -20.86  0.008199  -1.15  C8g  complement component 8, gamma polypeptide  -20.87  0.008193  -1.20  Dio1  deiodinase, iodothyronine, type I  -20.89  0.008139  -1.39  Acox1  acyl-Coenzyme A oxidase 1, palmitoyl  -20.90  0.008136  -1.22  Gene Symbol  Gene Name  Nnmt  P-valuec  142  Gene Symbol  Gene Name  Differential Scoreb  P-valuec  Fold Changed  Tmem134  transmembrane protein 134, transcript variant 1  -22.62  0.005473  -1.28  Clpx  caseinolytic peptidase X (E.coli), transcript variant 1  -22.72  0.005349  -2.10  Vkorc1  vitamin K epoxide reductase complex, subunit 1  -23.56  0.004406  -1.15  Psen2  presenilin 2  -24.09  0.003900  -1.20  Onecut2  one cut domain, family member 2  -24.74  0.003356  -1.65  Klb  klotho beta  -26.21  0.002394  -1.29  Prodh2  proline dehydrogenase (oxidase) 2  -27.27  0.001876  -1.25  EG13909  predicted gene, EG13909  -27.66  0.001713  -1.19  Pla2g6  phospholipase A2, group VI  -28.79  0.001322  -1.63  3300001P08Rik  RIKEN cDNA 3300001P08 gene  -31.03  0.000788  -1.21  Fxn  frataxin  -31.08  0.000780  -1.20  Prodh2  proline dehydrogenase (oxidase) 2  -31.80  0.000661  -1.37  Oprs1  opioid receptor, sigma 1  -32.49  0.000564  -1.26  Slc30a10  solute carrier family 30, member 10  -34.37  0.000365  -1.71  Ugcg  UDP-glucose ceramide glucosyltransferase  -37.62  0.000173  -1.43  Arhgap29  Rho GTPase activating protein 29  -38.19  0.000152  -1.26  Afmid  arylformamidase  -39.74  0.000106  -1.34  Cldn3  claudin 3  -39.77  0.000105  -1.32  Tfam  transcription factor A, mitochondrial nuclear gene encoding mitochondrial protein  -45.72  2.68E-05  -1.27  Gclc  glutamate-cysteine ligase, catalytic subunit  -49.19  1.20E-05  -1.55  143  Gene Symbol  Gene Name  Differential Scoreb  P-valuec  Fold Changed  Slc22a5  solute carrier family 22 (organic cation transporter), member 5  -49.78  1.05E-05  -1.28  BC004728  cDNA sequence BC004728  -51.51  7.06E-06  -1.24  Gsta4  glutathione S-transferase, alpha 4  -54.37  3.65E-06  -1.79  Pank1  pantothenate kinase 1, transcript variant 2  -56.68  2.15E-06  -1.22  Mtus1  mitochondrial tumor suppressor 1, nuclear gene encoding mitochondrial protein, transcript variant 4  -59.03  1.25E-06  -1.62  Cyp2d26  cytochrome P450, family 2, subfamily d, polypeptide 26  -64.95  3.20E-07  -1.23  Afm  afamin  -65.24  3.00E-07  -1.26  Slc25a16  solute carrier family 25 (mitochondrial carrier, Graves disease autoantigen), member 16, nuclear gene encoding mitochondrial protein  -65.24  3.00E-07  -1.27  Retsat  retinol saturase (all trans retinol 13,14 reductase)  -65.24  3.00E-07  -1.35  Hamp  hepcidin antimicrobial peptide  -65.26  2.98E-07  -1.50  A530050D06Rik  RIKEN cDNA A530050D06 gene  -81.61  6.91E-09  -1.42  Ppp1r10  protein phosphatase 1, regulatory subunit 10  -92.24  5.97E-10  -1.91  Akap1  A kinase (PRKA) anchor protein 1, nuclear gene encoding mitochondrial protein, transcript variant 2  -93.42  4.55E-10  -1.37  Nrp1  neuropilin 1  -93.91  4.06E-10  -1.36  Khk  ketohexokinase  -94.72  3.37E-10  -1.28  Socs2  suppressor of cytokine signalling 2  -132.04  6.25E-14  -3.65  Hhex  hematopoietically expressed homeobox  -141.66  6.83E-15  -2.96  144  aquaporin 8  Differential Scoreb -152.94  5.08E-16  Fold Changed -1.65  BC025446  cDNA sequence BC025446  -174.46  3.58E-18  -1.40  Rnf125  ring finger protein 125  -336.27  2.36E-34  -1.88  Gene Symbol  Gene Name  Aqp8  P-valuec  a  Two Leprflox/flox AlbCre+ and two Leprflox/flox AlbCre- males were fasted overnight and then liver tissue harvested into RNAlater®. RNA samples were then subjected to the Illumina BeadArray MouseRef-8 v2.0 Expression BeadChip according to the manufacturer’s instructions. b Differential Score=-10(sgn(IntensityCre+ - IntensityCre-))log10(p-value). Genes are arranged from highest to lowest differential score. c P-value cut-off was 0.01 d Fold change of Leprflox/flox AlbCre+ compared to Leprflox/flox AlbCre-  145  Table A2. Complete list of genes differentially expressed in overnight fasted/2-hour re-fed Leprflox/flox AlbCre+ male mice compared to Leprflox/flox AlbCre- littermate controls.a Differential Scorea  P-valueb  Fold Changec  coenzyme Q5 homolog, methyltransferase (yeast)  92.29  5.91E-10  1.63  Rbm13  RNA binding motif protein 13  74.16  3.84E-08  1.60  Mupcdh  mucin-like protocadherin  69.63  1.09E-07  1.52  Lcn2  lipocalin 2  67.71  1.69E-07  2.71  Rgs3  regulator of G-protein signalling 3, transcript variant 2  67.71  1.69E-07  1.85  Tubb5  tubulin, beta 5  61.22  7.55E-07  1.46  Itih4  inter alpha-trypsin inhibitor, heavy chain 4  56.36  2.31E-06  1.44  BC048546  cDNA sequence BC048546  48.68  1.36E-05  3.12  Pnpo  pyridoxine 5'-phosphate oxidase  44.85  3.27E-05  1.39  Lasp1  LIM and SH3 protein 1  40.67  8.57E-05  1.52  Actb  actin, beta, cytoplasmic  39.12  0.000122  1.36  Ttc39a  tetratricopeptide repeat domain 39A  38.25  0.000150  1.37  Apom  apolipoprotein M  37.20  0.000191  1.60  Pdlim1  PDZ and LIM domain 1 (elfin)  36.72  0.000213  1.44  Cct3  chaperonin containing Tcp1, subunit 3 (gamma)  35.91  0.000256  1.35  Fbxo6  F-box protein 6  34.73  0.000336  1.34  Hapln4  hyaluronan and proteoglycan link protein 4  31.80  0.000661  1.36  BC048546  cDNA sequence BC048546  30.89  0.000814  2.88  Cpb2  carboxypeptidase B2 (plasma)  29.07  0.001238  1.32  Mlkl  mixed lineage kinase domain-like  28.24  0.001498  1.43  Gene Symbol  Gene Name  Coq5  146  Gene Symbol  Gene Name  D9Wsu20e  transmembrane protein 30A  P-valueb  Differential Scorea 27.49  0.001781  Fold Changec 1.31  Smarcd2  SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 2  25.19  0.003024  1.36  Dnajc12  DnaJ (Hsp40) homolog, subfamily C, member 12  24.79  0.003319  1.34  Olfml1  olfactomedin-like 1  24.28  0.003730  1.30  Grn  granulin  23.82  0.004145  1.39  Cope  coatomer protein complex, subunit epsilon  23.27  0.004707  1.29  Chrd  chordin  22.93  0.005096  1.38  Atp6v1g1  ATPase, H+ transporting, lysosomal V1 subunit G1  21.83  0.006561  1.32  Midn  midnolin  21.83  0.006561  1.28  Dnajb2  DnaJ (Hsp40) homolog, subfamily B, member 2, transcript variant 2  21.46  0.007147  1.31  Acot1  acyl-CoA thioesterase 1  -19.62  0.010922  -1.27  Aes  amino-terminal enhancer of split  -20.28  0.009380  -1.38  BB128963  myotubularin related protein 10  -20.52  0.008879  -1.29  Slc25a10  solute carrier family 25 (mitochondrial carrier, dicarboxylate transporter), member 10, nuclear gene encoding mitochondrial protein  -20.93  0.008073  -1.27  Actr3  ARP3 actin-related protein 3 homolog (yeast)  -21.29  0.007428  -1.27  Ltbp4  latent transforming growth factor beta binding protein 4  -23.15  0.004838  -1.29  Kif1b  kinesin family member 1B, transcript variant 2  -23.58  0.004388  -1.30  147  Gene Symbol  Gene Name  BC038156  cDNA sequence BC038156  Peci  P-valueb  Differential Scorea -23.83  0.004145  Fold Changec -1.44  peroxisomal delta3, delta2-enoylCoenzyme A isomerase  -24.23  0.003776  -1.53  Arf3  ADP-ribosylation factor 3  -24.41  0.003626  -1.30  1810049H13Rik  RIKEN cDNA 1810049H13 gene  -25.14  0.003063  -1.29  Chkb  choline kinase beta  -25.19  0.003024  -1.29  Huwe1  HECT, UBA and WWE domain containing 1  -25.81  0.002626  -1.32  Tollip  toll interacting protein  -27.26  0.001879  -1.29  Atg3  autophagy-related 3 (yeast)  -27.60  0.001736  -1.29  Tank  TRAF family member-associated Nfkappa B activator  -28.76  0.001330  -1.32  Nr1d2  nuclear receptor subfamily 1, group D, member 2  -28.82  0.001313  -1.55  Sdhb  succinate dehydrogenase complex, subunit B, iron sulfur (Ip), nuclear gene encoding mitochondrial protein  -29.84  0.001038  -1.30  Rab9  RAB9, member RAS oncogene family  -29.94  0.001014  -1.36  Ids  iduronate 2-sulfatase, transcript variant 1  -30.85  0.000821  -1.32  Entpd5  ectonucleoside triphosphate diphosphohydrolase 5 , transcript variant 2  -30.89  0.000814  -1.48  D2Ertd391e  DNA segment, Chr 2, ERATO Doi 391, expressed  -31.79  0.000662  -1.37  Hint3  histidine triad nucleotide binding protein 3  -32.92  0.000510  -1.32  Aqp1  aquaporin 1  -34.10  0.000389  -1.32  Cldn2  claudin 2  -34.10  0.000389  -1.39  148  Gene Symbol  Gene Name  Txn1  thioredoxin 1  March6  P-valueb  Differential Scorea -34.73  0.000336  Fold Changec -1.32  membrane-associated ring finger (C3HC4) 6  -34.73  0.000336  -1.33  8430408G22Rik  RIKEN cDNA 8430408G22 gene  -34.73  0.000336  -1.90  Ugt2b34  UDP glucuronosyltransferase 2 family, polypeptide B34  -37.20  0.000191  -1.33  Spag9  sperm associated antigen 9, transcript variant 4  -39.32  0.000117  -1.62  Eif1a  eukaryotic translation initiation factor 1A  -39.87  0.000103  -1.35  Ugt2a3  UDP glucuronosyltransferase 2 family, polypeptide A3  -40.67  8.57E-05  -1.72  Cox6c  cytochrome c oxidase, subunit VIc  -40.71  8.49E-05  -1.35  Nfe2l1  nuclear factor, erythroid derived 2,-like 1  -40.97  8.00E-05  -1.36  Sdhb  succinate dehydrogenase complex, subunit B, iron sulfur (Ip), nuclear gene encoding mitochondrial protein  -42.18  6.06E-05  -1.36  F13b  coagulation factor XIII, beta subunit  -42.18  6.06E-05  -1.36  Aldh6a1  aldehyde dehydrogenase family 6, subfamily A1, nuclear gene encoding mitochondrial protein  -43.46  4.51E-05  -1.36  Acot4  acyl-CoA thioesterase 4  -61.13  7.70E-07  -1.91  Dcun1d4  DCN1, defective in cullin neddylation 1, domain containing 4 (S. cerevisiae)  -69.63  1.09E-07  -1.45  Cd74  CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated), transcript variant 2  -71.14  7.70E-08  -1.46  Slc25a33  solute carrier family 25, member 33  -74.16  3.84E-08  -1.66  149  Gene Symbol  Gene Name  Abcc3  ATP-binding cassette, sub-family C (CFTR/MRP), member 3  Differential Scorea  P-valueb  Fold Changec  -249.44  1.14E-25  -1.93  a  Two Leprflox/flox AlbCre+ and two Leprflox/flox AlbCre- males were fasted overnight and then refed for two hours. Liver tissue was then harvested into RNAlater®. RNA samples were subjected to the Illumina BeadArray MouseRef-8 v2.0 Expression BeadChip according to the manufacturer’s instructions. b Differential Score=-10(sgn(IntensityCre+ - IntensityCre-))log10(p-value). Genes are arranged from highest to lowest differential score. c P-value cut-off was 0.01 d Fold change of Leprflox/flox AlbCre+ compared to Leprflox/flox AlbCre-  150  Table A3. Complete list of downregulated genes in livers of random-fed leptin-treated Leprflox/flox AlbCre+ ob/ob mice compared to Leprflox/flox AlbCre- ob/ob littermate controls.a Gene Symbol  Gene Name  Gstt3  P-valuec  glutathione S-transferase, theta 3  Differential Scoreb -334.77  3.33E-34  Fold Changed -3.12  Apob  apolipoprotein B  -334.77  3.33E-34  -2.98  Cyp2c39  cytochrome P450, family 2, subfamily c, polypeptide 39  -334.77  3.33E-34  -2.98  Rnase4  ribonuclease, RNase A family 4, transcript variant 1  -334.77  3.33E-34  -2.82  Gsta3  glutathione S-transferase, alpha 3, transcript variant 2  -334.77  3.33E-34  -2.72  LOC673501  PREDICTED: hypothetical protein LOC673501  -334.77  3.33E-34  -2.52  Tegt  testis enhanced gene transcript  -334.77  3.33E-34  -2.39  Itih2  inter-alpha trypsin inhibitor, heavy chain 2  -334.77  3.33E-34  -2.23  Papss2  3'-phosphoadenosine 5'-phosphosulfate synthase 2  -334.77  3.33E-34  -1.92  Stard4  StAR-related lipid transfer (START) domain containing 4  -293.73  4.24E-30  -2.26  Cyp2a5  cytochrome P450, family 2, subfamily a, polypeptide 5  -264.98  3.18E-27  -2.24  Retsat  retinol saturase (all trans retinol 13,14 reductase)  -239.53  1.11E-24  -1.8  Ttpa  tocopherol (alpha) transfer protein  -208.13  1.54E-21  -2.03  LOC333331  PREDICTED: similar to medium-chain acyl-CoA dehydrogenase, misc RNA.  -180.95  8.04E-19  -1.93  C630022N07Rik  v-maf musculoaponeurotic fibrosarcoma oncogene family, protein G (avian)  -153.50  4.47E-16  -1.54  Cd1d1  CD1d1 antigen  -147.08  1.96E-15  -2.6  151  Gene Symbol  Gene Name  Gjb2  P-valuec  gap junction protein, beta 2  Differential Scoreb -143.94  4.04E-15  Fold Changed -1.76  mtDNA_COXII  cytochrome c oxidase subunit II  -141.77  6.66E-15  -2.03  2310051E17Rik  PREDICTED: RIKEN cDNA 2310051E17 gene  -141.77  6.66E-15  -1.84  Dhcr24  24-dehydrocholesterol reductase  -135.20  3.02E-14  -1.86  Acadm  acyl-Coenzyme A dehydrogenase, medium chain  -132.02  6.29E-14  -2.2  Cdo1  cysteine dioxygenase 1, cytosolic  -131.19  7.60E-14  -2.08  LOC100047937  PREDICTED: similar to Aldehyde dehydrogenase 1 family, member L1  -130.73  8.45E-14  -2.34  Agpat3  1-acylglycerol-3-phosphate Oacyltransferase 3  -130.35  9.23E-14  -1.74  Smoc1  SPARC related modular calcium binding 1  -122.85  5.18E-13  -2.43  1810055G02Rik  RIKEN cDNA 1810055G02 gene  -121.75  6.69E-13  -1.5  Ybx3  cold shock domain protein A  -116.58  2.20E-12  -2.33  Hsp90b1  heat shock protein 90, beta (Grp94), member 1  -110.47  8.98E-12  -2.48  Glud1  glutamate dehydrogenase 1  -108.52  1.41E-11  -1.83  Rhbdl2  rhomboid, veinlet-like 2 (Drosophila)  -108.16  1.53E-11  -1.58  Gns  glucosamine (N-acetyl)-6-sulfatase  -107.28  1.87E-11  -1.82  Idh2  isocitrate dehydrogenase 2 (NADP+), mitochondrial (Idh2), nuclear gene encoding mitochondrial protein  -105.96  2.53E-11  -1.93  Clec2d  C-type lectin domain family 2, member d  -105.08  3.11E-11  -1.9  LOC100041388  PREDICTED: similar to ORF1  -103.33  4.65E-11  -1.77  Serpind1  serine (or cysteine) peptidase inhibitor, clade D, member 1  -99.16  1.21E-10  -1.97  152  Gene Symbol  Gene Name  Dhcr24  P-valuec  24-dehydrocholesterol reductase  Differential Scoreb -98.83  1.31E-10  Fold Changed -2.16  Sord  sorbitol dehydrogenase  -98.83  1.31E-10  -2.07  Por  P450 (cytochrome) oxidoreductase  -98.27  1.49E-10  -1.54  Ghitm  growth hormone inducible transmembrane protein  -97.19  1.91E-10  -1.76  Mmd  monocyte to macrophage differentiation-associated  -94.99  3.17E-10  -2.24  Slco1b2  solute carrier organic anion transporter family, member 1b2, transcript variant 1  -90.31  9.30E-10  -2.73  Cp  ceruloplasmin, transcript variant 2  -89.65  1.08E-09  -2.62  Dbt  dihydrolipoamide branched chain transacylase E2  -85.49  2.82E-09  -1.51  BC044804  fatty acid desaturase domain family, member 6  -83.81  4.15E-09  -1.89  Terf2ip  telomeric repeat binding factor 2, interacting protein  -82.21  6.01E-09  -1.5  F5  coagulation factor V  -81.84  6.55E-09  -2.05  Serpina6  serine (or cysteine) peptidase inhibitor, clade A, member 6  -80.82  8.28E-09  -1.96  2310036D22Rik  transmembrane protein 106B  -78.91  1.29E-08  -1.66  2310043N10Rik  RIKEN cDNA 2310043N10 gene on chromosome 19.  -76.14  2.43E-08  -1.81  Trub2  TruB pseudouridine (psi) synthase homolog 2 (E. coli)  -74.68  3.40E-08  -2.05  Sqrdl  sulfide quinone reductase-like (yeast)  -74.32  3.70E-08  -1.67  Hsd3b2  hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2  -74.30  3.71E-08  -2.85  Gjb2  gap junction protein, beta 2  -72.75  5.31E-08  -2.33  153  Differential Scoreb  P-valuec  Fold Changed  ATP-binding cassette, sub-family A (ABC1), member 6  -70.66  8.58E-08  -1.53  Sec14l4  SEC14-like 4 (S. cerevisiae)  -69.44  1.14E-07  -1.5  Slco1b2  solute carrier organic anion transporter family, member 1b2, transcript variant 1  -68.53  1.40E-07  -2.52  Ugt2b34  UDP glucuronosyltransferase 2 family, polypeptide B34  -68.53  1.40E-07  -1.47  Sypl  synaptophysin-like protein, transcript variant 1  -64.14  3.85E-07  -1.72  Mat1a  methionine adenosyltransferase I, alpha  -64.09  3.90E-07  -2.36  Hacl1  2-hydroxyacyl-CoA lyase 1  -64.09  3.90E-07  -1.55  Lypla3  lysophospholipase 3  -61.74  6.69E-07  -1.69  Apol7a  apolipoprotein L 7a  -61.17  7.64E-07  -1.82  Reep3  receptor accessory protein 3  -61.17  7.64E-07  -1.62  Rpl23  ribosomal protein L23  -61.17  7.64E-07  -1.44  Rab7  RAB7, member RAS oncogene family  -60.45  9.02E-07  -1.66  Dbp  D site albumin promoter binding protein  -59.78  1.05E-06  -1.96  Drctnnb1a  family with sequence similarity 126, member A  -59.37  1.16E-06  -1.59  Atp6v0d1  ATPase, H+ transporting, lysosomal V0 subunit D1  -59.16  1.21E-06  -1.58  Ivd  isovaleryl coenzyme A dehydrogenase, nuclear gene encoding mitochondrial protein  -59.07  1.24E-06  -1.53  Flot2  flotillin 2, transcript variant 2  -58.10  1.55E-06  -1.36  Uox  urate oxidase  -57.99  1.59E-06  -2.05  Aldh1l1  aldehyde dehydrogenase 1 family, member L1  -57.27  1.87E-06  -2.07  Gene Symbol  Gene Name  Abca6  154  Differential Scoreb  P-valuec  Fold Changed  hydroxyprostaglandin dehydrogenase 15 (NAD)  -56.90  2.04E-06  -1.59  Cyp1a2  cytochrome P450, family 1, subfamily a, polypeptide 2  -56.90  2.04E-06  -1.52  Cyp2a5  cytochrome P450, family 2, subfamily a, polypeptide 5  -56.42  2.28E-06  -2.47  Flnb  filamin, beta  -54.50  4.16E-06  -1.56  Tspan12  tetraspanin 12  -52.91  5.12E-06  -1.38  LOC100047937  PREDICTED: similar to Aldehyde dehydrogenase 1 family, member L1  -52.09  6.18E-06  -2.06  Abcc2  ATP-binding cassette, sub-family C (CFTR/MRP), member 2  -51.99  6.32E-06  -1.47  C3  complement component 3  -51.97  6.35E-06  -2.21  Ahcy  S-adenosylhomocysteine hydrolase  -51.97  6.35E-06  -2.17  6330578E17Rik  RIKEN cDNA 6330578E17 gene  -51.88  6.49E-06  -1.74  Cyp4a14  cytochrome P450, family 4, subfamily a, polypeptide 14  -50.02  9.95E-06  -2.52  Srr  serine racemase  -49.61  1.15E-05  -1.83  D630004K10Rik  RIKEN cDNA D630004K10 gene  -49.58  1.10E-05  -1.43  Abcg5  ATP-binding cassette, sub-family G (WHITE), member 5  -49.31  1.17E-05  -1.94  Nrn1  neuritin 1  -45.52  2.81E-05  -2.13  Es1  esterase 1  -45.49  2.82E-05  -2.24  Ptprd  protein tyrosine phosphatase, receptor type, D, transcript variant a  -45.17  3.04E-05  -1.64  Aldh4a1  aldehyde dehydrogenase 4 family, member A1, nuclear gene encoding mitochondrial protein  -44.48  3.64E-05  -2.06  Gene Symbol  Gene Name  Hpgd  155  Differential Scoreb  P-valuec  Fold Changed  PREDICTED: hypothetical protein LOC100044218  -43.60  4.36E-05  -2.25  Ammecr1l  AMME chromosomal region gene 1like  -43.40  4.57E-05  -1.47  Ass1  argininosuccinate synthetase 1  -43.27  4.71E-05  -1.74  Es1  esterase 1  -42.35  5.82E-05  -2.32  Glud1  glutamate dehydrogenase 1  -40.88  8.17E-05  -2.38  Ptprd  protein tyrosine phosphatase, receptor type, D, transcript variant a  -39.65  0.000108  -1.57  D1Ertd471e  immunoglobulin-like domain containing receptor 2  -39.60  0.00011  -1.56  Cept1  choline/ethanolaminephosphotransferas e1  -39.57  0.000111  -2.31  Pabpc1  poly A binding protein, cytoplasmic 1  -39.41  0.000115  -1.96  Psmd1  proteasome (prosome, macropain) 26S subunit, non-ATPase, 1  -39.28  0.000118  -2.63  ENSMUSG0000 0074075  PREDICTED: predicted gene, ENSMUSG00000074075  -39.05  0.000125  -1.8  Cyp3a25  cytochrome P450, family 3, subfamily a, polypeptide 25  -38.17  0.000152  -2.3  Rab1  RAB1, member RAS oncogene family  -38.17  0.000152  -1.32  Mbnl2  muscleblind-like 2, transcript variant 1  -37.94  0.000161  -1.44  Ccrl1  chemokine (C-C motif) receptor-like 1  -37.53  0.000177  -1.87  Fads1  fatty acid desaturase 1  -37.31  0.000186  -3.44  Wwtr1  WW domain containing transcription regulator 1  -37.17  0.000192  -1.54  Rnf14  ring finger protein 14  -37.11  0.000195  -1.63  Eif2s2  eukaryotic translation initiation factor 2, subunit 2 (beta)  -36.86  0.000206  -1.4  Gene Symbol  Gene Name  LOC100044218  156  Differential Scoreb  P-valuec  Fold Changed  PREDICTED: transmembrane emp24 protein transport domain containing 7  -36.82  0.000208  -1.52  mtDNA_ND4  NADH dehydrogenase subunit 4  -36.61  0.000218  -1.46  Pon1  paraoxonase 1  -34.46  0.000358  -2.14  Itm2b  integral membrane protein 2B  -33.76  0.00042  -2.03  Flot2  flotillin 2, transcript variant 1  -33.76  0.000421  -1.7  Lpin2  lipin 2  -33.76  0.000421  -1.54  Dcn  decorin  -32.89  0.000514  -1.58  Fmo1  flavin containing monooxygenase 1  -31.92  0.000643  -1.94  Stard7  START domain containing 7  -31.45  0.000717  -1.38  Dars  aspartyl-tRNA synthetase  -31.32  0.000738  -1.44  Aldh1a1  aldehyde dehydrogenase family 1, subfamily A1  -31.05  0.000784  -2.48  Gorasp2  golgi reassembly stacking protein 2  -30.68  0.000856  -1.38  Gpt2  glutamic pyruvate transaminase (alanine aminotransferase) 2  -30.54  0.000883  -2.2  Stard7  START domain containing 7  -30.52  0.000887  -1.36  Rn18s  18S RNA on chromosome 17.  -30.38  0.000917  -2.24  Es22  esterase 22  -30.00  0.001001  -1.64  1110002B05Rik  RIKEN cDNA 1110002B05 gene  -29.95  0.001012  -1.44  Psmd1  proteasome (prosome, macropain) 26S subunit, non-ATPase, 1  -29.58  0.001102  -2.31  Slc29a1  solute carrier family 29 (nucleoside transporters), member 1  -29.58  0.001102  -1.44  LOC100044218  PREDICTED: hypothetical protein LOC100044218  -29.52  0.001117  -1.68  Gene Symbol  Gene Name  Tmed7  157  Differential Scoreb  P-valuec  Fold Changed  Slc39a7  solute carrier family 39 (zinc transporter), member 7, transcript variant 1  -29.46  0.001132  -1.35  Ngef  neuronal guanine nucleotide exchange factor  -29.14  0.00122  -1.43  Mbnl2  muscleblind-like 2, transcript variant 1  -29.06  0.001241  -1.82  Cyp2a5  cytochrome P450, family 2, subfamily a, polypeptide 5  -28.40  0.001446  -1.93  LOC100045617  PREDICTED: similar to Eukaryotic translation initiation factor 4A2  -28.38  0.001453  -1.86  Cyp2d22  cytochrome P450, family 2, subfamily d, polypeptide 22  -28.13  0.001539  -2.18  Slc27a5  solute carrier family 27 (fatty acid transporter), member 5  -27.85  0.001639  -2.64  Mug1  murinoglobulin 1  -27.80  0.00166  -2.07  Pls3  plastin 3 (T-isoform)  -27.69  0.001703  -1.86  Ces3  carboxylesterase 3  -27.11  0.001945  -3  Cyp2c50  cytochrome P450, family 2, subfamily c, polypeptide 50  -27.11  0.001945  -2.74  Gpr125  PREDICTED: G protein-coupled receptor 125, transcript variant 1  -27.11  0.001945  -1.4  Hs3st3b1  heparan sulfate (glucosamine) 3-Osulfotransferase 3B1  -26.92  0.00203  -1.6  Eif2s3y  eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked  -26.82  0.00208  -1.45  Btbd1  BTB (POZ) domain containing 1  -26.69  0.002141  -1.38  Sybl1  synaptobrevin like 1  -26.17  0.002418  -1.56  Gene Symbol  Gene Name  158  Differential Scoreb  P-valuec  Fold Changed  SMEK homolog 2, suppressor of mek1 (Dictyostelium)  -26.05  0.002485  -1.38  Fads2  fatty acid desaturase 2  -25.88  0.002582  -1.48  Rab43  RAB43, member RAS oncogene family, transcript variant 1  -25.69  0.002698  -2.17  Golga2  golgi autoantigen, golgin subfamily a, 2  -25.69  0.002698  -1.47  Adk  adenosine kinase  -25.09  0.003098  -2.32  Abca6  ATP-binding cassette, sub-family A, member 6  -24.96  0.00319  -1.45  Gulo  gulonolactone (L-) oxidase  -24.65  0.003428  -1.61  Dsg2  desmoglein 2  -24.40  0.003633  -2.51  Brd3  bromodomain containing 3  -24.15  0.00385  -1.35  2810407C02Rik  RIKEN cDNA 2810407C02 gene  -23.90  0.004072  -2.32  Stat3  signal transducer and activator of transcription 3, transcript variant 1  -23.90  0.004072  -1.75  Slc19a2  solute carrier family 19 (thiamine transporter), member 2  -23.76  0.004202  -1.89  Cyp2e1  cytochrome P450, family 2, subfamily e, polypeptide 1  -23.7  0.00427  -2.02  AW061290  expressed sequence AW061290  -23.59  0.00437  -1.82  Eif4a2  eukaryotic translation initiation factor 4A2  -23.59  0.00437  -1.38  Lamp2  lysosomal-associated membrane protein 2, transcript variant 1  -23.47  0.004496  -1.52  Creb3l3  cAMP responsive element binding protein 3-like 3  -23.24  0.004744  -1.52  mtDNA_ATP8  ATP synthase F0 subunit 8  -23.22  0.004767  -1.49  Retsat  retinol saturase (all trans retinol 13,14 reductase)  -23.01  0.004998  -2.17  Gene Symbol  Gene Name  Smek2  159  Differential Scoreb  P-valuec  Fold Changed  RIKEN cDNA 1810015C04 gene, transcript variant 2  -23.01  0.004998  -1.78  Slc38a4  solute carrier family 38, member 4  -22.99  0.005026  -1.6  Eif4ebp2  eukaryotic translation initiation factor 4E binding protein 2  -22.33  0.005854  -1.42  Gyk  glycerol kinase  -22.29  0.005898  -1.41  Sult1a1  sulfotransferase family 1A, phenolpreferring, member 1  -22.10  0.006162  -2.35  5730472N09Rik  RIKEN cDNA 5730472N09 gene  -22.10  0.006162  -1.66  Rn18s  18S RNA on chromosome 17.  -22.08  0.006195  -2.48  Tm7sf3  transmembrane 7 superfamily member 3  -22.01  0.006292  -1.38  Pdia4  protein disulfide isomerase associated 4  -21.5  0.007085  -1.73  Serinc1  serine incorporator 1  -21.49  0.007096  -2.13  Peci  peroxisomal delta3, delta2-enoylCoenzyme A isomerase  -21.49  0.007102  -1.88  Cfb  complement factor B  -21.39  0.007262  -1.81  Aldh6a1  aldehyde dehydrogenase family 6, subfamily A1, nuclear gene encoding mitochondrial protein  -21.17  0.007632  -1.69  Aqp9  aquaporin 9  -21.05  0.007853  -2.68  Sfrs2  splicing factor, arginine/serine-rich 2  -21.05  0.007857  -1.45  Depdc6  DEP domain containing 6, transcript variant 1  -20.92  0.008093  -1.56  4631416L12Rik  RIKEN cDNA 4631416L12 gene  -20.82  0.008277  -1.39  Dync1i2  dynein cytoplasmic 1 intermediate chain 2  -20.73  0.008452  -1.94  Gene Symbol  Gene Name  1810015C04Rik  160  Differential Scoreb  P-valuec  Fold Changed  AFG3(ATPase family gene 3)-like 1 (yeast)  -20.70  0.008503  -1.33  Mylk  myosin, light polypeptide kinase  -20.14  0.009691  -1.96  Slc27a2  solute carrier family 27 (fatty acid transporter), member 2  -19.88  0.010285  -1.95  LOC382044  liver carboxylesterase N-like  -19.85  0.010349  -1.95  Gene Symbol  Gene Name  Afg3l1  a  Two Leprflox/flox AlbCre+ ob/ob and two Leprflox/flox AlbCre- ob/ob males were treated with intraperitoneal injections of 1.5 µg/g leptin for two days and then liver tissue harvested into RNAlater® on the third day. RNA samples were then subjected to the Illumina BeadArray MouseRef-6 v2.0 Expression BeadChip according to the manufacturer’s instructions. b Differential Score=-10(sgn(IntensityCre+ - IntensityCre-))log10(p-value). Genes are arranged from lowest to highest differential score. c P-value cut-off was 0.01 d Fold change of leptin-treated Leprflox/flox AlbCre+ ob/ob compared to Leprflox/flox AlbCre- ob/ob males.  161  Figure A1. Plasma lipocalin-2 levels are unaffected by a loss of hepatic leptin signalling. Plasma samples from 30-week-old Leprflox/flox AlbCre male mice were obtained after an overnight fast, after 45 minutes of re-feeding, after a 2 hour fast, and after a 5 hour fast. Plasma was then assayed for lipocalin-2 by ELISA. Data are mean±SEM, n≥5.  162  Figure A2. Pathways affected by genes differentially expressed in Leprflox/flox AlbCre mice after an overnight fast. Leprflox/flox AlbCre mice were fasted overnight for 16 hours. Liver tissue was then harvested into RNAlater®. RNA samples were subjected to the Illumina BeadArray MouseRef-8 v2.0 Expression BeadChip according to the manufacturer’s instructions. Affected KEGG metabolic pathways are highlighted in red. KEGG metabolic pathway analysis was performed using the Web-based Gene Set Analysis Toolkit (WebGestalt) v2.0 [144, 145].  163  Figure A3. Biological processes affected by genes differentially expressed in Leprflox/flox AlbCre ob/ob mice after an overnight fast. Leprflox/flox AlbCre ob/ob males were treated with intraperitoneal injections of 1.5 µg/g leptin for two days and then liver tissue harvested into RNAlater® on the third day. RNA samples were then subjected to the Illumina BeadArray MouseRef-6 v2.0 Expression BeadChip according to the manufacturer’s instructions. Biological processes analysis was performed using the Web-based Gene Set Analysis Toolkit (WebGestalt) v2.0 [144, 145].  164  

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