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Mechanisms of fructose-induced hypertension Tran, Trinh Xuan 2009

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MECHANISMS OF FRUCTOSE-INDUCED HYPERTENSION  by  Trinh Xuan Tran B.Sc., McMaster University, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  March 2009  © Trinh Xuan Tran, 2009  ABSTRACT The metabolic syndrome is a cluster of cardiovascular risk factors and is a global health concern. The most accepted and unifying hypothesis proposes that insulin resistance is the major common underlying abnormality that describes the metabolic syndrome and links it to the development of cardiovascular disease. The fructose-fed rat is an animal model that exhibits several features observed in the metabolic syndrome including insulin resistance, hyperinsulinemia, hypertriglyceridemia and hypertension. This animal model is used to study the relationship between these metabolic disturbances and hypertension. Numerous mechanisms have been proposed to mediate the link between insulin resistance and hypertension. The objectives of this thesis were to further investigate the underlying mechanisms that have been proposed to contribute to the development of hypertension in fructose-fed rats through the use of various pharmacological agents. We demonstrated that chronic treatment with bosentan, a dual endothelin receptor antagonist, L-158,809, an angiotensin receptor antagonist, prazosin, an α1-adrenoceptor antagonist or etanercept, a soluble recombinant fusion protein consisting of the extracellular ligand binding domain of tumor necrosis factor receptor type 2, prevented the development of fructose-induced hypertension without affecting insulin levels or insulin sensitivity. These results suggest that increased production and/or activity of the endothelin system, renin angiotensin system or sympathetic nervous system contribute to the development of hypertension through insulinindependent mechanisms. Both the endothelin system and renin angiotensin system are crucial players in the development of fructose-induced hypertension, with endothelin-1 contributing its effects through modulation of angiotensin II.  Overactivation of the  sympathetic nervous system contributed to the development of hypertension, but does not  ii  appear to be an initial, precipitating mediator. Chronic etanercept treatment prevented the development of hypertension by improving vascular function and restoring endothelial nitric oxide synthase expression. Therefore, the pathogenesis of hypertension in fructose-fed rats is complex in nature and involves numerous pathways that do not necessarily function independently from one another.  iii  TABLE OF CONTENTS ABSTRACT......................................................................................................................... ii TABLE OF CONTENTS .................................................................................................... iv LIST OF TABLES .............................................................................................................. ix LIST OF FIGURES.............................................................................................................. x LIST OF ABBREVIATIONS............................................................................................ xiv ACKNOWLEDGEMENTS............................................................................................. xviii 1  INTRODUCTION.................................................................................................... 1 1.1  THE METABOLIC SYNDROME........................................................................ 1  1.1.1  Definitions of the metabolic syndrome .......................................................... 2  1.1.2  Insulin resistance is causally linked to hypertension ...................................... 5  1.1.3  Therapeutic approaches in the treatment of hypertension in the metabolic syndrome ...................................................................................................... 6  1.2  DIETARY FRUCTOSE........................................................................................ 7  1.3  THE FRUCTOSE-FED RAT: AN ANIMAL MODEL OF INSULIN RESISTANCE AND HYPERTENSION............................................................. 12  1.3.1  THE ROLE OF THE SYMPATHETIC NERVOUS SYSTEM.................... 15  1.3.2  THE ROLE OF THE ENDOTHELIUM AND ENDOTHELIUM-DERIVED FACTORS .................................................................................................. 17  1.3.2.1  THE ENDOTHELIN SYSTEM............................................................... 19  1.3.2.1.1 Synthesis of ET-1 .............................................................................. 19 1.3.2.1.2 Endothelin receptors and signaling .................................................... 22 1.3.2.1.3 ET-1 in hypertension ......................................................................... 26 1.3.2.2  THE RENIN ANGIOTENSIN SYSTEM ................................................ 28  1.3.2.2.1 Synthesis and effects of Ang II .......................................................... 28 1.3.2.2.2 Ang II receptors and signaling ........................................................... 31  iv  1.3.2.2.3 Ang II in hypertension ....................................................................... 35 1.3.2.3  THE NO SYSTEM.................................................................................. 37  1.3.2.3.1 NO in hypertension............................................................................ 40 1.3.3  2  THE ROLE OF TNF-α................................................................................ 41  1.3.3.1  TNF-α biology ........................................................................................ 41  1.3.3.2  Synthesis of TNF-α ................................................................................. 41  1.3.3.3  TNF-α receptors and signaling................................................................. 42  1.3.3.4  TNF-α in insulin resistance...................................................................... 45  RESEARCH OUTLINE ......................................................................................... 47 2.1  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension............ 47  2.1.1  Rationale ..................................................................................................... 47  2.1.2  Objective..................................................................................................... 48  2.1.3  Hypothesis .................................................................................................. 48 Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats. 49  2.2 2.2.1  Rationale ..................................................................................................... 49  2.2.2  Objective..................................................................................................... 49  2.2.3  Hypothesis .................................................................................................. 50  2.3  Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension ..................................................................... 51  2.3.1  Rationale ..................................................................................................... 51  2.3.2  Objective..................................................................................................... 54  2.3.3  Hypothesis .................................................................................................. 54  3  MATERIALS AND METHODS............................................................................ 55 3.1 3.1.1  General methodology.......................................................................................... 55 SBP measurements...................................................................................... 55  v  3.1.2  Oral glucose tolerance test and insulin sensitivity index............................... 55  3.1.3  Blood collection .......................................................................................... 57  3.1.4  Biochemical measurements ......................................................................... 57  3.1.5  Reagents...................................................................................................... 57  3.1.6  Statistical analysis ....................................................................................... 58  3.2  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension............ 59  3.2.1  Animals and experimental design ................................................................ 59  3.2.2  Immunohistochemistry and image analysis.................................................. 60 Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats. 61  3.3 3.3.1 3.4  Animals and experimental design ................................................................ 61 Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension ..................................................................... 62  3.4.1  Animals and experimental design ................................................................ 62  3.4.2  Vascular reactivity ...................................................................................... 63  3.4.3  Western blot analysis................................................................................... 63  4  RESULTS .............................................................................................................. 65 4.1  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension............ 65  4.1.1  General characteristics of rats following six weeks of bosentan or L-158,809 treatment ..................................................................................................... 65  4.1.2  SBP............................................................................................................. 67  4.1.3  OGTT ......................................................................................................... 67  4.1.4  Plasma Ang II levels ................................................................................... 72  4.1.5  ET-1-ir microscopy and semi-quantitative analysis...................................... 72  4.2 4.2.1  Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats. 75 General characteristics of rats following nine weeks of prazosin treatment .. 75  vi  4.2.2  SBP............................................................................................................. 75  4.2.3  OGTT ......................................................................................................... 78  4.2.4  Plasma NE levels ........................................................................................ 78  4.2.5  Plasma Ang II levels ................................................................................... 78  4.3  Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension ..................................................................... 83  4.3.1  General characteristics of rats following nine weeks of etanercept treatment 83  4.3.2  SBP............................................................................................................. 83  4.3.3  OGTT ......................................................................................................... 86  4.3.4  Vascular reactivity ...................................................................................... 86  4.3.5  Plasma TNF-α and IL-6 levels..................................................................... 91  4.3.6  Plasma NE levels ........................................................................................ 91  4.3.7  Plasma Ang II levels ................................................................................... 91  4.3.8  Aortic eNOS and iNOS protein expression .................................................. 96  4.3.9  Aortic RhoA, ROCK-1 and ROCK-2 protein expression ............................. 96  4.3.10  Fat pad TNF-α and TNFR1 protein expression ............................................ 96  4.3.11  Fat pad p-NFκB p65 and NFκB p100/p52 protein expression .................... 101  4.3.12  Aortic caspase-3 protein expression........................................................... 101  5  DISCUSSION AND CONCLUSIONS ................................................................. 104 5.1  DISCUSSION................................................................................................... 104  5.1.1  Overview of results for fructose-fed rats.................................................... 104  5.1.2  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension .. 106  5.1.3  Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats ............................................................................................................ 112  5.1.4  Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension............................................................ 117  vii  5.2  CONCLUSIONS .............................................................................................. 122  5.3  FURTHER WORK ........................................................................................... 124  REFERENCES................................................................................................................. 126 APPENDIX...................................................................................................................... 155  viii  LIST OF TABLES Table 1.1  Definitions of the metabolic syndrome .......................................................... 4  Table 4.1  General characteristics and fasted plasma parameters of control and fructosefed rats following six weeks of bosentan treatment ...................................... 66  Table 4.2  General characteristics and fasted plasma parameters of control and fructoserats following six weeks of L-158,809 treatment ......................................... 66  Table 4.3  General characteristics and fasted plasma parameters of control and fructosefed rats following nine weeks of prazosin treatment .................................... 76  Table 4.4  General characteristics and fasted plasma parameters of control and fructosefed rats following nine weeks of etanercept treatment.................................. 84  Table 4.5  pD2 and Emax values of superior mesenteric arteries from control and fructosefed rats following nine weeks of etanercept treatment.................................. 90  ix  LIST OF FIGURES Figure 1.1  Chemical structures of glucose, fructose and sucrose..................................... 8  Figure 1.2  Hepatic metabolism of glucose and fructose. ............................................... 10  Figure 1.3  Proposed mechanisms that link insulin resistance/hyperinsulinemia to hypertension in fructose-fed rats.................................................................. 14  Figure 1.4  A cross section of a blood vessel. ................................................................ 18  Figure 1.5  The vascular endothelin system. .................................................................. 20  Figure 1.6  ETA and ETB receptor mediated signal transduction in vascular smooth muscle cells............................................................................................................. 24  Figure 1.7  ETB receptor mediated signal transduction in endothelial cells. ................... 25  Figure 1.8  The classical renin angiotensin system. ....................................................... 29  Figure 1.9  AT1 receptor mediated signal transduction in vascular smooth muscle cells. 33  Figure 1.10  AT2 receptor mediated signal transduction................................................... 34  Figure 1.11  NO mediated signal transduction. ................................................................ 39  Figure 1.12  TNFR1 and TNFR2 mediated signaling pathways. ...................................... 44  Figure 2.1  Effect of high fructose-feeding on plasma TNF-α following one, two, seven and thirteen weeks of study. Values expressed as mean ± SEM, n=7-11. * p<0.05, vs. C. .............................................................................................. 53  Figure 3.1  A schematic illustration of an OGTT. .......................................................... 56  Figure 4.1  Effect of chronic (A) bosentan or (B) L-158,809 treatment on SBP in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8. * p<0.05, vs. C, CB; ‡ p<0.05, vs. C, CL; † p<0.05, vs. F. .... 68  Figure 4.2  Effect of chronic (A) bosentan or (B) L-158,809 treatment on plasma glucose response and AUC (inset) during an OGTT in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8. ..... 69  Figure 4.3  Effect of chronic (A) bosentan or (B) L-158,809 treatment on plasma insulin response and AUC (inset) during an OGTT in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8. ‡ p<0.05, vs. C, CL. ....................................................................................... 70  x  Figure 4.4  Insulin sensitivity index values obtained from OGTT data in control and fructose-fed rats following six weeks of (A) bosentan or (B) L-158,809 treatment. Values expressed as mean ± SEM, n = 8. * p<0.05, vs. C, CB; ‡ p<0.05, vs. C, CL. ....................................................................................... 71  Figure 4.5  Effect of chronic (A) bosentan or (B) L-158,809 treatment on plasma Ang II levels in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8. * p<0.05, vs. C, CB; † p<0.05, vs. F; # p<0.05, vs. C. .............................................................................................. 73  Figure 4.6  (A) Representative fluorescence images of immunohistochemical expression of ET-1 in superior mesenteric arteries from control and fructose-fed rats following six weeks of L-158,809 treatment. Arrows indicate positive immunostaining for ET-1 within the intimal and medial layers. (B) Semiquantitative analysis of vascular ET-1-ir from control and fructose-fed rats following six weeks of L-158,809 treatment. Values expressed as mean ± SEM, n = 8. † <0.05, vs. F; # p<0.05, vs. C. ................................................ 74  Figure 4.7  Effect of chronic prazosin treatment on SBP in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 8. † p<0.05, vs. C, CP; ‡ p<0.05, vs. F................................................................ 77  Figure 4.8  Effect of chronic prazosin treatment on plasma A) glucose response and AUC (inset) and B) insulin response and AUC (inset) during an OGTT in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 8...................................................................................... 79  Figure 4.9  Insulin sensitivity index values obtained from OGTT data in control and fructose-fed rats following nine weeks of prazosin treatment. Values expressed as mean ± SEM, n = 8. # p<0.05, vs. C. ...................................... 80  Figure 4.10  Effect of chronic prazosin treatment on plasma NE levels in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 8. # p<0.05, vs. C; ^ p<0.05, vs. FP.............................................. 81  Figure 4.11  Effect of chronic prazosin treatment on plasma Ang II levels in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM. † p<0.05, vs. C, CP; # p<0.05, vs. C. ................................................. 82  Figure 4.12  Effect of chronic etanercept treatment on SBP in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 20. * p<0.05, vs. C, CE; † p<0.05, vs. F................................................................ 85  Figure 4.13  Effect of chronic etanercept treatment on plasma A) glucose response and AUC (inset) and B) insulin response and AUC (inset) during an OGTT in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 20. # p<0.05, vs. C; * p<0.05, vs. C, CE...................... 87  xi  Figure 4.14  Insulin sensitivity index values obtained from OGTT data from control and fructose-fed rats following nine weeks of etanercept treatment. Values expressed as mean ± SEM, n = 20. * p<0.05, vs. C, CE............................... 88  Figure 4.15  Cumulative concentration-response curves to A) PE and B) ACh in superior mesenteric arteries from control and fructose-fed rats following nine weeks of etanercept treatment. Values expressed as mean ± SEM, n= 14. ................. 89  Figure 4.16  Effect of chronic etanercept treatment on plasma TNF-α in control and fructose-fed rats following A) two and B) nine weeks of study. Values expressed as mean ± SEM, n = 20. # p<0.5, vs. C; † p<0.05, vs. F............... 92  Figure 4.17  Effect of chronic etanercept treatment on plasma IL-6 levels in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 12. ............................................................................................... 93  Figure 4.18  Effect of chronic etanercept treatment on plasma NE in control and fructosefed rats following nine weeks of study. Values expressed as mean ± SEM, n = 12................................................................................................................ 94  Figure 4.19  Effect of chronic etanercept treatment on plasma Ang II in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 12. * p<0.05, vs. C, CE. .............................................................. 95  Figure 4.20  Representative Western blots of A) eNOS and C) iNOS from thoracic aorta from control and fructose-fed rats following nine weeks study. Effect of chronic etanercept treatment on the relative intensity of B) eNOS and D) iNOS protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 10. * p<0.05, vs. C, CE; † p<0.05, vs. F. .............................................................................................. 97  Figure 4.21  Representative Western blots of A) RhoA, C) ROCK-1 and E) ROCK-2 from thoracic aorta from control and fructose-fed rats following nine weeks of study. Effect of chronic etanercept treatment on the relative intensity of B) RhoA, D) ROCK-1 and F) ROCK-2 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=8-12. * p<0.05, vs. C, CE.............................................................. 98  Figure 4.22  Representative Western blots of A) transmembrane TNF-α (24 kDa) and C) soluble TNF-α (17 kDa) from epidydimal fat pads from control and fructosefed rats following nine weeks of study. Effect of chronic etanercept treatment on the relative intensity of B) transmembrane TNF (24kDa) and D) soluble TNF-α (17kDa) protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12. * p<0.05, vs. C, CE. ......................................................................................................... 99  Figure 4.23  A) Representative Western blot of TNFR1 from epidydimal fat pads from control and fructose-fed rats following nine weeks of study. B) Effect of xii  chronic etanercept on the relative intensity of TNFR1 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12. .............................................................................. 100 Figure 4.24  Representative Western blots of A) p-NFκB p65, C) NFκB p100 and E) NFκB p52 from epidydimal fat pads from control and fructose-fed rats following nine weeks of study. Effect of chronic etanercept treatment on the relative intensity of B) p-NFκB p65, D) NFκB p100 and F) NFκB p52 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12. # p<0.05, vs. C.......................... 102  Figure 4.25  A) Representative Western blot of caspase-3 from thoracic aorta from control and fructose-fed rats following nine weeks of study. B) Effect of chronic etanercept treatment on the relative intensity of caspase-3 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12. .............................................................. 103  Figure 5.1  Proposed mechanism of ET-1 modulating Ang II in the development of hypertension in fructose-fed rats................................................................ 111  xiii  LIST OF ABBREVIATIONS AA  arachidonic acid  AACE  American Association of Clinical Endocrinologists  AC  adenylate cyclase  ACE  angiotensin converting enzyme  acetyl CoA  acetyl coenzyme A  ACh  acetylcholine  AHA/NHLBI ANF  American Heart Association/National Heart, Lung and Blood Institute atrial natrietic factor  Ang I  angiotensin I  Ang II  angiotensin II  AT1  angiotensin II type 1 receptor  AT2  angiotensin II type 2 receptor  ATP III  National Cholesterol Education Program Adult Treatment Panel III  ATP  adenosine triphosphate  AUC  area under the curve  big-ET-1  big-endothelin-1  cAMP  cyclic adenosine monophosphate  cGMP  cyclic guanosine monophosphate  DAG  diacylglycerol  DOCA  deoxycorticosterone acetate  ECE  endothelin converting enzyme  xiv  EDHF  endothelium-dependent hyperpolarizing factor  EDRF  endothelium-derived relaxing factor  EDTA  ethylenediaminetetraacetic acid  EGIR  European Group for the Study of Insulin Resistance  Emax  maximum response  eNOS  endothelial nitric oxide synthase  ET-1  endothelin-1  ET-1-ir  ET-1-immunoreactivity  ETA  endothelin subtype A receptor  ETB  endothelin subtype B receptor  FAD  flavin adenine dinucleotide  FADD  Fas-associated death domain protein  FMN  flavin mononucleotide  fructose 1,6-bisP  fructose 1,6-bisphosphate  fructose 1-P  fructose 1-phosphate  fructose 6-P  fructose 6-phosphate  GC  guanylate cyclase  GLUT2  glucose transporter type 2  GLUT5  glucose transporter type 5  glyceraldehyde 3-P  glyceraldehyde 3-phosphate  glycerol 3-P  glycerol 3-phosphate  GTP  guanine triphosphate  IDF  International Diabetes Federation  xv  IKK  IκB kinase  IL-6  interleukin-6  iNOS  inducible nitric oxide synthase  IP3  inositol triphosphate  IP3R  inositol triphosphate receptor  IRS-1  insulin receptor substrate-1  NADPH  nicotinamide adenine dinucleotide phosphate  NE  norepinephrine  NFκB  nuclear factor κB  nNOS  neuronal nitric oxide synthase  NO  nitric oxide  NOS  nitric oxide synthase  OGTT  oral glucose tolerance test  pD2  negative log of the concentration producing 50% of the maximum response  PE  phenylephrine  PGI2  prostacyclin  PI3K  phosphoinositide-3 kinase  PLA2  phospholipase A2  PLC  phospholipase C  PLD  phospholipase D  prepro-ET-1  prepro-endothelin-1  RIP  receptor-interacting protein  ROCK-1  Rho-kinase-1  xvi  ROCK-2  Rho-kinase-2  SBP  systolic blood pressure  SDS  sodium dodecyl sulphate  SHR  spontaneously hypertensive rat  TACE  TNF-α converting enzyme  TBS-T  Tris-buffered saline-Tween  TNFR1  tumor necrosis factor receptor type 1  TNFR2  tumor necrosis factor receptor type 2  TNF-α  tumor necrosis factor-α  TRADD  tumor necrosis factor receptor-associated death domain protein  TRAF1  tumor necrosis factor receptor-associated factor 1  TRAF2  tumor necrosis factor receptor-associated factor 2  TxA2  thromboxane A2  VLDL  very low density lipoproteins  WHO  World Health Organization  xvii  ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor, Dr. John McNeill, for the guidance, encouragement and enthusiasm that he has provided throughout my graduate training. This thesis would not have been possible without his continued support. Thank you to my co-supervisor, Dr. Kathleen MacLeod, for her invaluable insight and constructive comments on my thesis. I am grateful to my supervisory committee members, Dr. Helen Burt (Chair), Dr. Ismail Laher, Dr. Brian Rodrigues and Dr. Judy Wong for their time, effort and suggestions. Thank you to the McNeill lab members, Mary Battell, Moira Greaven, Dr. Jihong Jiang, Prabhakara Nagareddy, Dr. Vijay Sharma, Harish Vasudevan, Dr. Linfu Yao, Dr. Zhengyuan Xia and Violet Yuen, and to Hisham Ismail, Geetu Kharmate, Dr. Guorong Lin, Kate Potter, Padmesh Rajput and Rui Zhang for their technical assistance, advice and support. I would like to thank Dr. Gary Kargacin, Dr. Ujendra Kumar and Dr. Lucy Marzban for consultation on the immunohistochemistry studies and to Dr. Bruce Verchere for the use of the microscope to conduct them. Thank you to my fellow graduate students and members of the Faculty of Pharmaceutical Sciences who have made my time here a memorable one. I am especially thankful to Kevin Letchford for his willingness to listen and his continued encouragement. Thank you to my parents, Kathy and Tom, and my brothers, John and Charlie, for their constant support throughout my education. Thank you to the Heart and Stroke Foundation of British Columbia and Yukon for providing financial support for this research.  I am grateful to the Health Research  Foundation of Canada’s Research-Based Pharmaceutical Companies & the Canadian  xviii  Institute for Health Research and the Pacific Century Graduate Scholarship and University of British Columbia for providing financial support throughout my studies.  xix  1  INTRODUCTION  1.1  THE METABOLIC SYNDROME The metabolic syndrome is a cluster of clinical and biochemical features that includes  abdominal obesity, insulin resistance, hypertension, accelerated atherosclerosis and dyslipidemia (Reaven, 1988; Wajchenberg et al., 1994). It is well documented that each metabolic disturbance is an important risk factor for the development of cardiovascular disease, defined as any disease or injury to the heart and blood vessel system. According to the Heart and Stroke Foundation of Canada, 80% of Canadian individuals have at least one of the above risk factors; nearly 33% have two; and 11% have three or more risk factors (Heart and Stroke Foundation of Canada, 2003).  As a result, individuals afflicted with the  metabolic syndrome are at an increased risk for developing Type 2 diabetes (Hanson et al., 2002; Laaksonen et al., 2002; Lorenzo et al., 2003) and cardiovascular disease (Hunt et al., 2004; Isomaa et al., 2001; Lakka et al., 2002; Malik et al., 2004). Cardiovascular disease is the major cause of morbidity and mortality in the developed world (Braunwald, 1997; Ginsberg, 2000) and claims more lives than any other disease. In Canada, cardiovascular disease was responsible for more than 70 000 deaths in 2002 (Statistics Canada, 2004). The worldwide prevalence of the metabolic syndrome has been estimated to be between 10-25% of the adult population (Wild and Byrne, 2005). Modifiable risk factors for the development of cardiovascular disease include smoking, physical inactivity, dyslipidemia, obesity, hypertension and diabetes.  In order to reduce and prevent the  occurrence of cardiovascular disease, it is important to further our understanding of its underlying risk factors as well as to effectively manage them.  1  1.1.1  Definitions of the metabolic syndrome In 1999, the World Health Organization (WHO) recognized the need for defining the  metabolic syndrome (Organization, 1999). Insulin resistance was emphasized as the major underlying factor for this syndrome; therefore evidence for the presence of insulin resistance was a required criterion along with two additional parameters for diagnosis. That same year, the European Group for the Study of Insulin Resistance (EGIR) proposed a modified definition to be used in non-diabetic individuals that placed importance on the presence of abdominal obesity (Balkau and Charles, 1999).  The EGIR’s definition also required  evidence of insulin resistance, as reflected by elevated plasma insulin levels, and the presence of two additional parameters (Balkau and Charles, 1999). In 2001, the National Cholesterol Education Program Adult Treatment Panel III (ATP III) adapted their criteria for the metabolic syndrome for easier use in a clinical setting. Disadvantages in the ability to directly measure insulin resistance allowed for a diagnosis to be made without evidence of insulin resistance (2001). An individual presenting with any three of the parameters was considered to have the metabolic syndrome. In 2003, the American Association of Clinical Endocrinologists (AACE) modified the ATP III definition to refocus on the presence of insulin resistance as a required criterion, but excluded abdominal obesity and insulin values from their definition (Einhorn et al., 2003). Other inclusion criteria, such as a family history of Type 2 diabetes, polycystic ovary syndrome and hyperuricemia, were incorporated into their definition. As a result of the numerous definitions for the metabolic syndrome, the International Diabetes Federation (IDF) formulated a worldwide definition that can be used in both the clinical and research setting (Alberti et al., 2005). Since abdominal obesity is highly correlated with insulin resistance and cardiovascular disease, the presence of  2  abdominal obesity was included as a fundamental criterion, with ethnic-specific waist circumference values incorporated into this definition (Alberti et al., 2005). More recently, the American Heart Association/National Heart, Lung and Blood Institute (AHA/NHLBI) has put forth their definition of the metabolic syndrome (Grundy et al., 2005). The various definitions of the metabolic syndrome are summarized in Table 1.1. With the numerous definitions of the metabolic syndrome currently in use, there is debate on which set of criteria is most appropriate. Despite the controversy surrounding the various definitions of the metabolic syndrome, it is well established that the greater number of metabolic disturbances that present, the greater the risk of developing Type 2 diabetes and/or cardiovascular disease. Implementing and utilizing a universally accepted definition of the metabolic syndrome will allow clinicians to accurately diagnose the presence of this syndrome in order to identify, reduce and prevent the risk of developing Type 2 diabetes and/or cardiovascular disease. It also provides researchers with the necessary tools for comparing the prevalence of the metabolic syndrome among different subpopulations.  3  Table 1.1  Definitions of the metabolic syndrome WHO (Organization, 1999)  Required criteria  Diabetes, IGT or insulin resistance* Plus two or more of the following  EGIR (Balkau and Charles, 1999) Insulin resistance or fasting hyperinsulinemia Plus two or more of the following  ATP III (2001)  AACE (Einhorn et al., 2003)  IDF (Alberti et al., 2005)  AHA/NHLBI (Grundy et al., 2005)  IGT or IFG  Central obesity  Three or more of the following  Plus any of the following based on clinical judgement  Plus two or more of the following  Three of more of the following  Waist-hip ratio > 0.9 (male) > 0.85 (female) or BMI > 30 kg/m2 TG > 1.7 mmol/L  Waist circumference ≥ 94 cm (male) ≥ 80 cm (female)  Waist circumference ≥ 102 cm (male) ≥ 88 cm (female)  BMI ≥ 25 kg/m2  Waist circumference (ethnic-specific) ≥ 94 cm (male) ≥ 80 cm (female)  Waist circumference ≥ 102 cm (male) ≥ 88 cm (female)  TG > 2.0 mmol/L  TG > 1.7 mmol/L  TG > 1.7 mmol/L  and/or HDL-C < 0.9 mmol/L (male) < 1.0 mmol/L (female)  and/or HDL-C < 1.0 mmol/L or on drug treatment for dyslipidemia  HDL-C < 1.0 mmol/L (male) < 1.3 mmol/L (female)  and HDL-C < 1.0 mmol/L (male) < 1.3 mmol/L (female)  Blood pressure  BP ≥ 140/90 mmHg  BP ≥ 140/90 mmHg or on drug treatment for hypertension  SBP ≥ 130 mmHg DBP ≥ 85 mmHg  BP ≥ 135/85 mmHg  Glucose  Diabetes, IGT or insulin resistance  FPG ≥ 6.1 mmol/L (but not diabetes)  FPG ≥ 6.1 mmol/L  IGT or IFG (but not diabetes)  TG > 1.7 mmol/L or on drug treatment for hypertriglyceridemia HDL-C < 1.0 mmol/L (male) < 1.3 mmol/L (female) or on drug treatment for low HDL-C SBP ≥ 130 mmHg DBP ≥ 85 mmHg or on drug treatment for hypertension FPG ≥ 5.6 mmol/L or previous diagnosis of Type 2 diabetes  TG ≥ 1.7 mmol/L or on drug treatment for hypertriglyceridemia HDL-C < 1.0 mmol/L (male) < 1.3 mmol/L (female) or on drug treatment for low HDL-C SBP ≥ 130 mmHg DBP ≥ 85 mmHg or on drug treatment for hypertension FPG ≥ 5.6 mmol/L or on drug treatment for hyperglycemia  Central obesity  Lipids  Other  4  Albumin excretion Other features of insulin resistance† rate ≥ 20 µg/min BMI = body mass index; BP = blood pressure; DBP = diastolic blood pressure; FPG = fasting plasma glucose; HDL-C = high density lipoprotein-cholesterol; IFG = impaired fasting glucose; IGT = impaired glucose tolerance; SBP = systolic blood pressure; TG = triglycerides * Insulin resistance (under hyperinsulinemic, euglycemic conditions, glucose uptake below lowest quartile) † Includes family history of Type 2 diabetes, polycystic ovary syndrome, sedentary lifestyle, ethnic groups susceptible to Type 2 diabetes.  1.1.2  Insulin resistance is causally linked to hypertension Both hereditary and environmental factors contribute to the development of the  metabolic syndrome; however the exact cause of this disorder remains uncertain. The most accepted and unifying hypothesis proposes that insulin resistance is the major common underlying abnormality that describes the metabolic syndrome and links it to the development of cardiovascular disease (Ginsberg, 2000; Reaven, 1988). Insulin resistance occurs when there is a defect in insulin action resulting in fasting hyperinsulinemia. The "insulin hypothesis" proposes that insulin resistance and compensatory hyperinsulinemia contribute causally toward the pathogenesis of hypertension (Bhanot and McNeill, 1996; DeFronzo, 1992; Reaven, 1988).  In both humans and animal models, hypertension is  associated with hyperinsulinemia and insulin resistance (DeFronzo and Ferrannini, 1991; Reaven, 1991; Verma, 2000).  Studies have demonstrated that both obese and lean  hypertensive patients exhibited insulin resistance (Reaven, 1991). In addition, normotensive offspring of hypertensive parents also exhibited insulin resistance (Grunfeld et al., 1994). Several hypertensive rodent models exhibit similar defects in insulin action, including the spontaneously hypertensive rat (SHR) (Bhanot et al., 1995; Hulman et al., 1991; Mondon and Reaven, 1988), Dahl rat (Kotchen et al., 1991; Somova and Channa, 1999), and fructosefed rat (Bhanot et al., 1995; Dall'Aglio et al., 1995; Hwang et al., 1987). Given the presence of these metabolic defects in rodent models that are etiologically distinct suggests a strong relationship  between insulin resistance/hyperinsulinemia and the development  of  hypertension.  5  1.1.3  Therapeutic approaches in the treatment of hypertension in the metabolic syndrome Hypertension is a major risk factor for cardiovascular morbidity and mortality, such  as stroke, coronary artery disease, peripheral vascular disease and congestive heart failure (Heart and Stroke Foundation of Canada, 2003; Himmelmann et al., 1998; Kannel, 1993). An individual with hypertension is two or three times more likely to suffer from a cardiovascular event as compared to an individual with normal blood pressure (Heart and Stroke Foundation of Canada, 2003). In 2000, 26% of the worldwide adult population was estimated to have hypertension and projected numbers are expected to increase by 60% to 1.56 billion by the year 2025 (Kearney et al., 2005). The adequate control of blood pressure in hypertensive individuals is associated with a significant reduction in the incidence of cardiovascular events such as stroke, myocardial infarction, heart failure and death (Heart and Stroke Foundation of Canada, 2003; Hansson et al., 1998; Neal et al., 2000). Therapeutic approaches for the treatment of hypertension in individuals with the metabolic syndrome consists of monotherapy or combination therapy with angiotensin converting enzyme inhibitors/angiotensin receptor blockers, β-blockers, calcium channel blockers or diuretics (Hanefeld and Schaper, 2005). Despite an increased awareness, the implementation of guidelines for the treatment of hypertension, the availability of antihypertensive medications and knowledge of preventative measures, the proportion of individuals that are being treated for hypertension and have adequate control of their blood pressure has remained low (Chobanian et al., 2003). Having an incomplete understanding of the pathogenesis of hypertension may be partially responsible.  6  1.2  DIETARY FRUCTOSE Fructose is a monosaccharide that is present in many fruits and vegetables. Although  fructose has the same chemical formula (C6H12O6) as glucose, it differs in its chemical structure.  Fructose is a five-membered ring whereas glucose is a six-membered ring.  Fructose also exists in foods as a disaccharide in the form of sucrose, which is composed of one molecule of glucose linked to one molecule of fructose through a 1-2 glycoside bond. The chemical structures of glucose, fructose and sucrose are shown in Figure 1.1. The metabolism of fructose differs from the metabolism of glucose and occurs through an insulin-independent mechanism. Fructose absorption is mediated through the actions of two transporters, glucose transporter type 5 (GLUT5) and glucose transporter type 2 (GLUT2) (Helliwell et al., 2000; Helliwell et al., 2000). Following ingestion, fructose is rapidly transported across the brush border of enterocytes via the fructose-specific glucose transporter, GLUT5 (Burant et al., 1992). GLUT2, located on the basolateral membranes of enterocytes, transports both glucose and fructose into the circulation (Cheeseman, 1993). With chronic carbohydrate consumption, GLUT2 can traffic between the basolateral membrane to the brush border to facilitate fructose absorption (Kellett et al., 2008). In the liver, fructose is phosphorylated by fructokinase, a fructose-specific enzyme that is highly expressed in the liver, to form fructose 1-phosphate (fructose 1-P), which is further converted to three-carbon phosphate intermediates glyceraldehyde, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (glyceraldehyde 3-P), as illustrated in Figure 1.2. A portion of these intermediates can enter the gluconeogenic pathway and be converted to glucose. These intermediates can also be metabolized to lactate or formed into triglycerides.  7  GLUCOSE  FRUCTOSE  SUCROSE  Figure 1.1  Chemical structures of glucose, fructose and sucrose.  8  The rate-limiting step of glucose metabolism involves the phosphorylation of fructose 6-phosphate (fructose 6-P) to fructose 1,6-bisphosphate (fructose 1,6-bisP), which is catalyzed by the enzyme phosphofructokinase. This reaction is negatively regulated through a feedback mechanism in which citrate and adenosine triphosphate (ATP) allosterically inhibit phosphofructokinase, thereby preventing further glucose uptake into the liver (Havel, 2005). Hepatic metabolism of glucose and fructose merge at the three-carbon phosphate intermediates.  Given  that  fructose  can  bypass  the  rate-limiting  reaction  of  phosphofructokinase, fructose-derived intermediates can enter the glycolytic pathway downstream of this enzyme. As these intermediates accumulate, they can be converted to glycerol 3-phosphate (glycerol 3-P), the glycerol moiety of triglycerides. Metabolism to pyruvate followed by conversion to acetyl coenzyme A (acetyl CoA) through pyruvate dehydrogenase allows for de novo lipogenesis and formation of long chain fatty acids, which can be esterified to form triglycerides (Mayes, 1993). Therefore, consumption of high levels of fructose functions as an unregulated source for triglyceride production.  9  FRUCTOSE  GLUCOSE glucokinase  GLUCOSE 6-P  fructokinase  phosphoglucoisomerase  FRUCTOSE 6-P fructose 1,6-bisphosphatase  FRUCTOSE 1-P  phosphofructokinase  FRUCTOSE 1,6-bisP  ATP CITRATE  aldolase  GLYCERALDEHYDE  DIHYDROXYACETONE PHOSPHATE  GLYCERALDEHYDE 3-P  triokinase  PYRUVATE GLYCEROL 3-P Acyl glycerols  Acyl CoA  VLDL  10  Figure 1.2  Hepatic metabolism of glucose and fructose.  LACTATE Acetyl CoA CO2 + ATP  CITRATE  The hormonal effects of fructose also differ from those of glucose.  Following  ingestion of fructose, there is no surge in insulin levels and this occurs for two reasons. Although the pancreas does not express GLUT5 (Curry, 1989), fructose uptake is mediated by GLUT2. Due to a lack of fructokinase, phosphorylation of fructose is limited (Sener et al., 1984).  Secondly, fructose does not stimulate gastric inhibitory polypeptide release  (Ganda et al., 1979; Rayner et al., 2000; Teff et al., 2004), which stimulates insulin secretion. In contrast to glucose, the levels of leptin, a hormone that signals satiety, are not increased following fructose consumption (Adams et al., 2008; Teff et al., 2004). Interestingly, chronic fructose feeding has been reported to increase leptin levels in rats (Alzamendi et al., 2009; Lee et al., 2006; Roglans et al., 2007), which may indicate the presence of leptin resistance. Fructose has also been reported to increase plasma adiponectin (Alzamendi et al., 2009; Kamari et al., 2007; Roglans et al., 2007), a hormone produced by adipose tissue that regulates various metabolic processes.  The increase in circulating  adiponectin may indicate resistance to adiponectin since beneficial effects, such as increased insulin sensitivity and lowered blood pressure, were not observed.  11  1.3  THE FRUCTOSE-FED RAT: AN ANIMAL MODEL OF INSULIN RESISTANCE AND HYPERTENSION The fructose-fed rat represents an animal model of acquired systolic hypertension.  Hwang et al first reported that rats fed a diet high in fructose exhibited insulin resistance, hyperinsulinemia, hypertriglyceridemia and hypertension (Hwang et al., 1987). Substituting the starch carbohydrate content in laboratory rodent diet with fructose resulted in elevated blood pressure within a period of 6-8 weeks (Reaven et al., 1989; Vasudevan et al., 2005; Verma et al., 1994).  The effects of high fructose feeding have been reported to be  concentration- and time-dependent (Dai and McNeill, 1995).  Given that high fructose  feeding does not result in weight gain during this time period, the fructose-fed rat is used to study the relationship between these metabolic defects and the development of hypertension independent of obesity or genetic contributions. Interventions that either reduce plasma insulin levels or improve insulin sensitivity prevent the development of hypertension in fructose-fed rats. Exercise training reduced plasma insulin levels, improved insulin sensitivity and normalized blood pressure (Reaven et al., 1988). Somatostatin, a hormone that suppresses the release of insulin, also lowered blood pressure (Reaven et al., 1989).  In addition, improving insulin sensitivity with  metformin (Verma et al., 1994; Verma et al., 2000), vanadium compounds (Bhanot et al., 1994; Bhanot et al., 1995) or thiazolidinediones (Chen et al., 1996; Kotchen et al., 1997; Lee et al., 1994) prevented the increase in blood pressure in these rats. These findings support the hypothesis that insulin resistance/hyperinsulinemia are causally linked to the development of hypertension in this animal model.  12  As shown in Figure 1.3, several mechanisms have been proposed to mediate the link between insulin resistance/hyperinsulinemia and hypertension, including continued activation of the sympathetic nervous system (Penicaud et al., 1998; Rosen et al., 1997; Verma et al., 1999), increased production and/or activity of vasoconstrictors, such as endothelin-1 (ET-1) (Cosenzi et al., 1999; Juan et al., 1998; Verma et al., 1995; Verma et al., 1997), angiotensin II (Ang II) (Iimura et al., 1995; Iyer and Katovich, 1994; Kobayashi et al., 1993; NavarroCid et al., 1995) or thromboxane A2 (TxA2) (Galipeau et al., 2001) and impaired endothelium-dependent relaxation (Katakam et al., 1998; Miller et al., 1998; Miller et al., 1999; Takagawa et al., 2001; Verma et al., 1996). Each of the proposed mechanisms can contribute to an increase in vascular tone, which may result in endothelial dysfunction and an increase in blood pressure.  13  GENETICS  ENVIRONMENT  INSULIN RESISTANCE HYPERINSULINEMIA  ↓ VASODILATION TO INSULIN  ↑ SYMPATHETIC NERVOUS SYSTEM ACTIVITY  ↑ VASOCONSTRICTORS (ET-1, Ang II, TxA2)  ↑ VASCULAR TONE  ↑ BLOOD PRESSURE  14  Figure 1.3  Proposed mechanisms that link insulin resistance/hyperinsulinemia to hypertension in fructose-fed rats.  1.3.1  THE ROLE OF THE SYMPATHETIC NERVOUS SYSTEM Insulin-induced stimulation of the sympathetic nervous system is a proposed  mechanism that links insulin resistance to hypertension. Stimulation of α1-adrenoceptors on vascular smooth muscle cells can activate phospholipase C (PLC) and increase the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3), resulting in increased calcium concentrations. Agonists, such as norepinephrine (NE), ET-1 or Ang II, that activate G-protein coupled receptors can also increase the calcium sensitivity of the contractile machinery independent of changes in intracellular calcium concentrations, a process known as calcium sensitization (Loirand et al., 2006). The resulting increase in intracellular calcium concentrations and/or increase in calcium sensitization lead to vascular smooth muscle contraction. In the setting of hyperinsulinemia, insulin may chronically activate the sympathetic nervous system, resulting in increased peripheral vascular tone and elevated blood pressure. In support of this hypothesis, plasma NE levels and sympathetic nerve activity are increased in response to insulin infusion (Anderson et al., 1991; Lembo et al., 1992; Liang et al., 1982; Rowe et al., 1981), with no change in blood pressure (Anderson et al., 1991; Berne et al., 1992). The lack of effect on blood pressure despite the increase in sympathetic nerve activity may result from vasodilation in the vasculature of skeletal muscle, which results in the compensatory redistribution of cardiac output (Baron, 1993; Sartori et al., 1999). As a consequence of continued α1-adrenoceptor activation, an increase in the activity of the sympathetic nervous system may contribute to insulin resistance through enhanced vasoconstriction, reducing blood flow and glucose delivery to insulin-sensitive tissues (Rattigan et al., 1999). The compensatory hyperinsulinemia in response to insulin resistance  15  can then act as a continued stimulus to activate the sympathetic nervous system. The issue of which abnormality occurs first has been raised. Specifically, do elevated levels of insulin activate the sympathetic nervous system or does activation of the sympathetic nervous system exacerbate insulin resistance? As a result of the cyclical relationship between insulin resistance and elevated sympathetic activity, determining the primary event has been a difficult task. Chronic activation of the sympathetic nervous system has been suggested as an initial, precipitating event in the development of hypertension (Verma et al., 1999). In support of this hypothesis, it has been demonstrated that sympathetic nerve hyperactivity preceded hyperinsulinemia and subsequent elevations in blood pressure in a population of young, non-obese Japanese individuals (Masuo et al., 1997). In fructose-fed rats, chemical sympathectomy prevented the development of hyperinsulinemia and hypertension, demonstrating that a functional sympathetic nervous system is necessary for the development of hyperinsulinemia and hypertension in this animal model (Verma et al., 1999). Treatment with moxonidine (Rosen et al., 1997) or rilmenidine (Penicaud et al., 1998), imidazoline receptor agonists that centrally reduce sympathetic outflow, also prevented the development of insulin resistance and hypertension. Vessels from fructose-fed rats exhibited decreased vascular responses to NE, suggesting an impaired sensitivity to NE (Bunnag et al., 1997). This effect may be explained as a compensatory change following increased activity of the sympathetic nervous system, which can lead to receptor downregulation or desensitization (Hogikyan and Supiano, 1994; Sun and Hanig, 1983).  16  1.3.2  THE ROLE OF THE ENDOTHELIUM AND ENDOTHELIUMDERIVED FACTORS Within the cardiovascular system, blood vessels function to transport and supply  blood throughout the body. Figure 1.4 illustrates the various layers within a blood vessel. The adventitia is the outermost layer composed of connective tissue. The media is the middle layer and consists of vascular smooth muscle cells. The innermost layer is the intima and it consists of a single layer of endothelial cells that make up the endothelium. The endothelium is involved in both physiological and pathophysiological processes. It acts as a protective layer and a permeability barrier to the movement of substances throughout blood vessels. As well, it plays a key role in regulating vascular tone through the synthesis and release of numerous relaxing and contracting factors.  Examples of  endothelium-derived relaxing factors include nitric oxide (NO), endothelium-dependent hyperpolarizing factor (EDHF) and prostacyclin (PGI2). Endothelium-derived contracting factors include ET-1, Ang II and TxA2. These factors are released in response to various stimuli including neurohumoral factors and mechanical forces (Luscher and Barton, 1997; Luscher and Noll, 1995; Schiffrin, 1994) and can act directly on endothelial cells or surrounding vascular smooth muscle cells. The endothelium also releases a number of factors involved in coagulation, maintenance of blood fluidity, and inhibition and promotion of vascular growth (Luscher and Barton, 1997; Luscher and Noll, 1995; Schiffrin, 1994). Disturbances in the balance between these relaxing and contracting factors may play a role in the initiation or progression of vascular diseases and may contribute to the development of hypertension.  17  endothelial cell  intima  media  adventitia smooth muscle cell  Figure 1.4  A cross section of a blood vessel.  18  1.3.2.1  THE ENDOTHELIN SYSTEM  ET-1 was originally isolated and identified from the supernatant of porcine endothelial cells (Yanagisawa et al., 1988). It is the major peptide primarily produced by endothelial cells and is preferentially released toward vascular smooth muscle cells to produce sustained increases in vascular tone (Perez del Villar et al., 2005). There are three isoforms of endothelin, ET-1, ET-2 and ET-3. Each of these isoforms is encoded by a separate gene and is differentially distributed in tissues and cells (Inoue et al., 1989). The actions of ET-1 are mediated by its two receptors, ETA and ETB. A schematic illustration of the vascular endothelin system is shown in Figure 1.5.  1.3.2.1.1  Synthesis of ET-1  Biosynthesis of ET-1 begins with processing of prepro-endothelin-1 (prepro-ET-1), a 200 amino acid precursor peptide, to pro-endothelin-1. Pro-endothelin-1 is converted within the cell to a 38-41 amino acid long biologically inactive intermediate, big-endothelin-1 (bigET-1) (Blais et al., 2002). Big-ET-1 is further processed by endothelin converting enzyme (ECE) into mature and active ET-1 (D'Orleans-Juste et al., 2003; Schiffrin and Touyz, 1998). ECE can process big-ET-1 intracellularly as well as on the surface of the cell (Barnes et al., 1998; Kedzierski and Yanagisawa, 2001; Xu et al., 1994). Two isoforms of ECE exist, ECE1 and ECE-2. ECEs are membrane-bound zinc metalloproteases that cleave big-ET-1 with greater efficiency than either big-ET-2 or big-ET-3 (Schneider et al., 2007). Alternative splicing of both ECE-1 and ECE-2 produces subisoforms that are differentially expressed in subcellular sites within endothelial cells (Schneider et al., 2007).  19  endothelial cell NO ANF PGI2  Big-ET-1  Prepro-ET-1 Ang II NE cytokines thrombin  ETB  ECE  release of NO  ET-1  ETA  ETB  ?  contraction  clearance  vascular smooth muscle cell  Figure 1.5  The vascular endothelin system.  20  Since cells that produce ET-1 lack storage vesicles or regulatory secretory pathways, regulation of ET-1 occurs through changes in gene expression and/or ECE activity (Brunner et al., 2006) and any increase in ET-1 secretion above basal levels requires 2 to 5 hours (Perez del Villar et al., 2005).  Many factors can regulate the levels of prepro-ET-1.  Upregulation of prepro-ET-1 mRNA occurs in response to substances such as NE (Yanagisawa et al., 1988), Ang II (Dohi et al., 1992; Emori et al., 1991), vasopressin (Bakris et al., 1991; Emori et al., 1991), thrombin (Kohno et al., 1990; Kohno et al., 1992) and tumor necrosis factor-α (TNF-α) (Marsden and Brenner, 1992; Orisio et al., 1992). Vasodilators such as NO (Boulanger and Luscher, 1990), PGI2 (Prins et al., 1994), bradykinin (Ohde et al., 1991) and atrial natrietic factor (ANF) (Kohno et al., 1992) downregulate transcription of ET-1. Plasma ET-1 levels are considered to indicate an excess in the local release of ET-1, therefore circulating levels of ET-1 may not necessarily reflect local tissue levels for two main reasons. Firstly, nearly 80% of ET-1 produced by endothelial cells is secreted toward the underlying vascular smooth muscle cells (Perez del Villar et al., 2005). Secondly, ET-1 binds with high affinity to its receptors and has a slow rate of dissociation (Waggoner et al., 1992). Plasma concentrations of ET-1 range between 1-10 pM (Haynes and Webb, 1998), which is considerably lower than the pharmacological threshold of 0.05-1 nM (Frelin and Guedin, 1994; Haynes and Webb, 1998). With a half-life of less than 90 seconds, ET-1 is rapidly cleared from the circulation (Perez del Villar et al., 2005) primarily through the lungs, as well as the kidneys, liver and heart (Brunner et al., 2006). Clearance of ET-1 occurs through an ETB receptor-mediated pathway (Fukuroda et al., 1994) in which the ET1-ETB receptor complex is internalized and targeted to lysosomes for degradation (Brunner et  21  al., 2006). This clearance process is important in maintaining low concentrations of tissue ET-1 since impaired clearance may lead to enhanced local effects of ET-1, such as vasoconstriction (Brunner et al., 2006).  1.3.2.1.2  Endothelin receptors and signaling  ETA and ETB receptors belong to the family of G-protein coupled receptors and are classified based on their rank order of potencies for the endothelins (Davenport, 2002). ETA receptors bind ET-1 and ET-2 with greater affinity than ET-3 whereas ETB receptors bind all three isoforms with equal affinity (Davenport, 2002). In the vasculature, both ETA and ETB receptors are located on vascular smooth muscle cells and binding of ET-1 mediates vasoconstriction (Rubanyi and Polokoff, 1994). Activation of ETA and ETB receptors on vascular smooth muscle cells leads to activation of PLC, resulting in the generation of second messengers DAG and IP3. Binding of IP3 to its receptor, IP3R, results in a transient increase in intracellular calcium concentrations. Binding of ET-1 also activates receptor-operated and voltage-gated calcium channels in the plasma membrane, allowing for the entry of calcium from extracellular stores, which results in sustained elevations in calcium and contributes to the prolonged vasoconstriction that is elicited by ET-1. Activation of ETA receptors can also result in calcium sensitization (Evans et al., 1999) and may contribute to the development of hypertension (Masumoto et al., 2001; Uehata et al., 1997). ET-1 mediated vasoconstriction in vascular smooth muscle cells is shown in Figure 1.6. ETB receptors on endothelial cells stimulate the production of NO and vasodilator cyclooxygenase metabolites, which exert vasodilatory effects on underlying vascular smooth muscle cells (Kedzierski and Yanagisawa, 2001). Activation of ETB receptors on endothelial  22  cells activates soluble phospholipase A2 (PLA2) and guanylate cyclase (GC), increasing intracellular cyclic guanosine monophosphate (cGMP), which stimulates NO and prostaglandin release mediating vascular smooth muscle relaxation. ETB receptor mediated vasodilation is shown in Figure 1.7. ET-1 can also promote the growth and proliferation of vascular smooth muscle cells through ETA receptor mediated pathways which involves the activation of mitogen-activated protein kinases and transactivation of epidermal growth factor receptors (Schneider et al., 2007). Within the vasculature, ET-1 has also been reported to stimulate oxidative stress through ETA receptors (Callera et al., 2003; Loomis et al., 2005).  23  vascular smooth muscle cell ET-1 Ca2+  ETA  PIP2  ETB  PLC  DAG +  IP3  IP3R  PKC  Ca2+ ↑ Ca2+ Ca 2+ sensitization  VASOCONSTRICTION  Figure 1.6  ETA and ETB receptor mediated signal transduction in vascular smooth muscle cells.  24  endothelial cell ET-1  Membrane phospholipids  ETB  PIP2  PLA2  PLC  AA  DAG +  IP3  IP3R  cGMP  L-Arginine prostaglandins eNOS  ↑ Ca2+  RELEASE OF NO  Figure 1.7  ETB receptor mediated signal transduction in endothelial cells.  25  1.3.2.1.3  ET-1 in hypertension  Increasing evidence suggests a role for ET-1 in the pathogenesis of hypertension. In humans (Vierhapper et al., 1990) and animals (Hinojosa-Laborde et al., 1989), infusion of ET-1 increased blood pressure, while blockade of the endothelin system decreased blood pressure (Haynes et al., 1996). Intravenous infusion of ET-1 caused a rapid and transient vasodilation, followed by a prolonged, sustained and dose-dependent increase in blood pressure (Haynes and Webb, 1998; Rubanyi and Polokoff, 1994). The initial decrease in blood pressure occurs as a result of ETB receptor activation, which releases NO and PGI2 mediating vasodilation (Hirata et al., 1993). The prolonged vasoconstriction results from ETA receptor activation and the slow dissociation of ET-1 from its receptors (Perez del Villar et al., 2005). ET-1 has been implicated in the development of hypertension in numerous experimental models of hypertension, including the Dahl salt-sensitive rat (Ikeda et al., 1999; Kassab et al., 1997), deoxycorticosterone acetate (DOCA)-salt hypertensive rat (Lariviere et al., 1993; Lariviere et al., 1993; Nguyen et al., 1992), DOCA-salt-treated SHR (Suzuki et al., 1990) and fructose-fed rat (Verma et al., 1995; Verma et al., 1997). Insulin has been reported to simulate the secretion and expression of both ET-1 and its receptor. In response to insulin, ET-1 levels are increased in vitro (Frank et al., 1993; Oliver et al., 1991) and in vivo (Wolpert et al., 1993). In addition, ETA receptor expression and binding of ET-1 are increased in vascular smooth muscle cells (Frank et al., 1993) and rats exposed to chronic hyperinsulinemia exhibited increased ET-1 levels (Verma et al., 1995). It has been proposed that chronic hyperinsulinemia may continually stimulate the release of ET-1 and subsequently alter circulating or local levels of ET-1, resulting in  26  increased blood pressure (Verma et al., 1995). The results of Juan and colleagues support this hypothesis by demonstrating that chronic insulin infusion increased ET-1 content and induced hypertension through the actions of ET-1 (Juan et al., 2004). Verma and coworkers demonstrated that fructose-fed rats exhibited elevated systolic blood pressure (SBP), greater amounts of ET-1 in mesenteric arteries (Verma et al., 1995) and decreased vascular reactivity to ET-1 (Verma et al., 1997). The diminished response to ET-1 may be associated with receptor downregulation as a consequence of increased ET-1 production (Nguyen et al., 1992). In contrast, overexpression of vascular ET-1 and ETA receptor mRNA has also been reported in fructose-fed rats (Juan et al., 1998) and given that ET-1 normally downregulates ETA receptors in vascular smooth muscle cells (Yu and Davenport, 1995), this overexpression of ETA receptor mRNA may occur as a result of receptor or post receptor defects that allows for continued vasoconstriction and elevated blood pressure (Juan et al., 1998). It has also been reported that mesenteric arteries from fructose-fed rats exhibited increased maximal binding of ET-1 and enhanced ET-1-induced vasoconstriction (Katakam et al., 2001). Chronic endothelin receptor antagonism in fructosefed rats lowered blood pressure and restored the altered vascular reactivity to ET-1, suggesting that ET-1 may link insulin resistance/hyperinsulinemia and hypertension (Verma et al., 1995; Verma et al., 1997).  27  1.3.2.2  THE RENIN ANGIOTENSIN SYSTEM  The renin angiotensin system maintains cardiovascular homeostasis by regulating fluid and electrolyte balance, as well as vascular tone (Dinh et al., 2001). A schematic illustration depicting the effects of the classical renin angiotensin system on blood pressure regulation is shown in Figure 1.8. Ang II is the biologically active component of the renin angiotensin system and can be produced systemically through the classical renin angiotensin system, locally through tissue renin angiotensin systems (Berry et al., 2001) or intracellularly (Kumar et al., 2007). As a result, the renin angiotensin system can function in an endocrine, paracrine or autocrine manner.  1.3.2.2.1  Synthesis and effects of Ang II  In the classical renin angiotensin system, angiotensinogen, which is produced in the liver, is enzymatically cleaved by renin, which is released from juxtaglomerular cells of the kidney, to form angiotensin I (Ang I), an inactive decapeptide (Dinh et al., 2001). Within the pulmonary circulation, Ang I is further converted by angiotensin converting enzyme (ACE) to Ang II, the active octapeptide. Ang II can also be synthesized through renin- and ACEindependent pathways (Kramkowski et al., 2006). Degradation of Ang II occurs through enzymatic cleavage by aminopeptidases producing angiotensin III and angiotensin IV (Lavoie and Sigmund, 2003). The biological effects of Ang II are mediated through two receptors, angiotensin II type 1 (AT1) or type 2 (AT2) receptors.  28  blood pressure increases salt retention  aldosterone  angiotensin II angiotensin converting enzyme angiotensin I  renin  angiotensinogen  blood pressure decreases  Figure 1.8  The classical renin angiotensin system.  29  Regulation of Ang II occurs through AT1 receptors. Acute exposure to Ang II results in increased activation of AT1 receptors, whereas chronic exposure downregulates AT1 receptors (Gunther et al., 1980).  Continued Ang II stimulation leads to receptor  desensitization, which can occur via two mechanisms, receptor phosphorylation or receptor internalization. In vascular smooth muscle cells, Ang II-induced activation of AT1 receptors resulted in phosphorylation of AT1 receptors at serine and tyrosine residues (Kai et al., 1994), therefore preventing downstream targets from being activated. Ang II-AT1 receptor complexes also undergo endocytosis in which a proportion of receptors are recycled to the plasma membrane while the majority of receptors are degraded in lysosomes (Gunther et al., 1980; Ullian and Linas, 1989). The processes of receptor phosphorylation and receptor internalization are responsible for desensitizing tissues following continued exposure to Ang II. AT1 receptors can also be upregulated or downregulated by a number of factors, such as insulin (Takayanagi et al., 1992) and NO (Ichiki et al., 1998), respectively. Activation of AT1 receptors is responsible for most biological actions of Ang II that are involved in the regulation of blood pressure and fluid homeostasis. The regulation of blood pressure occurs through vascular and renal effects of Ang II. In the vasculature, Ang II elicits vasoconstriction resulting in increased vascular tone and blood pressure (Berry et al., 2001). In the kidney, there are direct and indirect effects of Ang II. Binding to renal AT1 receptors stimulates renal sodium and water reabsorption (Bottari et al., 1993). Activation of AT1 receptors on the adrenal gland stimulates the production and release of aldosterone, which also stimulates renal sodium and water reabsorption (Aquitlera and Marusic, 1971). Ang II also stimulates sympathetic transmission by enhancing the release of catecholamines (Reid, 1992). The cardiovascular effects of AT2 receptor activation are less well understood,  30  but are thought to antagonize those of the AT1 receptors and be involved in processes such as anti-proliferation, apoptosis, differentiation and vasodilation (Widdop et al., 2003). Given that components of the renin angiotensin system are localized in tissues that are not part of the classical system (Ganten et al., 1971; Ganten et al., 1971), the concept of local and intracellular renin angiotensin systems has emerged. All components of the renin angiotensin system can be found in various tissues, including the brain (Bader and Ganten, 2008; Paul et al., 2006), heart (Bader and Ganten, 2008; Paul et al., 2006), vasculature (Bader and Ganten, 2008; Paul et al., 2006), adipose tissue (Engeli et al., 2000), gonads (Paul et al., 2006; Speth et al., 1999), pancreas (Paul et al., 2006), placenta (Nielsen et al., 2000) and kidney (Bader and Ganten, 2008). These local systems do not appear to function independently and are thought to interact with the classical renin angiotensin system (Paul et al., 2006). Therefore, local production of Ang II may also contribute to the pathogenesis of cardiovascular disease.  1.3.2.2.2  Ang II receptors and signaling  AT1 and AT2 receptors belong to the family of G-protein coupled receptors. Given that both receptors bind Ang II with equal affinity (de Gasparo et al., 1995), AT1 and AT2 receptors are classified on the basis of selective antagonism. AT1 receptors are selectively antagonized by biphenylimidazoles, such as losartan, whereas AT2 receptors are selectively inhibited by tetrahydroimidazopyridines, such as PD123319 (Ardaillou, 1999). In the vasculature, AT1 receptors are highly expressed on smooth muscle cells (Mehta and Griendling, 2007) and activation of AT1 receptors can lead to five different signaling cascades, of which activation of PLC is the most described (Dinh et al., 2001). Similar to ETA receptor activation, activation of PLC results in the generation of secondary messengers,  31  DAG and IP3. Binding of IP3 to its receptor, IP3R, results in an increase in intracellular calcium concentrations. Activation of PLA2 and phospholipase D (PLD) stimulates the release of arachidonic acid (AA), resulting in prostaglandin synthesis. Activation of AT1 receptor coupled to Gi leads to inhibition of adenylate cyclase (AC) and prevents the production of cyclic adenosine monophosphate (cAMP). Given that increased levels of cAMP mediates vasodilation, preventing the increase in cAMP results in vasoconstriction. Lastly, opening of L-type calcium channels allows for the influx of extracellular calcium into the cell, increasing the calcium concentration and resulting in vasoconstriction. Binding of Ang II can also activate the RhoA/Rho-kinase pathway (Somlyo and Somlyo, 2003), another important mechanism involved in regulating vascular tone.  Activation of this pathway  increases the contractile response of smooth muscle cells to an increase in intracellular calcium (Sward et al., 2003) and may play a role in the development of hypertension (Masumoto et al., 2001; Uehata et al., 1997). AT1 receptor mediated signaling in vascular smooth muscle cells is illustrated in Figure 1.9. Depending on the cell type, various intracellular signaling cascades have been described for AT2 receptors, including stimulation of the NO-cGMP pathway (Johren et al., 2004), activation of potassium channels (Kang et al., 1993) or protein phosphatases (Dinh et al., 2001), or stimulation of PLA2 leading to the production of AA (Dinh et al., 2001). AT2 receptor mediated signaling pathways are illustrated in Figure 1.10.  32  vascular smooth muscle cell Ang II Ca2+  PIP2  AT1  Membrane phospholipids  AT1  PLA2  PLC  PLD DAG  PKC  AC  + IP3  prostaglandins IP3R  RhoA ↓ cAMP ROCK  Ca2+  ↑ Ca2+  VASOCONSTRICTION  Figure 1.9  AT1 receptor mediated signal transduction in vascular smooth muscle cells.  33  vascular smooth muscle cell  Ang II Ca2+  AT2  K+  Membrane phospholipids  NO  PLA2  phosphatases  K+  cGMP HYPERPOLARIZATION  VASORELAXATION  Figure 1.10  AT2 receptor mediated signal transduction.  34  1.3.2.2.3  Ang II in hypertension  Since the renin angiotensin system is an important mediator in the maintenance of blood pressure, overactivation of this system has been proposed to contribute to the development of hypertension following insulin resistance. Increasing evidence indicates that Ang II inhibits the action of insulin in vascular smooth muscle cells by interfering with the insulin signaling cascade through the phosphoinositide-3 kinase (PI3K)/protein kinase B (Akt) signaling pathway, leading to inhibition of vasodilatory mechanisms (Sowers, 2004). In hypertensive individuals, treatment with either an ACE inhibitor or angiotensin receptor antagonist reduced the incidence of diabetes (Scheen, 2004). In rats, infusion of Ang II led to hepatic insulin resistance and elevated blood pressure (Rao, 1994), as well as endothelial dysfunction (Diep et al., 2002). Therefore, elevations in Ang II may contribute to insulin resistance through its vasoconstrictor actions, which reduces blood flow and glucose uptake into insulin-sensitive tissues (Iyer and Katovich, 1996). Fructose-fed rats exhibited elevated Ang II (Iyer et al., 1996) and altered AT1 receptor expression (Giacchetti et al., 2000; Iyer et al., 1996; Nyby et al., 2007), whereas blockade of the renin angiotensin system with either an ACE inhibitor or angiotensin receptor antagonist prevented the development of hypertension (Iimura et al., 1995; Iyer and Katovich, 1994; Kamide et al., 2002; Navarro-Cid et al., 1995; Uchida et al., 2002). In conjunction with reduced blood pressure, improvements in insulin sensitivity were also observed (Iimura et al., 1995; Kobayashi et al., 1993; Navarro-Cid et al., 1995; Uchida et al., 2002).  Blockade of the renin angiotensin system also improved the altered vascular  reactivity associated with fructose feeding (Navarro-Cid et al., 1995; Uchida et al., 2002).  35  The mechanism for improved insulin sensitivity following blockade of the renin angiotensin system is dependent on which class of drug used.  Angiotensin receptor  antagonists prevent Ang II from binding to AT1 receptors, thereby inhibiting vasoconstriction and increasing muscle flow through vasodilation.  ACE inhibitors can improve insulin  sensitivity via two mechanisms. ACE inhibitors reduce ACE-mediated generation of Ang II and prevent the degradation of bradykinins, which function to relax blood vessels through endothelium-dependent mechanisms (Wiemer et al., 1991).  36  1.3.2.3  THE NO SYSTEM  The discovery of NO emerged as a result of two significant observations. Firstly, Katsuki and coworkers reported that the vasodilatory effects of nitrovasodilator compounds, such as sodium nitroprusside and nitroglycerine, activated soluble GC and that this activation was proposed to occur through the formation of NO (Katsuki et al., 1977). Secondly, Furchgott and Zawadzki observed that acetylcholine (ACh)-induced relaxation of blood vessels required an intact endothelium and hypothesized that a compound, referred to as endothelium-derived relaxing factor (EDRF), was released by endothelial cells to act on smooth muscle cells (Furchgott and Zawadzki, 1980).  Later, Palmer and colleagues  demonstrated that EDRF was in fact NO (Palmer et al., 1987) and that NO is synthesized from L-arginine (Palmer et al., 1988; Palmer et al., 1988). NO is one of the most important vasodilators and determinants of basal vascular tone (Luscher and Barton, 1997).  NO is synthesized from L-arginine through nitric oxide  synthase (NOS). Three forms of NOS exist: endothelial (eNOS or NOS III), neuronal (nNOS or NOS I) and inducible (iNOS or NOS II). All NOS isoforms are homodimeric enzymes that require oxygen and nicotinamide adenine dinucleotide phosphate (NADPH) as cosubstrates and flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), tetrahydrobiopterin and heme as co-factors. eNOS and nNOS are constitutively expressed and are calcium-calmodulin-dependent whereas iNOS is inducible and calcium-calmodulinindependent (Schiffrin, 1994). In the endothelium, the predominantly expressed isoform is eNOS. NO is produced in response to shear stress and various factors, such as ACh and bradykinin (Schiffrin, 1994). With a half-life of only a few seconds (Hakim et al., 1996), NO can readily diffuse through  37  the cytoplasm and plasma membrane to act in an autocrine or paracrine manner. NO can react with oxygen and water to form nitrates and nitrites or with superoxide anions to form peroxynitrite, an oxidizing free radical that fragments DNA and oxidizes lipids. In the vasculature, NO stimulates soluble GC by binding to the heme moiety of the enzyme. Activation of soluble GC converts guanine triphosphate (GTP) to cGMP resulting in smooth muscle relaxation and vasodilation (Forstermann et al., 1986; Rapoport and Murad, 1983), as shown in Figure 1.11.  38  endothelial cell L-Arginine eNOS  NO  RELAXATION ↓ Ca2+  NO  K+ GTP sGC cGMP vascular smooth muscle cell  Ca2+  Figure 1.11  NO mediated signal transduction.  39  1.3.2.3.1  NO in hypertension  Disturbances in the NO pathway along with subsequent impairments in endotheliumdependent relaxation have been proposed as a link between insulin resistance and hypertension (De Meyer and Herman, 1997; Schiffrin, 1994). In support of this hypothesis, inhibition of NO synthesis has been shown to elevate blood pressure (Rees et al., 1989). In several animal models of hypertension (Lockette et al., 1986; Luscher and Vanhoutte, 1986) and in hypertensive individuals (Panza et al., 1993; Panza et al., 1990), impaired endothelium-dependent relaxation has been reported. Reductions in NO levels have been demonstrated to be associated with endothelial dysfunction, which may occur as a result of decreased eNOS activity or reduced bioavailability of NO (Endemann and Schiffrin, 2004). Along with several laboratories, we have demonstrated that vascular tissues from fructose-fed rats exhibited impaired endothelium-dependent relaxation (Kamata and Yamashita, 1999; Miller et al., 1998; Verma et al., 1996). We have previously demonstrated the endothelium-dependent vasodilation response to insulin was abolished in aortas of fructose-fed rats (Verma et al., 1997). This impairment has been attributed to defects in vasodilatory mechanisms associated with NO (Kamata and Yamashita, 1999) and EDHF (Miller et al., 1998).  Thus during hypertension, a diminished synthesis, release or  responsiveness of vascular smooth muscle cells to NO may cause these cells to become more sensitive to the effects of vasoconstrictors, leading to enhanced vascular tone and elevated blood pressure  40  1.3.3  THE ROLE OF TNF-α In recent years, researchers have suggested that chronic inflammation may play an  important role in the development of insulin resistance and Type 2 diabetes (Pradhan et al., 2003; Pradhan et al., 2001). Numerous epidemiological studies reported strong positive associations between systemic markers of inflammation with features of the metabolic syndrome and/or cardiovascular risk (Festa et al., 2000; Frohlich et al., 2000; Pickup et al., 1997).  Specifically, systemic markers of inflammation were associated with the  development of hypertension (Bautista et al., 2005; Chrysohoou et al., 2004; Sesso et al., 2003), suggesting that an inflammatory component may contribute to this disorder. Increasing evidence has implicated TNF-α as a key mediator involved in the development of insulin resistance (Borst, 2004; Moller, 2000).  1.3.3.1  TNF-α biology  TNF-α is a major proinflammatory cytokine that elicits a broad range of immunological and metabolic responses. The biological effects of TNF-α depend on its relative concentration, the length of exposure and presence of other mediators (Camussi et al., 1991).  At low concentrations, TNF-α is beneficial in improving host defence  mechanisms against infections, while at high concentrations it can lead to excessive inflammation and organ injury (Tracey et al., 2008).  1.3.3.2  Synthesis of TNF-α  Biosynthesis of TNF-α is induced by many stimuli, including bacteria, viruses and cytokines such as interleukin-6 (IL-6), and is tightly regulated at the transcriptional and post-  41  transcriptional level (Tracey et al., 2008). TNF-α is synthesized de novo by many cells and is expressed as a 26 kDa transmembrane precursor, which is biologically active when assembled as a homotrimer (Tracey et al., 2008).  The extracellular domain of  transmembrane TNF-α is enzymatically cleaved by TNF-α converting enzyme (TACE), forming a 17 kDa soluble protein (Moller, 2000). Homotrimerization of these monomers is required for TNF-α to be biologically active. The effects of TNF-α are mediated by two receptors: tumor necrosis factor receptor type 1 (TNFR1) or type 2 (TNFR2) (Tracey et al., 2008), with the majority of its biological actions occurring through TNFR1 activation.  1.3.3.3  TNF-α receptors and signaling  TNFR1 and TNFR2 are membrane glycoproteins that bind TNF-α with approximately equal affinity (Tracey et al., 2008). TNFR1 is constitutively expressed on most cell types while TNFR2 is inducible and its expression is limited (Hehlgans and Mannel, 2002). Depending on the state of the cell, ligand binding to TNFR1 can result in either apoptosis or inflammation. Prior to ligand binding, trimers of receptor chains are preassembled on the cell surface (Chan et al., 2000). Upon ligand binding, the TNF-αTNFR1 complex is internalized and the adaptor molecule TNFR-associated death domain protein (TRADD) is recruited to the intracellular ‘death domain’ of TNFR1.  Further  recruitment of the Fas-associated death domain protein (FADD) activates caspase-8, which eventually leads to DNA degradation and cell death. Alternatively, TRADD can recruit receptor-interacting protein (RIP), which recruits tumor necrosis factor receptor-associated factor 2 (TRAF2). The IκB kinase (IKK) complex then interacts with RIP and TRAF2. Activation of the IKK complex results in phosphorylation of one of the catalytic subunits, IκB, causing its degradation and releasing the transcription factor nuclear factor κB, (NFκB).  42  NFκB translocates into the nucleus where it binds to DNA and activates the transcription of numerous proinflammatory genes, including cytokines, chemokines and growth factors (Hayden and Ghosh, 2004; Perkins, 2007). Ligand binding of TNFR1 can also induce TACE-mediated cleavage and shedding of the extracellular portion of TNFR1 and TNFR2 (Higuchi and Aggarwal, 1994; Tracey et al., 2008). The shed forms of TNFR1 and TNFR2 are soluble receptors that act as natural antagonists by binding to and inhibiting TNF-α (Camussi et al., 1991). These soluble receptors can also prevent circulating TNF-α from being cleared, prolonging or enhancing the activity of TNF-α (Aderka et al., 1992). Transmembrane TNF-α bound to TNFR1 can also induce a process referred to as reverse signaling, in which transmembrane TNF-α can stimulate cell activation, cytokine suppression or apoptosis (Eissner et al., 2000; Eissner et al., 2004). The extracellular domain of TNFR2 is similar to that of TNFR1, but differs in its intracellular domain and signal transduction pathway. TNFR2 lacks an intracellular ‘death domain’ and is more efficiently activated by transmembrane TNF-α. Upon ligand binding of TNF-α, TNFR2 homotrimerizes and recruits adaptor proteins TRAF2 and tumor necrosis factor receptor-associated factor 1 (TRAF1), which activates the NFκB pathway. TNFR2 also functions in concentrating TNF-α at the cell membrane and passing it to TNFR1, a concept known as ‘ligand passing’. TNFR1 and TNFR2 mediated signaling pathways are shown in Figure 1.12.  43  TNF-α  TNFR1  TNFR2  TRAF2 TRADD  FADD  RIP  caspase 8  TRAF2  APOPTOSIS  Figure 1.12  TRAF1  NFκB  NFκB  INFLAMMATION  INFLAMMATION  TNFR1 and TNFR2 mediated signaling pathways.  44  1.3.3.4  TNF-α in insulin resistance  TNF-α is constitutively expressed in adipocytes and its expression is elevated in the adipose tissue of obese animals (Hofmann et al., 1994; Hotamisligil et al., 1993) and humans (Kern et al., 1995).  Studies have demonstrated that TNF-α treatment induced insulin  resistance (Hofmann et al., 1994; Hotamisligil et al., 1994; Hotamisligil et al., 1993; Uysal et al., 1997) while inhibition of TNF-α improved insulin sensitivity in obese rodents (Hotamisligil et al., 1993; Uysal et al., 1997). Elevated levels of TNF-α have also been associated with the development of hypertension (Bautista et al., 2005; Furumoto et al., 2002; Ito et al., 2001; Zinman et al., 1999). TNF-α has been reported to directly inhibit the insulin signaling pathway at various sites.  TNF-α induced serine phosphorylation of insulin receptor substrate-1 (IRS-1), a  downstream effector of the insulin signaling cascade, and rendered IRS-1 resistant to insulin stimulated tyrosine phosphorylation (Borst, 2004). This modification results in reduced PI3K docking and impaired insulin-stimulated glucose transport. TNF-α interrupted insulin signaling through phosphorylation and activation of SH-PTPase (Ahmad and Goldstein, 1997), a protein tyrosine phosphatase that dephosphorylates IRS-1. TNF-α phosphorylated and inactivated protein phosphatase-1, a protein phosphatase that stimulates glycogen synthase and glucose storage when activated (Ragolia and Begum, 1998). Each of these molecular modifications is involved in impairing the insulin signal and may contribute to the development of insulin resistance. TNF-α has been demonstrated to directly interfere with triglyceride metabolism in both adipose tissue and liver. TNF-α stimulated lipolysis and increased the concentration of free fatty acids (Feingold et al., 1990). Free fatty acids are released into the circulation and  45  are substrates for triglyceride synthesis. TNF-α also reduced the clearance of very low density lipoproteins (VLDL) from the circulation (Feingold et al., 1994), contributing to increased levels of triglycerides.  As hypertriglyceridemia is associated with insulin  resistance, the effects of TNF-α on lipid metabolism may also contribute to the development of insulin resistance. Several investigators have reported that TNF-α inhibited endothelium-dependent relaxation (Aoki et al., 1989; Greenberg et al., 1993; Liu et al., 1992; Wang et al., 1994; Wimalasundera et al., 2003). The inhibitory effect of TNF-α on endothelium-dependent relaxation has also been observed in humans. Nakamura and co-workers demonstrated that TNF-α infusion increased forearm vascular resistance (Nakamura et al., 2000). Fichtlscherer and colleagues also reported that administration of etanercept, a soluble recombinant tumor necrosis factor receptor that binds to and inactivates TNF-α, improved endotheliumdependent relaxation (Fichtlscherer et al., 2001).  Improved endothelium-dependent  relaxation following etanercept treatment has also been reported in rats (Arenas et al., 2005; Arenas et al., 2006; Arenas et al., 2006; Csiszar et al., 2007).  46  2  RESEARCH OUTLINE  2.1  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension  2.1.1  Rationale In conditions where both the endothelin system and renin angiotensin system are  activated, interrelationships between them have been proposed to contribute to the development of hypertension. For example, chronic infusion of subpressor doses of both Ang II and ET-1 induced significant increases in SBP, whereas a subpressor dose of either Ang II or ET-1 had no effect in conscious rats, leading the authors to suggest that the combined actions of both peptides may be involved in blood pressure regulation (Yoshida et al., 1992). In fructose-fed rats, it has been reported that blood pressure was reduced by a greater degree in the presence of a combination treatment consisting of an ACE inhibitor, trandolapril, and an endothelin receptor antagonist, LU-135252, as compared to either treatment alone (Ezra-Nimni et al., 2003). However, whether an interrelationship exists between these systems that contribute to the development of hypertension in fructose-fed animals is currently unknown. Bosentan is a dual endothelin receptor antagonist. It competitively and reversibly antagonizes the specific binding of ET-1 more strongly at the ETA receptor than at the ETB receptor (Clozel et al., 1994). At a dose of 100 mg/kg/day, bosentan has been reported to exhibit antihypertensive properties in rats (Clozel et al., 1994). In isolated rat aorta, bosentan selectively inhibited ET-1 binding and blocked ET-1-induced contraction (Clozel et al.,  47  1994). L-158,809 is a potent and selective AT1 receptor antagonist that has similar affinity for AT1 receptors as Ang II (Chang et al., 1992). The affinity of L-158,809 for AT1 receptors is greater than other reported angiotensin receptor antagonists (Chang et al., 1992). L-158,809 binds in a reversible and competitive manner and in vivo experiments demonstrated antihypertensive effects at doses as low as 0.1-0.3 mg/kg in rats (Siegl et al., 1992). For these reasons, we utilized bosentan and L-158,809 as pharmacological tools to investigate the role of ET-1 and Ang II in the development of fructose-induced hypertension.  2.1.2  Objective The aim of this study was to investigate and define the existence of an interaction  between the endothelin system and renin angiotensin system that may play a role in the development of hypertension in insulin resistant fructose-fed rats. We determined the effects of a dual endothelin receptor antagonist, bosentan, or an AT1 receptor antagonist, L-158,809, on plasma levels of insulin, glucose and triglycerides, SBP and insulin sensitivity.  In  addition, we assessed the effect of bosentan treatment on plasma Ang II levels and the effect of L-158,809 treatment on vascular ET-1-immunoreactivity (ET-1-ir).  2.1.3  Hypothesis We hypothesized that both the endothelin system and renin angiotensin system  contribute to the development of hypertension in fructose-fed rats and that these systems are interconnected. If the endothelin system elicits its effects upstream of the renin angiotensin system, we expected to observe reduced or normalized Ang II levels in the presence of bosentan treatment. Likewise, if the renin angiotensin system acts through stimulation of the endothelin system, we expected downregulation of vascular ET-1-ir to occur.  48  2.2  Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats  2.2.1  Rationale In fructose-fed rats, chemical sympathectomy prevented the development of  hyperinsulinemia and hypertension (Verma et al., 1999), demonstrating that a functional sympathetic nervous system is necessary for the development of hyperinsulinemia and hypertension in this model. Treating fructose-fed rats with imidazoline receptor agonists that centrally reduce sympathetic outflow, such as moxonidine (Rosen et al., 1997) or rilmenidine (Penicaud et al., 1998), also prevented the development of insulin resistance and hypertension. Since blockade of the sympathetic nervous system prevented both insulin resistance/hyperinsulinemia and hypertension, it has been suggested that chronic overactivation of the sympathetic nervous system is an early precipitating event in the pathogenesis of fructose-induced hypertension. There has not been a study reporting the effect of chronic blockade of the sympathetic nervous system on other proposed mediators of fructose-induced hypertension, such as the renin angiotensin system.  2.2.2  Objective The aim of this study was to determine whether chronic overactivation of the  sympathetic nervous system is an initial defect that contributes to the development of hypertension in fructose-fed rats.  We determined the effects of an α1-adrenoceptor  antagonist, prazosin, on the general characteristics of rats, including plasma levels of insulin, glucose, triglycerides, and NE, SBP and insulin sensitivity. To determine whether chronic  49  activation of the sympathetic nervous system is an initial event that affects the renin angiotensin system, we assessed the effect of chronic prazosin treatment on plasma levels of Ang II.  2.2.3  Hypothesis We hypothesized that the development of hypertension in fructose-fed rats is  dependent on chronic activation of α1-adrenoceptors that acts as a precipitating event, which contributes to an increase in Ang II and is involved in the development of fructose-induced hypertension. If an increase in sympathetic drive occurs as an initiating event, we anticipated Ang II levels to reduced or normalized in the presence of prazosin treatment.  50  2.3  Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension  2.3.1  Rationale Togashi and co-workers have reported that inhibition of the TACE improved insulin  sensitivity in fructose-fed rats, however no accompanying reduction in blood pressure was observed (Togashi et al., 2002). Given that a relationship between insulin resistance and hypertension has been proposed (Bhanot and McNeill, 1996; DeFronzo, 1992; Reaven, 1988), we found the lack of effect on blood pressure regulation surprising. This effect may be due to the experimental design utilized as treatment with a TACE inhibitor occurred only during the final two weeks of the six weeks of fructose feeding. Preliminary studies were conducted in which we collected plasma from rats following one, two, seven and thirteen weeks of fructose-feeding for the measurement of plasma TNF-α as described in sections 3.1.3 and 3.1.4. As shown in Figure 2.1, our preliminary results suggest that elevations in plasma TNF-α occurred following two weeks of high fructose feeding, suggesting that an inflammatory insult may act as an initiating event in the development of fructose-induced hypertension. TNF-α has been reported to modulate the pathways of various vasoactive mediators including the endothelin system (Corder et al., 1995; Kanse et al., 1991; Marsden and Brenner, 1992), renin angiotensin system (Antonipillai et al., 1990; Brasier et al., 1996; Gurantz et al., 1999) and NO system (Davis et al., 2002; Giardina et al., 2002; Yoshizumi et al., 1993).  Perturbations in any of these pathways may promote the development of  endothelial dysfunction and contribute to hypertension; however the exact mechanism(s) by  51  which TNF-α modulates vasoactive mediators and contributes to the development of hypertension secondary to insulin resistance remains to be clarified.  52  PLASMA TNF- α (pg/mL)  60  C F  * 40  *  20  0 1  2  7  13  WEEKS  Figure 2.1  Effect of high fructose-feeding on plasma TNF-α following one, two, seven and thirteen weeks of study. Values expressed as mean ± SEM, n=7-11. * p<0.05, vs. C.  53  2.3.2  Objective The aim of the present study was to investigate the effects of chronic etanercept  treatment in insulin resistant fructose-fed rats. Etanercept is a soluble recombinant fusion protein consisting of the extracellular ligand binding domain of TNFR2. Etanercept binds to and inactivates circulating TNF-α, thereby acting as a competitive inhibitor of TNF-α. We determined the effects of etanercept on plasma parameters, SBP, insulin sensitivity and vascular reactivity. We examined the protein expression of various downstream targets of TNF-α, such as eNOS, iNOS, NFκB and caspase-3, a downstream effector of TNFR1induced apoptosis. We also investigated the role of calcium sensitization in the development of hypertension in fructose-fed rats by determining the protein expression of RhoA, and its downstream effectors, Rho-kinase-1 (ROCK-1) and Rho-kinase-2 (ROCK-2).  2.3.3  Hypothesis We hypothesized that elevated levels of TNF-α contribute to the development of  hypertension in fructose-fed rats by promoting an imbalance between vasoconstriction and vasodilation factors and that treatment with etanercept will prevent the development of hypertension by improving vascular function.  54  3  MATERIALS AND METHODS  3.1  General methodology  3.1.1  SBP measurements Prior to obtaining blood pressure measurements, rats were preconditioned to the  procedure. SBP was measured in conscious rats using the indirect non-invasive tail-cuff method without external preheating as previously described (Bunag, 1973; Hwang et al., 1987).  3.1.2  Oral glucose tolerance test and insulin sensitivity index At the end of the study, all rats were fasted overnight (15 hours) and subjected to an  oral glucose tolerance test (OGTT). A schematic illustrating an OGTT is shown in Figure 3.1. A 40% glucose solution was prepared and administered by oral gavage (1 g/kg) to conscious animals. Blood samples were obtained at 0, 10, 20, 30, 60 and 90 minutes following the glucose challenge. Plasma was separated and stored at -20°C until further analysis. The insulin sensitivity index was calculated for each animal using data obtained from the OGTT and applied to the formula of Matsuda and DeFronzo (1999) where insulin sensitivity index = 100/square root [(mean plasma glucose × mean plasma insulin) × (fasting plasma glucose × fasting plasma insulin)] (Matsuda and DeFronzo, 1999). Values obtained with this methodology correlate highly with results obtained from the euglycemic hyperinsulinemic clamp technique (Matsuda and DeFronzo, 1999).  55  PLASMA GLUCOSE (mmol/L)  10 9 8 7 6 0  30  60  90  PLASMA INSULIN (ng/mL)  TIME (min) 4  3  2  1 0  20  40  60  80  TIME (min)  collect basal blood sample  Figure 3.1  oral glucose challenge  10 min 20 min 30 min  60 min  90 min  collect blood samples  A schematic illustration of an OGTT.  56  3.1.3  Blood collection Blood samples were collected from the tail vein for determination of plasma glucose,  insulin, triglycerides, TNF-α and IL-6 levels. Plasma samples were separated, aliquoted and stored at -20°C until further analysis. At termination, blood was collected via cardiac puncture. For determination of plasma Ang II levels, blood was placed into plastic tubes containing 0.44 mM ο–phenanthroline, 25 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ρ–hydroxymercuribenzoic acid and 0.12 mM pepstatin A. For determination of plasma NE levels, blood was aliquoted into heparinized tubes. Plasma samples were separated and stored at -20°C until analysis.  3.1.4  Biochemical measurements Plasma glucose levels were determined through a Beckman Glucose Analyzer II  (Beckman, Fullerton, CA). Plasma triglycerides were measured using an enzymatic colourimetric assay from Boehringer Mannheim (Germany). Plasma insulin levels were determined using a radioimmunoassay kit from Linco Research (St. Charles, MO). Plasma Ang II and NE levels were measured using an enzyme immunoassay kit from Cedarlane (Hornby, Ontario) and IBL Hamburg (Toronto, Ontario), respectively. Plasma TNF-α and IL-6 were determined using enzyme immunoassay kits from Biosource (Camarillo, CA).  3.1.5  Reagents All chemicals were reagent grade and purchased from Sigma (St. Louis, MO).  Bosentan, L-158,809, prazosin and etanercept were generous gifts from Actelion Ltd.  57  (Allschwil, Switzerland), Merck Research Laboratories (Rahway, NJ), Apotex (Toronto, ON) and Amgen (Seattle, WA), respectively.  3.1.6  Statistical analysis All data are expressed as mean ± SEM. Statistical analysis of all data was performed  using the Number Cruncher Statistical Software 2000 (NCSS, Kaysville, UT). Data with multiple time points were analyzed by General Linear Model ANOVA and inter-group comparisons of dependent variables were analyzed by one-way ANOVA. For all results, the Newman-Keuls test for post-hoc analysis was applied. A value of p<0.05 was taken as the level of significance.  58  3.2  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension  3.2.1  Animals and experimental design Forty-eight male Wistar rats were obtained from Charles River Laboratories (St-  Constant, Quebec) at five weeks of age and randomly divided into six experimental groups: control (C, n=8), control bosentan-treated (CB, n=8), control L-158,809-treated (CL, n=8), fructose (F, n=8), fructose bosentan-treated (FB, n=8) and fructose L-158,809-treated (FL, n=8).  At six weeks of age, fasted (5 hours) plasma parameters (glucose, insulin and  triglycerides) and SBP were measured in all groups. At seven weeks of age, rats in fructosefed groups (F, FB and FL) were started on a 60% fructose diet (Teklad Laboratory Diets, Madison, WI) for six weeks, whereas rats in control groups (C, CB and CL) were maintained on standard laboratory rat chow containing 30% carbohydrate in the form of starch for the same period. Bosentan (CB and FB) or L-158,809 (CL and FL) treatment was initiated concurrently at a dose of 100 mg/kg or 1 mg/kg, respectively, suspended in 1% gum arabic administered via daily oral gavage for the duration of the study. The dose of bosentan (Clozel et al., 1994; Verma et al., 1995) and L-158,809 (Gillies et al., 1998; Tamura et al., 2002) was chosen based on effective blood pressure lowering as reported in previous studies. Rats were housed on a 12 hour light-dark cycle and received food and water ad libitum. At the end of the study, rats from all groups were euthanized with an overdose of pentobarbital (65 mg/kg, i.p.).  Superior mesenteric arteries were isolated, cleaned of  adherent connective tissue and fixed in formalin to assess vascular ET-1-ir.  This  investigation conforms with the Canadian Council on Animal Care Guidelines on the Care  59  and Use of Experimental Animals. All protocols were approved by the University of British Columbia Animal Care Committee. Given that we were interested in comparing either bosentan or L-158,809 treatment against vehicle-treated animals and not bosentan treatment against L-158,809 treatment, statistical analysis for this study consisted of using animals from the C and F groups to analyze the effects of bosentan treatment against vehicle treatment, as well as the effects of L-158,809 treatment against vehicle treatment.  3.2.2  Immunohistochemistry and image analysis Superior mesenteric arteries were fixed in formalin, embedded in paraffin and cut into  10 µm thick sections on a vibratome. Sections were mounted on slides, deparaffinized and immunostained for ET-1-ir. Briefly, sections were incubated in 5% normal goat serum for 1 hour at room temperature. Sections were incubated overnight in a humid atmosphere at 4°C with a rabbit anti-ET-1 specific antibody (1:500, Peninsula Laboratories, San Carols, CA) diluted in phosphate buffered saline (PBS) containing 1% normal goat serum. Following three washes in PBS, sections were incubated with an Alexa 594-conjugated goat anti-rabbit IgG antibody (1:1600 diluted in PBS, Molecular Probes, Eugene, OR) for 90 minutes at room temperature. Negative controls were stained by omitting the primary antibody incubation. Immunoreactivity was not detected in the absence of primary antibody. Fluorescent images were captured using an Olympus Fluoview BX61 confocal microscope and analyzed using the Image Pro Analyzer 6.2 software. For semi-quantitative analysis of vascular ET-1-ir, the area of interest was chosen by outlining the intimal and medial layers of each section. Vascular ET-1-ir was evaluated within the area of interest and was based on the amount of immunopositive staining expressed as a proportion of the total area of interest.  60  3.3  Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats  3.3.1  Animals and experimental design Male Wistar rats were obtained from Charles River Laboratories (St-Constant,  Quebec) at five weeks of age and randomly divided into four experimental groups: control (C, n=8), control prazosin-treated (CP, n=8), fructose (F, n=8) and fructose prazosin-treated (FP, n=8). At six weeks of age, fasted (5 hours) plasma parameters (glucose, insulin and triglycerides) and SBP were measured in all groups. At seven weeks of age, rats in fructosefed groups (F and FP) were started on a 60% fructose diet (Teklad Laboratory Diets, Madison, WI) for nine weeks, whereas rats in control groups (C and CP) were maintained on standard laboratory rat chow containing 30% carbohydrate in the form of starch for the same period. Prazosin (CP and FP) treatment was initiated concurrently at a dose of 1 mg/kg administered via daily oral gavage for the duration of the study. Rats were housed on a 12 hour light-dark cycle and received food and water ad libitum. At the end of the study, rats from all groups were euthanized with an overdose of pentobarbital (65 mg/kg, i.p.). This investigation conforms with the Canadian Council on Animal Care Guidelines on the Care and Use of Experimental Animals. All protocols were approved by the University of British Columbia Animal Care Committee.  61  3.4  Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension  3.4.1  Animals and experimental design Male Wistar rats were obtained from Charles River Laboratories (St-Constant,  Quebec) at five weeks of age and randomly divided into four experimental groups: control (C, n=20), control etanercept-treated (CE, n=20), fructose (F, n=20) and fructose etanercepttreated (FE, n=20). At six weeks of age, fasted (5 hours) plasma parameters (glucose, insulin and triglycerides) and SBP were measured in all groups. At seven weeks of age, rats in fructose-fed groups (F and FE) were started on a 60% fructose diet (Teklad Laboratory Diets, Madison, WI) for nine weeks, whereas rats in control groups (C and CE) were maintained on standard laboratory rat chow containing 30% carbohydrate in the form of starch for the same period. Etanercept (CE and FE) treatment was initiated concurrently at a dose of 0.3 mg/kg administered via s.c. injection three times per week for the duration of the study. This dose of etanercept was used based on previous studies (Arenas et al., 2005; Arenas et al., 2006; Joussen et al., 2002). Rats were housed on a 12 hour light-dark cycle and received food and water ad libitum. At the end of the study, rats from all groups were euthanized with an overdose of pentobarbital (65 mg/kg, i.p.). Thoracic aorta, superior mesenteric arteries and epidydimal fat pads were isolated and cleaned of adherent connective tissue for further analysis. This investigation conforms with the Canadian Council on Animal Care Guidelines on the Care and Use of Experimental Animals. All protocols were approved by the University of British Columbia Animal Care Committee.  62  3.4.2  Vascular reactivity Isolated superior mesenteric arteries were cut into 3 mm rings and suspended on wire  hooks in isolated tissue baths containing modified Krebs-Ringer bicarbonate solution pH 7.4 (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 0.026 mM EDTA, 11.1 mM glucose and 25 mM NaHCO3) aerated with 95% O2: 5% CO2 at 37ºC as previously described (Ginsberg, 2000; Reaven, 1988). Resting tension was adjusted to 1.0 g for each ring to allow for maximal active force generated. Changes in vascular tension were recorded using a force transducer on a Grass polygraph machine (model 79D). After an equilibration period, rings were challenged with 40 mM KCl and endothelial integrity was assessed.  Vessels were subjected to a cumulative concentration-response curve to  phenylephrine (PE; 10-9 to 10-4 mol/L) followed by a cumulative concentration-response curve to acetylcholine (ACh; 10-9 to 10-4 mol/L) in arteries pre-contracted with the ED70 of PE. Vessels were equilibrated to baseline between concentration-response curves. PE-induced responses were expressed as a percentage of the maximal response to KCl. Changes in ACh-induced responses were expressed as a percentage of the response to PE in each tissue. The maximum response (Emax) and negative log of the concentration producing 50% of the maximum response (pD2) were obtained by nonlinear regression analysis of individual concentration-response curves using GraphPad Prism, version 5.0 (GraphPad Software, Inc., San Diego, CA).  3.4.3  Western blot analysis Thoracic aorta and epidydimal fat pads were frozen in liquid nitrogen, powdered and  homogenized in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM  63  EDTA, 10 µg/mL aprotinin, 5 µg/mL leupeptin, 2.5 µg/mL pepstatin, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)). Homogenates were centrifuged at 12 000 × g for 15 min and supernatants were collected. Total protein content was determined using the Bradford protein assay.  Equal amounts of protein were subjected to SDS-  polyacrylamide gel electrophoresis. Resolved proteins were transferred onto polyvinylidene difluoride membrane. Membranes were blocked with 5% non-fat milk in Tris-buffered saline-Tween (TBS-T) and incubated with the appropriate primary antibody.  Immune  complexes were detected using horseradish peroxidase conjugated secondary antibody for 1 hr at room temperature (5% milk in TBS-T) and a chemiluminescence detection kit (Amersham Pharmacia). Band intensity was analyzed using densitometry and represented as a percent of the control mean.  64  4  RESULTS  4.1  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension  4.1.1  General characteristics of rats following six weeks of bosentan or L-158,809 treatment General characteristics of rats following six weeks of bosentan or L-158,809  treatment are summarized in Table 4.1 and Table 4.2, respectively. The body weight of rats did not differ among the experimental groups. Food intake was slightly, but significantly, reduced in fructose-fed (F, FB and FL) animals as compared to control (C, CB and CL) animals. In addition, food intake was significantly reduced in the CL group as compared to the C group. Plasma insulin levels of the F and FL groups were significantly elevated as compared to the C and CL groups. Plasma glucose levels were slightly, but significantly, elevated in fructose-fed animals as compared to control animals. Treatment with either bosentan or L-158,809 had no effect on glucose or insulin levels in either fructose-fed or control animals. Plasma triglycerides were significantly elevated in the F group. Chronic bosentan treatment significantly reduced elevated levels of triglycerides in fructose-fed rats, while chronic L-158,809 treatment had no effect.  65  Table 4.1  General characteristics and fasted plasma parameters of control and fructosefed rats following six weeks of bosentan treatment C  CB  F  FB  Body weight (g)  545 ± 17  550 ± 13  547 ± 18  566 ± 26  Food intake (g/day)  34.8 ± 0.9  32.3 ± 0.8  29.3 ± 0.7 *  29.3 ± 1.3 *  Plasma insulin (ng/mL)  1.42 ± 0.20  1.44 ± 0.17  2.16 ± 0.14  2.14 ± 0.42  Plasma glucose (mmol/L)  7.24 ± 0.13  7.38 ± 0.11  7.99 ± 0.14 *  8.11 ± 0.25 *  Plasma triglycerides (mmol/L)  0.85 ± 0.13  0.48 ± 0.10  2.27 ± 0.21 *  0.87 ± 0.15 †  Values expressed as mean ± SEM. * p<0.05, vs. C, CB; † p<0.05, vs. F.  Table 4.2  General characteristics and fasted plasma parameters of control and fructoserats following six weeks of L-158,809 treatment C  CL  F  FL  Body weight (g)  545 ± 17  541 ± 12  547 ± 18  564 ± 20  Food intake (g/day)  34.8 ± 0.9  31.9 ± 0.2 #  29.3 ± 0.7  Plasma insulin (ng/mL)  1.42 ± 0.20  1.19 ± 0.16  2.16 ± 0.14  ‡  2.72 ± 0.30  ‡  Plasma glucose (mmol/L)  7.24 ± 0.13  7.36 ± 0.16  7.99 ± 0.14  ‡  8.09 ± 0.30  ‡  Plasma triglycerides (mmol/L)  0.85 ± 0.13  0.42 ± 0.05  2.27 ± 0.21  ‡  1.91 ± 0.34  ‡  Values expressed as mean ± SEM.  #  ‡  29.3 ± 0.8  ‡  ‡ p<0.05, vs. C; p<0.05, vs. C, CL.  66  4.1.2  SBP SBP was significantly elevated in the F group following six weeks of high fructose  feeding (Figure 4.1). Chronic treatment with either bosentan or L-158,809 significantly attenuated the increase in blood pressure in fructose-fed rats. Treatment with either bosentan or L-158,809 had no effect on blood pressure in control rats.  4.1.3  OGTT Plasma glucose and insulin profiles following an oral glucose challenge are shown in  Figure 4.2 and Figure 4.3, respectively. High fructose feeding or treatment with either bosentan or L-158,809 had no effect on the glucose area under the curve (AUC). The insulin AUC of the F and FL groups were significantly elevated as compared to the C and CL groups. Comparisons of the insulin sensitivity index demonstrated that high fructose feeding significantly impaired insulin sensitivity (Figure 4.4).  Chronic treatment with either  bosentan or L-158,809 did not alter insulin sensitivity in either control or fructose-fed animals.  67  A 150  *  SBP (mmHg)  140 130 120  †  110 100 90 C  CB  F  FB  B 150  ‡  SBP (mmHg)  140 130 120 110  †  100 90 C  Figure 4.1  CL  F  FL  Effect of chronic (A) bosentan or (B) L-158,809 treatment on SBP in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8. * p<0.05, vs. C, CB; ‡ p<0.05, vs. C, CL; † p<0.05, vs. F.  68  A GLUCOSE AUC (mmol/L/90 min)  PLASMA GLUCOSE (mmol/L)  1000  12  800 600 400 200 0  10  C  CB  F  FB  C CB F FB  8  6 0  20  40  60  80  100  TIME (min)  B 12  GLUCOSE AUC (mmol/L/90 min)  PLASMA GLUCOSE (mmol/L)  1000  10  800 600 400 200 0 C  CL  F  FL  C CL F FL  8  6 0  20  40  60  80  100  TIME (min)  Figure 4.2  Effect of chronic (A) bosentan or (B) L-158,809 treatment on plasma glucose response and AUC (inset) during an OGTT in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8.  69  A INSULIN AUC (ng/mL/90 min)  PLASMA INSULIN (ng/mL)  500  12 10 8  400 300 200 100 0 C  CB  F  FB  6  C CB F FB  4 2 0 0  20  40  60  80  100  TIME (min)  B  500  INSULIN AUC (ng/mL/90 min)  PLASMA INSULIN (ng/mL)  ‡  12 10 8  400  ‡  300 200 100 0 C  6  CL  F  FL  C  4  CL  2  F FL  0 0  20  40  60  80  100  TIME (min)  Figure 4.3  Effect of chronic (A) bosentan or (B) L-158,809 treatment on plasma insulin response and AUC (inset) during an OGTT in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8. ‡ p<0.05, vs. C, CL.  70  INSULIN SENSITIVITY INDEX  15  INSULIN SENSITIVITY INDEX  A  15  10  *  *  F  FB  5  0 C  CB  B  Figure 4.4  10  ‡  ‡  5  0 C  CL  F  FL  Insulin sensitivity index values obtained from OGTT data in control and fructose-fed rats following six weeks of (A) bosentan or (B) L-158,809 treatment. Values expressed as mean ± SEM, n = 8. * p<0.05, vs. C, CB; ‡ p<0.05, vs. C, CL.  71  4.1.4  Plasma Ang II levels Plasma Ang II levels were significantly elevated following six weeks of high fructose  feeding (Figure 4.5).  Chronic bosentan treatment completely prevented the increase in  plasma Ang II levels in fructose-fed rats, while it had no effect in control animals. In contrast, treatment with L-158,809 resulted in significantly elevated plasma Ang II levels in both control and fructose-fed rats.  4.1.5  ET-1-ir microscopy and semi-quantitative analysis Semi-quantitative analysis of vascular ET-1-ir in superior mesenteric arteries revealed  significant elevations in the proportion of ET-1-ir in animals in the CL and FL groups as compared to arteries from the C and F groups (Figure 4.6). Vascular ET-1-ir was slightly increased in arteries from fructose-fed rats, although this difference did not reach statistical significance.  72  PLASMA ANG II (pg/mL)  A 2000 1500  * 1000  †  500 0 C  CB  F  FB  PLASMA ANG II (pg/mL)  B 2000  #†  #†  1500 1000 500 0 C  Figure 4.5  CL  F  FL  Effect of chronic (A) bosentan or (B) L-158,809 treatment on plasma Ang II levels in control and fructose-fed rats following six weeks of study. Values expressed as mean ± SEM, n = 8. * p<0.05, vs. C, CB; † p<0.05, vs. F; # p<0.05, vs. C.  73  A  PROPORTION OF ET-1-ir (% control)  B  400 300 200 100 0 C  Figure 4.6  #†  #†  CL  F  FL  (A) Representative fluorescence images of immunohistochemical expression of ET-1 in superior mesenteric arteries from control and fructose-fed rats following six weeks of L-158,809 treatment. Arrows indicate positive immunostaining for ET-1 within the intimal and medial layers. (B) Semiquantitative analysis of vascular ET-1-ir from control and fructose-fed rats following six weeks of L-158,809 treatment. Values expressed as mean ± SEM, n = 8. † <0.05, vs. F; # p<0.05, vs. C.  74  4.2  Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats  4.2.1  General characteristics of rats following nine weeks of prazosin treatment General characteristics of rats following nine weeks of prazosin treatment are  summarized in Table 4.3.  Body weight and food intake did not differ among the  experimental groups. Plasma insulin levels were significantly elevated in animals from the F and FP groups. Fructose-fed rats treated with prazosin were slightly, but significantly, hyperglycemic. Plasma triglycerides were significantly elevated in fructose-fed rats and chronic prazosin treatment significantly exacerbated the hypertriglyceridemia in fructose-fed rats. Treatment with prazosin had no effect on any parameters measured in control rats.  4.2.2  SBP SBP was significantly elevated in the F group following nine weeks of high fructose  feeding (Figure 4.7). Chronic prazosin treatment significantly attenuated the increase in blood pressure in fructose-fed rats, while treatment with prazosin had no effect on blood pressure in control rats.  75  Table 4.3  General characteristics and fasted plasma parameters of control and fructosefed rats following nine weeks of prazosin treatment C  CP  F  FP  Body weight (g)  478 ± 14  511 ± 15  498 ± 11  507 ± 18  Food intake (g/day)  29.5 ± 1.3  31.4 ± 0.9  27.3 ± 0.5  27.9 ± 1.5  Plasma insulin (ng/mL)  1.46 ± 0.15  1.31 ± 0.18  2.29 ± 0.30 †  2.24 ± 0.24 †  Plasma glucose (mmol/L)  7.89 ± 0.19  8.61 ± 0.22  8.07 ± 0.20  8.92 ± 0.34 †  Plasma triglycerides (mmol/L)  0.93 ± 0.15  0.74 ± 0.13  1.69 ± 0.19 †  Values expressed as mean ± SEM.  †  2.67 ± 0.36  ‡  p<0.05, vs. C, CP; ‡ p<0.05, vs. F.  76  150  †  SBP (mmHg)  140 130 120  ‡  110 100 C  Figure 4.7  CP  F  FP  Effect of chronic prazosin treatment on SBP in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 8. † p<0.05, vs. C, CP; ‡ p<0.05, vs. F.  77  4.2.3  OGTT Plasma glucose and insulin profiles following an oral glucose challenge are shown in  Figure 4.8. High fructose feeding or treatment with prazosin had no significant effect on the glucose AUC or insulin AUC. Comparisons of the insulin sensitivity index demonstrated that high fructose feeding significantly impaired insulin sensitivity (Figure 4.9). Chronic treatment with prazosin did not alter insulin sensitivity in either control or fructose-fed rats.  4.2.4  Plasma NE levels High fructose feeding significantly increased plasma NE levels (Figure 4.10).  Chronic prazosin treatment normalized plasma NE in fructose-fed rats. Control rats treated with prazosin also had elevated levels of plasma NE.  4.2.5  Plasma Ang II levels High fructose feeding significantly increased plasma Ang II levels (Figure 4.11).  Fructose-fed rats treated with prazosin also exhibited elevated levels of plasma Ang II. Prazosin treatment did not alter plasma Ang II levels in control rats.  78  A  PLASMA GLUCOSE (mmol/L/90 min)  PLASMA GLUCOSE (mmol/L)  1000  14 12  800 600 400 200 0 C  CP  F  FP  10  C CP F FP  8 6 0  20  40  60  80  100  TIME (minutes) 300  INSULIN AUC (ng/mL/90 min)  PLASMA INSULIN (ng/mL)  B 10 8  200  100  0  6  C  CP  F  FP  C CP F FP  4 2 0 0  20  40  60  80  100  TIME (minutes)  Figure 4.8  Effect of chronic prazosin treatment on plasma A) glucose response and AUC (inset) and B) insulin response and AUC (inset) during an OGTT in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 8.  79  INSULIN SENSITIVITY INDEX Figure 4.9  20 15 #  #  F  FP  10 5 0 C  CP  Insulin sensitivity index values obtained from OGTT data in control and fructose-fed rats following nine weeks of prazosin treatment. Values expressed as mean ± SEM, n = 8. # p<0.05, vs. C.  80  PLASMA NE (ng/mL)  20 #  15  ^  #  ^  10 5 0 C  Figure 4.10  CP  F  FP  Effect of chronic prazosin treatment on plasma NE levels in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 8. # p<0.05, vs. C; ^ p<0.05, vs. FP.  81  PLASMA ANG II (pg/mL)  400  † 300  #  200 100 0 C  Figure 4.11  CP  F  FP  Effect of chronic prazosin treatment on plasma Ang II levels in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM. † p<0.05, vs. C, CP; # p<0.05, vs. C.  82  4.3  Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension  4.3.1  General characteristics of rats following nine weeks of etanercept treatment General characteristics of rats following nine weeks of etanercept treatment are  summarized in Table 4.4. No differences in body weight or fasted plasma glucose were observed among the experimental groups.  Food intake was slightly, but significantly,  reduced in fructose-fed (F and FE) animals as compared to control (C and CE) animals. Fasted plasma insulin and triglyceride levels were significantly elevated in fructose-fed animals. Treatment with etanercept had no effect on any parameters measured in either fructose-fed or control animals.  4.3.2  SBP Following nine weeks of study, animals fed a high fructose diet had significantly  elevated SBP (Figure 4.12). Chronic treatment with etanercept significantly attenuated the increase in blood pressure in fructose-fed rats. Treatment with etanercept had no effect on blood pressure in control rats.  83  Table 4.4  General characteristics and fasted plasma parameters of control and fructosefed rats following nine weeks of etanercept treatment C  CE  F  FE  Body weight (g)  524 ± 12  491 ± 10  520 ± 14  519 ± 11  Food intake (g/day)  31.9 ± 0.5  30.7 ± 0.4  27.1 ± 0.7 *  27.9 ± 0.4 *  Plasma insulin (ng/mL)  1.68 ± 0.14  1.78 ± 0.16  2.49 ± 0.20 *  2.50 ± 0.21 *  Plasma glucose (mmol/L)  7.19 ± 0.14  7.31 ± 0.14  7.66 ± 0.11  7.52 ± 0.11  Plasma triglycerides (mmol/L)  1.07 ± 0.06  1.00 ± 0.05  1.85 ± 0.16 *  1.87 ± 0.13 *  Values expressed as mean ± SEM. * p<0.05, vs. C, CE.  84  150  *  SBP (mmHg)  140 130 120  †  110 100 C  Figure 4.12  CE  F  FE  Effect of chronic etanercept treatment on SBP in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 20. * p<0.05, vs. C, CE; † p<0.05, vs. F.  85  4.3.3  OGTT Plasma glucose and insulin profiles following an oral glucose challenge are shown in  Figure 4.13. The glucose AUC (inset) were slightly, but significantly, elevated in the F and FE groups. Plasma insulin profiles following an oral glucose challenge are shown in Figure 4.13B. Fructose-fed animals (F and FE) had significantly elevated insulin AUC (inset) as compared to control animals (C and CE). High fructose feeding resulted in significantly impaired insulin sensitivity as demonstrated by the decreased insulin sensitivity index values (Figure 4.14). Chronic etanercept treatment did not alter insulin sensitivity in either control or fructose-fed rats.  4.3.4  Vascular reactivity PE-induced contractile responses of superior mesenteric arteries from control and  fructose-fed rats are shown in Figure 4.15A. There were no differences in the maximum contractile response (Emax) or sensitivity (pD2) to PE (Table 4.5).  Chronic etanercept  treatment had no effect on vessels from either control or fructose-fed rats. ACh-induced relaxation of mesenteric arteries from animals following nine weeks of treatment are shown in Figure 4.15B.  Arteries from fructose-fed rats had significantly impaired relaxation  responses to ACh as compared to arteries from control rats. Etanercept treatment improved Emax values of arteries from fructose-fed rats without altering the sensitivity to ACh (Table 4.5). Etanercept treatment had no effect on vessels from control animals.  86  1000  GLUCOSE AUC (mmol/L/90 min)  PLASMA GLUCOSE (mmol/L)  A 11 10  #  *  F  FE  800 600 400 200 0 C  9  CE  C CE F FE  8 7 6 0  20  40  60  80  100  TIME (minutes) 400  INSULIN AUC (ng/mL/90 min)  PLASMA INSULIN (ng/mL)  B 8 6  300  *  *  F  FE  200 100 0 C  CE  4  C CE F FE  2 0 0  20  40  60  80  100  TIME (minutes)  Figure 4.13  Effect of chronic etanercept treatment on plasma A) glucose response and AUC (inset) and B) insulin response and AUC (inset) during an OGTT in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 20. # p<0.05, vs. C; * p<0.05, vs. C, CE.  87  INSULIN SENSITIVITY INDEX Figure 4.14  15  10  *  *  F  FE  5  0 C  CE  Insulin sensitivity index values obtained from OGTT data from control and fructose-fed rats following nine weeks of etanercept treatment. Values expressed as mean ± SEM, n = 20. * p<0.05, vs. C, CE.  88  CONSTRICTION (% KCl)  A 400  C CE F FE  300 200 100 0 10  9  8  7  6  5  4  3  -LOG [PE]  B % RELAXATION  80  C CE F FE  60 40 20 0 10  9  8  7  6  5  4  3  -LOG [ACh]  Figure 4.15  Cumulative concentration-response curves to A) PE and B) ACh in superior mesenteric arteries from control and fructose-fed rats following nine weeks of etanercept treatment. Values expressed as mean ± SEM, n= 14.  89  Table 4.5  pD2 and Emax values of superior mesenteric arteries from control and fructosefed rats following nine weeks of etanercept treatment C  CE  F  FE  pD2  6.55 ± 0.12  6.49 ± 0.12  6.75 ± 0.10  6.77 ± 0.09  Emax  195 ± 16.4  202.8 ± 26.0  270.8 ± 58.6  189.5 ± 21.8  pD2  6.68 ± 0.18  6.73 ±0.20  6.46 ± 0.25  6.61 ± 0.16  Emax  70.7 ± 3.7  71.9 ± 4.3  54.3 ± 5.6 *  71.2 ± 4.5 †  PE  ACh  Values expressed as mean ± SEM, n=14. * p<0.05, vs. C, CE; † p<0.05, vs. F.  90  4.3.5  Plasma TNF-α and IL-6 levels Plasma TNF-α levels were significantly elevated in control and fructose-fed rats  treated with etanercept following two and nine weeks of treatment (Figure 4.16). There was a trend for animals from the F group to have slightly elevated levels of plasma TNF-α as compared to animals in the C group at both time points, although this increase did not reach statistical significance. No difference was observed in plasma levels of IL-6 after two and nine weeks of treatment (Figure 4.17).  4.3.6  Plasma NE levels Statistical analysis using a one-way ANOVA determined that plasma NE levels were  significantly different among the experimental groups; however, the Newman-Keuls post-hoc test was unable to determine where the differences were (Figure 4.18). Chronic etanercept treatment had a tendency to increase NE levels in both control and fructose-fed animals.  4.3.7  Plasma Ang II levels Plasma Ang II levels were significantly elevated following nine weeks of high  fructose feeding (Figure 4.19). Chronic etanercept treatment had no effect on plasma Ang II in either control or fructose-fed rats.  91  PLASMA TNF- α (pg/mL)  A  #†  15  #†  10  5 4 3 2 1 0 C  CE  F  FE  PLASMA TNF- α (pg/mL)  B #†  200  #†  140 80 20 9 6 3 0 C  Figure 4.16  CE  F  FE  Effect of chronic etanercept treatment on plasma TNF-α in control and fructose-fed rats following A) two and B) nine weeks of study. Values expressed as mean ± SEM, n = 20. # p<0.5, vs. C; † p<0.05, vs. F.  92  A PLASMA IL-6 (pg/mL)  100 80 60 40 20 0 C  CE  F  FE  C  CE  F  FE  B PLASMA IL-6 (pg/mL)  100 80 60 40 20 0  Figure 4.17  Effect of chronic etanercept treatment on plasma IL-6 levels in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 12.  93  PLASMA NE (ng/mL)  15  10  5  0 C  Figure 4.18  CE  F  FE  Effect of chronic etanercept treatment on plasma NE in control and fructosefed rats following nine weeks of study. Values expressed as mean ± SEM, n = 12.  94  PLASMA ANG II (pg/mL)  600  *  500  *  400 300 200 100 0 C  Figure 4.19  CE  F  FE  Effect of chronic etanercept treatment on plasma Ang II in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 12. * p<0.05, vs. C, CE.  95  4.3.8  Aortic eNOS and iNOS protein expression Arteries from fructose-fed rats had significantly reduced protein expression of eNOS  as compared to arteries from rats in the C and CE groups (Figure 4.20). Animals from the FE group showed significantly increased eNOS expression as compared to arteries from the F group. No significant differences were observed in the expression of iNOS among the experimental groups.  4.3.9  Aortic RhoA, ROCK-1 and ROCK-2 protein expression Arteries from fructose-fed rats had significantly increased protein expression of  ROCK-1 as compared to arteries from rats in the C and CE groups (Figure 4.21). Chronic treatment with etanercept did not affect the increased expression of ROCK-1 in fructose-fed rats. No significant differences were observed in the expression of RhoA or ROCK-2 among the experimental groups.  4.3.10  Fat pad TNF-α and TNFR1 protein expression There was a trend for fat pads from fructose-fed rats to have increased protein  expression of transmembrane TNF-α, although this difference did not reach statistical significance (Figure 4.22A). Fat pads from animals in the FE group exhibited significant elevations in transmembrane TNF-α as compared to animals from the C and CE groups. Although there was a trend for fat pads from the F group to have increased protein expression of soluble TNF-α (Figure 4.22B) or TNFR1 (Figure 4.23), this difference did not reach statistical significance.  96  A  eNOS GAPDH  B RELATIVE INTENSITY (% control)  150  †  100  * 50  0 C  CE  F  FE  C  iNOS GAPDH  D RELATIVE INTENSITY (% control)  150  100  50  0 C  Figure 4.20  CE  F  FE  Representative Western blots of A) eNOS and C) iNOS from thoracic aorta from control and fructose-fed rats following nine weeks study. Effect of chronic etanercept treatment on the relative intensity of B) eNOS and D) iNOS protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n = 10. * p<0.05, vs. C, CE; † p<0.05, vs. F.  97  A  RhoA  B  GAPDH  RELATIVE INTENSITY (% control)  500 400 300 200 100 0 C  CE  F  FE  C  ROCK-1 GAPDH  D RELATIVE INTENSITY (% control)  500  * 400 300 200 100 0 C  CE  F  FE  E  ROCK-2 GAPDH  F RELATIVE INTENSITY (% control)  500 400 300 200 100 0 C  Figure 4.21  CE  F  FE  Representative Western blots of A) RhoA, C) ROCK-1 and E) ROCK-2 from thoracic aorta from control and fructose-fed rats following nine weeks of study. Effect of chronic etanercept treatment on the relative intensity of B) RhoA, D) ROCK-1 and F) ROCK-2 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=8-12. * p<0.05, vs. C, CE.  98  A  TNF-α 26 kDa GAPDH C  B  CE  F  RELATIVE INTENSITY (% control)  300  FE  *  200  100  0 C  CE  F  FE  TNF-α 17 kDa  C  GAPDH  D RELATIVE INTENSITY (% control)  300  C  CE  F  FE  C  CE  F  FE  200  100  0  Figure 4.22  Representative Western blots of A) transmembrane TNF-α (24 kDa) and C) soluble TNF-α (17 kDa) from epidydimal fat pads from control and fructosefed rats following nine weeks of study. Effect of chronic etanercept treatment on the relative intensity of B) transmembrane TNF (24kDa) and D) soluble TNF-α (17kDa) protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12. * p<0.05, vs. C, CE.  99  TNFR1  A  GAPDH  B RELATIVE INTENSITY (% control)  250 200 150 100 50 0 C  Figure 4.23  CE  F  FE  A) Representative Western blot of TNFR1 from epidydimal fat pads from control and fructose-fed rats following nine weeks of study. B) Effect of chronic etanercept on the relative intensity of TNFR1 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12.  100  4.3.11  Fat pad p-NFκB p65 and NFκB p100/p52 protein expression Fat pads from animals in the CE, F and FE groups had significantly reduced  phosphorylation of the p65 subunit of NFκB; indicating a decreased activation of NFκB p65, as compared to animals in the C group (Figure 4.24). There were no significant differences observed in the expression of the p100 subunit of NFκB, which is the precursor to the p52 subunit of NFκB. No differences were observed in the expression of p52 subunit of NFκB, which is a necessary subunit to NFκB, among the experimental groups.  4.3.12  Aortic caspase-3 protein expression There were no significant differences observed in aortic protein expression of  caspase-3, a downstream effector of TNFR1-induced caspase activation, among the experimental groups (Figure 4.25).  101  A  p-NFκB p65 NFκB p65  B p-NFκ κ B/NFκ κB (% control)  150  100  # #  # 50  0 C  CE  F  FE  C  NFκB p100 GAPDH  D RELATIVE INTENSITY (% control)  150  100  50  0 C  CE  F  FE  E  NFκB p52 GAPDH  F RELATIVE INTENSITY (% control)  150  100  50  0 C  Figure 4.24  CE  F  FE  Representative Western blots of A) p-NFκB p65, C) NFκB p100 and E) NFκB p52 from epidydimal fat pads from control and fructose-fed rats following nine weeks of study. Effect of chronic etanercept treatment on the relative intensity of B) p-NFκB p65, D) NFκB p100 and F) NFκB p52 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12. # p<0.05, vs. C.  102  Caspase-3  A  GAPDH  B RELATIVE INTENSITY (% control)  200 150 100 50 0 C  Figure 4.25  CE  F  FE  A) Representative Western blot of caspase-3 from thoracic aorta from control and fructose-fed rats following nine weeks of study. B) Effect of chronic etanercept treatment on the relative intensity of caspase-3 protein expression in control and fructose-fed rats following nine weeks of study. Values expressed as mean ± SEM, n=12.  103  5  DISCUSSION AND CONCLUSIONS  5.1  DISCUSSION  5.1.1  Overview of results for fructose-fed rats Rats fed a high fructose diet for a period of 6-9 weeks exhibited several of the risk  factors observed in the metabolic syndrome, including insulin resistance, hyperinsulinemia, hypertriglyceridemia and hypertension. In all studies performed, fructose feeding did not affect body weight. Fructose-fed rats exhibited elevated levels of plasma Ang II and NE, suggesting that overactivation of the renin angiotensin system and α-adrenergic system may be involved in the development of fructose-induced hypertension. It has been proposed that insulin resistance and compensatory hyperinsulinemia are causally linked to the development of hypertension (Ginsberg, 2000; Reaven, 1988). Numerous mechanisms have been proposed that link insulin resistance/hyperinsulinemia and hypertension, and endothelial dysfunction has been suggested as a common underlying mechanism (Miller et al., 1998). In addition to the fructose-fed rat (Katakam et al., 1998; Miller et al., 1999; Takagawa et al., 2001; Verma et al., 1996), other animal models of hypertension (Konishi and Su, 1983; Lockette et al., 1986; Luscher et al., 1987) as well as humans with hypertension (Panza et al., 1990) exhibited impaired endothelium-dependent relaxation. The relationship between insulin resistance and endothelial dysfunction has been debated. Specifically, does insulin resistance precede endothelial dysfunction or is it a consequence of endothelial dysfunction? There is evidence that insulin resistance within the endothelium can contribute to endothelial dysfunction (Scherrer et al., 1994; Zeng and Quon, 1996), which in turn can contribute to the development of hypertension through increased  104  peripheral vascular resistance.  In contrast, there are data to support the concept that  endothelial dysfunction within the vasculature of skeletal muscle can cause insulin resistance by decreasing blood flow to insulin-sensitive tissues (Pinkney et al., 1997). It has been postulated that insulin resistance precedes endothelial dysfunction as Katakam and colleagues have reported that hyperinsulinemia occurred after 3 days of fructose feeding, while endothelial dysfunction was detected following 18 days and hypertension after 28 days (Katakam et al., 1998). Furthermore, normotensive offspring of hypertensive parents have impaired endothelium-dependent relaxation as compared to offspring of normotensive parents (Taddei et al., 1992).  Endothelial dysfunction may occur as a primary and  irreversible process given that antihypertensive therapy did not improve endothelial function in hypertensive individuals (Panza et al., 1993). In the fructose-fed rat, several mechanisms have been postulated to mediate the link between insulin resistance/hyperinsulinemia and hypertension. Our studies were designed with two primary objectives.  1) To determine the effects of chronic blockade of the  endothelin system, renin angiotensin system or α-adrenergic system in fructose-fed rats and to investigate whether blockade of one system would alter another system thereby indicating a potential interaction between them. 2) To investigate the effects of chronic etanercept treatment in fructose-fed rats to determine whether chronic inflammation, as reflected by elevated levels of TNF-α, is present and contributes to the development of fructose-induced hypertension.  105  5.1.2  Investigation of an interrelationship between the endothelin system and renin angiotensin system in the development of fructose-induced hypertension Treatment with either a dual endothelin receptor antagonist or an AT1 receptor  antagonist prevented the development of hypertension, indicating that both the endothelin system and renin angiotensin system play important roles in the development of fructoseinduced hypertension. Chronic blockade of the endothelin system normalized Ang II levels, while blockade of the renin angiotensin system increased ET-1-ir. These data suggest that there is an interaction between the endothelin system and renin angiotensin system, and that ET-1 may exert its effect through the renin angiotensin system.  To the best of our  knowledge, this is the first study that has concurrently investigated the role of both the endothelin system and renin angiotensin system in hypertension in fructose-fed rats. We have demonstrated that ET-1 contributes to the development of fructose-induced hypertension through modulation of Ang II levels. The results of this study extend previous reports showing that blood pressure regulation is dependent on an interrelationship between the endothelin system and renin angiotensin system.  Studies in normotensive (Gomez-Alamillo et al., 2003) and  hypertensive dogs (Massart et al., 1998) and various rat models of experimental hypertension (Bohlender et al., 2000; Ikeda et al., 2000; Pollock et al., 2000) have shown a greater hypotensive effect under conditions where both the endothelin system and renin angiotensin system were antagonized as compared to the effect seen with antagonists of either system alone. Furthermore, treatment of rats with an endothelin receptor antagonist prevented the development of hypertension induced by Ang II infusion (d'Uscio et al., 1997; Herizi et al.,  106  1998), while treatment with an ACE inhibitor prevented the development of hypertension induced by ET-1 infusion (Mortensen and Fink, 1992). Although these studies provide evidence for a potential link between the endothelin system and renin angiotensin system in the maintenance of normal or elevated blood pressure, the nature of this interaction has not been clear. Our data suggest that in the setting of fructose-induced hypertension, elevations in Ang II are dependent on the actions of ET-1. Recently, we demonstrated that bosentan treatment prevented the increase in cyclooxygenase-2 expression and normalized elevated levels of plasma thromboxane B2, a stable metabolite of TxA2, in fructose-fed rats (Jiang et al., 2007). These findings suggest that ET-1 also acts upstream of TxA2, a concept that has been previously suggested (Lariviere et al., 2004). Further evidence suggests that ET-1 stimulates oxidative stress, another mediator thought to contribute to the pathogenesis of hypertension (Pollock, 2005). In DOCA-salt hypertension, selective blockade of ETA receptors normalized vascular superoxide production (Callera et al., 2003) and decreased plasma thiobarbituric acid reacting substances, a marker of systemic oxidative stress (Callera et al., 2006). Given that oxidative stress is another proposed mechanism of fructose-induced hypertension (Delbosc et al., 2005), a potential role for ET-1 in stimulating oxidative stress in fructose-fed rats remains to be clarified. As plasma levels of ET-1 are thought to reflect excess release from local tissue, the use of this parameter may not accurately indicate the status of the endothelin system. Studies have reported no difference in plasma ET-1 levels in fructose-fed rats (Huang et al., 1997; Juan et al., 1998), while one reported an increase (Juan et al., 2004). Therefore, tissue levels may more accurately reflect local ET-1 production and regulation. For this reason, we  107  utilized immunohistochemical techniques to assess the level of immunoreactive ET-1 in superior mesenteric arteries. Animals in the CL and FL groups showed significant increases in vascular ET-1-ir as compared to arteries from animals in the C and F groups. As expected, chronic blockade of AT1 receptors resulted in significant elevations in plasma Ang II. As well, significant increases in vascular ET-1-ir were observed in both control and fructose-fed rats treated with L-158,809, suggesting a potential positive feedback loop between Ang II and ET-1 (Figure 5.1). We believe that during chronic blockade of AT1 receptors, elevations in Ang II result in a compensatory increase in ET-1. Arteries from animals in the F group showed a slight increase in vascular ET-1-ir as compared to arteries from animals in the C group, although this increase did not reach statistical significance, perhaps due to the sample size and larger variability observed in the F group. The increasing trend in vascular ET-1 is consistent with previous studies. Verma and colleagues observed an increase in the total content of ET-1 in superior mesenteric arteries from fructose-fed rats (Verma et al., 1995). Likewise, aortic tissue from fructose-fed rats exhibited an upregulation in ET-1 mRNA expression (Kang et al., 2004; Lee et al., 2001). Taken together, our results provide further evidence that ET-1 may play a central role in the development of fructose-induced hypertension through modulation of various vasoactive mediators. Interestingly, we did not observe improvements in insulin sensitivity in fructose-fed rats treated with L-158,809, a finding that differs from previously published reports. Treatment with either an ACE inhibitor or AT1 receptor antagonist have been reported to reduce elevated insulin levels and/or improve insulin sensitivity in fructose-fed rats (Higashiura et al., 2000; Iyer and Katovich, 1996). The reason for this discrepancy is  108  unclear, but may be due to differences in the duration of fructose feeding and/or drug treatment or a result of dose-dependent effects of the drugs used. Hypertriglyceridemia, and not insulin resistance/hyperinsulinemia, has been proposed to be a causal link in the development of fructose-induced hypertension (Si et al., 1999). Hypertriglyceridemia has been postulated to alter the composition of cellular membranes and increase membrane fluidity (Muzulu et al., 1995).  Changes in membrane fluidity can  contribute to altered permeability of calcium ions (Dominiczak and Bohr, 1989; Orlov and Postnov, 1982) and affect vascular reactivity through abnormal calcium handling (Si et al., 1999).  In support of this hypothesis, Holness reported that elevations in triglycerides  preceded the development of insulin resistance during fructose-feeding (Holness, 1994). Furthermore, fructose-fed rats treated with bezafibrate, a peroxisome proliferator-activated receptor α agonist, lowered triglycerides and attenuated the rise in blood pressure (Si et al., 1999). Our data do not support this hypothesis given that the development of hypertension was attenuated in fructose-fed animals chronically treated with either bosentan or L-158,809, despite the presence of hypertriglyceridemia in fructose-fed animals treated with L-158,809. Similar observations have been made in fructose-fed rats treated with the AT1 receptor antagonists, losartan (Navarro-Cid et al., 1995) or TCV-116 (Chen et al., 1996), or thromboxane synthase inhibitor, dazmegral (Galipeau et al., 2001), in which the development of hypertension was prevented despite the presence of hypertriglyceridemia. Furthermore, a reduction in elevated triglyceride levels did not reduce elevated blood pressure in fructosefed rats (Fujioka et al., 2003; Galipeau et al., 2002), supporting the concept of a triglycerideindependent component involved in the development of fructose-induced hypertension.  109  Conflicting reports exist on the interrelationship between ET-1 and Ang II (Rossi et al., 1999). As a result, it is unclear in what direction this interaction may occur. Differences in results may be due to the use of in vitro versus in vivo experiments, the use of different animal models of experimental hypertension, the duration of treatment, acute versus chronic effects of receptor blockade or species differences that may exist in the regulation of the endothelin system and/or renin angiotensin system. Although we were able to demonstrate a relationship between these two systems, this study was limited in its ability to determine whether the endothelin system affects the renin angiotensin system through direct or indirect mechanisms. Bosentan may act to suppress Ang II levels by preventing the actions of ET-1 on ACE activity and aldosterone production given that previous reports demonstrated that ET-1 stimulated ACE activity (Barton et al., 2000; Barton et al., 2003) and increased aldosterone production (Nussdorfer et al., 1997). Alternatively, bosentan may indirectly affect Ang II levels by decreasing blood pressure and subsequently reducing activation of the renin angiotensin system.  The  compensatory increase in Ang II, secondary to L-158,809 treatment, may regulate vascular ET-1 expression at various levels. Ang II has been shown to stimulate transcription of the prepro-ET-1 gene (Rossi et al., 1999), increase the activity of the ECE (Barton et al., 2003) and increase the release of ET-1 from vascular smooth muscle cells (Sung et al., 1994) and endothelial cells (Emori et al., 1989). As it remains unclear where the specific site(s) of interaction(s) may occur, further investigation on the exact nature of this relationship is required.  110  INSULIN RESISTANCE HYPERINSULINEMIA  + ↑ ET-1  +  +  ↑ Ang II  + ↑ BLOOD PRESSURE  Figure 5.1  Proposed mechanism of ET-1 modulating Ang II in the development of hypertension in fructose-fed rats.  111  5.1.3  Effect of chronic α1-adrenoceptor blockade on Ang II levels in fructose-fed rats In fructose-fed rats, treatment with prazosin, an α1-adrenoceptor antagonist, prevented  the development of hypertension without affecting insulin levels, insulin sensitivity or elevated Ang II levels. Surprisingly, chronic prazosin treatment normalized plasma NE levels while exacerbating hypertriglyceridemia in fructose-fed rats. These data suggest the involvement of the sympathetic nervous system, specifically α1-adrenoceptors, in the development of fructose-induced hypertension. Using plasma NE levels as a marker of adrenergic function, we observed that fructose-fed rats had elevated adrenergic function as reflected by an increase in plasma NE. This finding is in agreement with a meta-analysis that determined circulating levels of plasma NE were significantly increased in hypertensive individuals as compared to agematched normotensive individuals (Goldstein, 1983).  Numerous animal models of  hypertension have been reported to exhibit increased sympathetic nerve activity, including the SHR (Judy et al., 1976), sucrose-fed rat (Freitas et al., 2007), Dahl salt-sensitive rat (Saavedra et al., 1983) and DOCA salt hypertensive rat (Takeda and Bunag, 1980). These observations provide evidence that an overactivated sympathetic nervous system may contribute to the development of hypertension. Insulin-induced stimulation of the sympathetic nervous system has been suggested to link hyperinsulinemia and hypertension (Muntzel et al., 1995).  Insulin can induce  sympathetic nervous system activity through direct or indirect mechanisms. Insulin can directly stimulate NE release from adrenergic nerve endings (Edwards and Tipton, 1989), which in the vasculature of skeletal muscle may result in vasoconstriction, reducing glucose  112  delivery and contributing to insulin resistance. Indirectly, insulin can increase sympathetic nervous system activity by stimulating  carbohydrate metabolism and  oxidation  (Vollenweider et al., 1993) or by inducing baroreceptor-mediated increases in sympathetic activity following insulin-induced vasodilation (Muntzel et al., 1995). Hyperinsulinemia may excessively stimulate the sympathetic nervous system or increase catecholamine release from sympathetic nerve endings in the kidney, heart or vasculature, which can elevate blood pressure by stimulating sodium and water reabsorption, increasing cardiac output or increasing peripheral vascular resistance (Landsberg, 1986).  In support of an insulin-  stimulated sympathetic nervous system, both animals (Liang et al., 1982; Tomiyama et al., 1992) and humans (Anderson et al., 1991; Berne et al., 1992; Lembo et al., 1992; Rowe et al., 1981) exhibited increased plasma NE levels following insulin infusion. Previous studies have demonstrated that central blockade of the sympathetic nervous system, either through chemical sympathectomy (Verma et al., 1999) or the use of imidazole receptor agonists (Penicaud et al., 1998; Rosen et al., 1997), prevented the development of insulin resistance/hyperinsulinemia and hypertension in fructose-fed rats. The results of this study support the concept that an overactivated sympathetic nervous system may contribute to the development of hypertension as it is in agreement with studies that demonstrated no increase in blood pressure during α1-adrenoceptor antagonism in fructose-fed rats (Kamide et al., 2002), as well as in other models of hypertension, such as DOCA salt hypertensive rats (Saiz et al., 1986) and SHR (Pegram et al., 1984). Although consistent with results reported by Kamide and co-workers, who reported that bunazosin, an α1-adrenoceptor antagonist, had no effect on the elevated levels of insulin following an OGTT (Kamide et al., 2002), the lack of effect of prazosin on insulin sensitivity  113  in fructose-fed rats is puzzling given that central blockade of the sympathetic nervous system prevented insulin resistance (Penicaud et al., 1998; Rosen et al., 1997; Verma et al., 1999). These findings differ from those reported in hypertensive individuals, in which insulin sensitivity was improved following treatment with an α1-adrenoceptor antagonist (Eriksson et al., 1996; Suzuki et al., 1992; Swislocki et al., 1989). The reason for this inconsistency is unclear, but may have occurred as a result of the mechanism used to inhibit the sympathetic nervous system, the type and/or dose of α1-adrenoceptor antagonist or the treatment regimen that was followed. In contrast to a report that demonstrated unchanged urinary epinephrine excretion following bunazosin treatment in fructose-fed rats (Kamide et al., 2002), we observed normalized plasma NE levels in fructose-fed rats treated with prazosin. Since prazosin selectively antagonizes α1-adrenoceptors and α2-adrenoceptors function as autoreceptors to mop up excess NE from the circulation, it is possible that α2-adrenoceptors are upregulated to compensate for the increase in NE. This effect appears to occur in the setting of insulin resistance and/or hypertension since control animals treated with prazosin exhibited elevated levels of NE. We believe that the increase in plasma NE that occurred in the CP and F group occurred through different mechanisms.  We propose that the elevated levels of NE in  fructose-fed animals resulted from adrenergic overdrive while prazosin treated control animals exhibited increased NE levels due to a compensatory increase in catecholamine following receptor blockade. One effect of α1-adrenoceptor blockade is the ability to lower triglyceride levels through changes in lipoprotein lipase activity.  Lipoprotein lipase is the major enzyme  responsible for hydrolyzing triglycerides (Mead et al., 2002).  Stimulation of α1-  114  adrenoceptors decreases lipoprotein lipase activity (Jansen and Baggen, 1987), while α1adrenoceptor antagonists appear to increase lipoprotein lipase activity (Ferrara et al., 1986). An increase in the activity of lipoprotein lipase hydrolyzes triglycerides into glycerol and free fatty acids, which can be resynthesized and stored in adipose tissue. Previous studies have demonstrated that chronic treatment with an α1-adrenoceptor antagonist decreased triglyceride levels in sucrose-fed rats (Belahsen and Deshaies, 1993; Deshaies et al., 1991) and hypertensive individuals (Dell'Omo et al., 2005; Derosa et al., 2005), although no difference was also observed in hypertensive humans (Ferrara et al., 1986). Surprisingly, our study showed that prazosin treatment exacerbated hypertriglyceridemia in fructose-fed rats. This observation is inconsistent with previously published reports and may have occurred as a result of a dysfunction between the α1-adrenoceptors and lipoprotein lipase that results in the setting of fructose-induced insulin resistance and hypertension. Overactivation of the sympathetic nervous appears to contribute to the development of fructose-induced hypertension, however it does not appear to be the initial, precipitating event as previously proposed (Verma et al., 1999).}. If overactivation of the sympathetic nervous system occurred as an initial event following fructose feeding and this disturbance contributed to the overactivation of the renin angiotensin system, reduced or normalized Ang II levels following prazosin treatment would be expected. However, we did not observe changes to plasma Ang II levels following chronic prazosin treatment. Several mechanisms are involved in this form of hypertension and although blocking a specific pathway prevents the development of hypertension, it does not necessarily normalize all abnormalities present in this animal model.  Given that there is considerable evidence for a bi-directional  interaction between the sympathetic nervous system and renin angiotensin system (Grassi,  115  2001), determining the nature of this relationship remains a difficult task due to potential compensatory mechanisms that may exist between them.  116  5.1.4  Effect of chronic etanercept treatment on the development of fructose-induced insulin resistance and hypertension In fructose-fed rats, chronic treatment with etanercept prevented the rise in blood  pressure without affecting insulin levels, insulin sensitivity or triglyceride levels. The results of this study suggest that treatment with etanercept, a soluble recombinant fusion protein consisting of the extracellular ligand binding domain of TNFR2, prevented the development of hypertension by improving vascular function and restoring eNOS expression. Given that these effects occurred in the absence of significant elevations in TNF-α, we propose two potential mechanisms that may explain these findings. The first is that it is possible an elevation in TNF-α occurred as an initiating event that subsequently activated other vasoactive mediators, contributing to the eventual development of hypertension. Alternatively, etanercept may be acting through a mechanism independent of TNF-α inhibition. Following nine weeks of study, fructose-fed rats did not exhibit elevations in inflammatory mediators, TNF-α, IL-6 and iNOS, which suggests that the development of fructose-induced hypertension is not associated with a maintained inflammatory mediated process. Activation of NFκB stimulates transcription of various inflammatory genes and indicates an increased inflammatory state (Perkins, 2007).  Elevations in TNF-α may  contribute to the development of hypertension by acting as an initiating event to stimulate other vasoactive factors. Once this insult has occurred, compensatory mechanisms may be activated to negatively regulate secretion of TNF-α into the circulation. The concept of reverse signaling has recently emerged and proposes that binding of transmembrane TNF-α to its receptor can transduce a reverse signal through the cytoplasmic tail of transmembrane  117  TNF-α to trigger cell activation, cytokine suppression or apoptosis of the cell containing the transmembrane TNF-α (Eissner et al., 2000). Our data indicate that following nine weeks of study, there was a significant decrease in NFκB activation in fructose-fed animals as compared to control animals, which may have resulted as a consequence of reverse signaling leading to cytokine suppression.  A downstream effector of TNFR1 activation is the  induction of apoptosis through caspase-3. Given that we observed no change in aortic caspase-3 protein expression, apoptosis may not play a significant role in the pathogenesis of fructose-fed rats. As expected, etanercept treatment reduced NFκB activation and increased plasma TNF-α in both control and fructose-fed animals. Similar increases in TNF-α have been observed in humans treated with etanercept (Madhusudan et al., 2004; Nowlan et al., 2006; Tsimberidou et al., 2003) and may be attributed to the ability of etanercept to prolong the half-life of TNF-α by acting as a tumor necrosis factor carrier (Evans et al., 1994; Mohler et al., 1993). Eason and colleagues have demonstrated that elevated levels of TNF-α during etanercept treatment were associated with concomitant low levels of TNF-α bioactivity (Eason et al., 1996), which may explain why we observed increased TNF-α paralleled with decreased activation of NFκB. In contrast to the work of Togashi and co-workers (Togashi et al., 2002), we did not observe improvements in insulin sensitivity in fructose-fed rats treated with etanercept. It has previously been reported that inhibition of TNF-α improved insulin sensitivity in rodents (Cheung et al., 1998; Hotamisligil et al., 1993; Uysal et al., 1997). In contrast to animal studies, etanercept treatment in humans resulted in no improvements in insulin sensitivity in patients with the metabolic syndrome (Bernstein et al., 2006; Lo et al., 2007), insulin  118  resistance (Ofei et al., 1996; Paquot et al., 2000) or Type 2 diabetes (Dominguez et al., 2005). The reason for the inconsistency between the effects of TNF-α inhibition/etanercept treatment on insulin sensitivity in rodents versus humans is unclear. It is possible that in humans, etanercept is not capable of altering the site at which TNF-α interrupts the insulin signaling pathway (Dominguez et al., 2005), or as a result of the autocrine/paracrine actions of TNF-α, neutralizing circulating TNF-α may be insufficient to improve insulin action (Paquot et al., 2000).  Alternatively, the dissociation of soluble TNF-α from etanercept may  allow soluble TNF-α to remain biologically active and able to signal through its cell surface receptors (Scallon et al., 2002). The ability of etanercept to improve vascular function despite the lack of effect on insulin resistance may be of clinical importance in conditions such as the metabolic syndrome or Type 2 diabetes and requires further investigation. On the other hand, the effects of etanercept on blood pressure, vascular reactivity and eNOS expression may have resulted through a TNF-α-independent mechanism. Similar to TNF-α, Ang II also exhibits proinflammatory actions and can activate NFκB (Marchesi et al., 2008). Therefore, it is surprising that fructose-fed rats treated with etanercept had elevated Ang II levels, decreased NFκB activation and were normotensive.  It is possible that  etanercept interacts with the renin angiotensin system independent of TNF-α inhibition to prevent NFκB activation and maintain normal blood pressure. It is also interesting that etanercept treatment tended to increase plasma NE in both control and fructose-fed rats, leading us to speculate about a potential interaction between etanercept treatment and α1adrenoceptors.  119  To the best of our knowledge, this is the first study to demonstrate that etanercept treatment prevented the development of hypertension, normalized endothelium-dependent relaxation and restored eNOS expression in fructose-fed rats. TNF-α has been proposed as a contributing factor in the development of endothelial dysfunction (Wimalasundera et al., 2003). In addition, TNF-α has been shown to downregulate eNOS (Aljada et al., 2002; Cardaropoli et al., 2003) by shortening its half-life through an increased rate in mRNA degradation (Yoshizumi et al., 1993). Our findings are consistent with previous studies that have reported improved endothelium-dependent relaxation with etanercept treatment (Arenas et al., 2005; Arenas et al., 2006; Arenas et al., 2006; Csiszar et al., 2007; Fichtlscherer et al., 2001) and improved eNOS expression following treatment with a neutralizing antibody to TNF-α (Picchi et al., 2006). More recently, the role of increased oxidative stress as a proposed mechanism in the development of fructose-induced hypertension has gained attention (Delbosc et al., 2005; Song et al., 2005). Interestingly, in a rodent model of Ang IIinduced hypertension, etanercept not only prevented an increase in vascular oxidative stress, it also prevented the development of hypertension (Guzik et al., 2007). We propose that the beneficial effects of etanercept on blood pressure, vascular reactivity and eNOS protein expression may have occurred through a reduction in oxidative stress, a potential TNF-αindependent mechanism. As previously discussed in section 5.1.2, hypertriglyceridemia, and not insulin resistance/hyperinsulinemia, has been proposed as a causal link in the development of fructose-induced hypertension (Si et al., 1999). Our data do not support this hypothesis given that the development of hypertension was prevented in fructose-fed rats treated with etanercept, despite the presence of hypertriglyceridemia. Similar observations have been  120  made in fructose-fed rats treated with the AT1 receptor antagonists, losartan (Navarro-Cid et al., 1995) or TCV-116 (Chen et al., 1996), or thromboxane synthase inhibitor, dazmegral (Galipeau et al., 2001). In addition, a reduction in elevated triglyceride levels did not reduce elevated blood pressure in fructose-fed rats (Fujioka et al., 2003; Galipeau et al., 2002), supporting the concept of a triglyceride-independent component involved in the development of fructose-induced hypertension. The RhoA/Rho-kinase signaling pathway is involved in calcium sensitization of vascular smooth muscle cells and has been proposed as a contributing factor in the development of hypertension (Masumoto et al., 2001; Uehata et al., 1997). We observed increased ROCK-1 protein expression in thoracic aorta from fructose-fed rats, which suggests that calcium sensitization may contribute to the pathogenesis of fructose-induced hypertension. It is unclear whether the increase in ROCK-1 expression was a result of increased activity of the RhoA/Rho-kinase pathway or due to the elevated levels of NE and Ang II. Therefore, further studies are required to elucidate a specific role for the RhoA/Rhokinase signaling pathway in the development of hypertension in fructose-fed rats.  121  5.2  CONCLUSIONS  1. The pathogenesis of fructose-induced hypertension is complex in nature and involves numerous pathways that do not necessarily function independently from one another. We have demonstrated that chronic treatment with bosentan, a dual endothelin receptor antagonist, L-158,809, an AT1 receptor antagonist, prazosin, an α1adrenoceptor antagonist or etanercept, a soluble recombinant fusion protein consisting of the extracellular ligand binding domain of TNFR2, prevented the development of fructose-induced hypertension without affecting insulin levels or insulin sensitivity.  2. In the presence of chronic endothelin receptor blockade, Ang II levels were normalized, whereas an upregulation in vascular ET-1 levels was observed following AT1 receptor blockade. These data suggest that both the endothelin system and renin angiotensin system are crucial players in the development of fructose-induced hypertension, with ET-1 contributing its effects through modulation of Ang II. The significance of identifying a relationship between two of the most potent vasoconstrictor systems is that it provides further information on the pathogenesis of fructose-induced hypertension and may suggest potential usefulness as a therapeutic strategy.  3. Overactivation of the sympathetic nervous contributes to the development of fructoseinduced hypertension, however it does not appear to be an initial, precipitating event given that no effect on Ang II levels were observed during prazosin treatment.  122  4. Chronic etanercept treatment prevented the development of hypertension by improving vascular function and restoring eNOS expression in fructose-fed rats. These effects do not appear to be associated with a maintained state of chronic inflammation and occurred in an insulin-independent manner.  123  5.3  FURTHER WORK Given that several pathways have been proposed to mediate the link between insulin  resistance/hyperinsulinemia and hypertension and that blocking any one of the proposed mechanisms prevents the development of hypertension in fructose-fed rats, potential interrelationships between these pathways may exist that contribute to the development of fructose-induced hypertension. Substantial evidence exists for an interaction between the sympathetic nervous system and renin angiotensin system, which is considered to occur in both directions (Grassi, 2001).  Although we investigated the effect of chronic α1-  adrenoceptor blockade on plasma Ang II levels, whether the renin angiotensin system influences sympathetic nervous system activity in fructose-fed rats remains to be determined. Further studies to investigate whether this crosstalk plays an important role in fructose-fed rats may provide additional information on the pathophysiology of this animal model. As well, the ability to distinguish between the role of the endocrine and local renin angiotensin systems is another important aspect that needs to be investigated as locally produced Ang II within cardiovascular tissues may be involved in the development of hypertension. This interaction may be of clinical importance in conditions where both the sympathetic nervous system and renin angiotensin system are activated, such as in the metabolic syndrome. Another interaction that may play a role in fructose-fed rats is between the endothelin system and sympathetic nervous system. Questions remain regarding the existence of such a relationship, whether ET-1 can facilitate NE release or if increased sympathetic nervous system activity can enhance ET-1 secretion. Determining the effects of chronic endothelin receptor blockade on parameters of the sympathetic nervous system may shed light on the complex nature of fructose-induced hypertension and provide insight into potential  124  therapeutic strategies. Other factors, including adipokines such as leptin and adiponectin, oxidative stress, advanced glycation end products and hyperuricemia, have been proposed as causative mediators in the development of insulin resistance, hypertension and/or the metabolic syndrome. Further studies are needed to examine the role of these proposed mediators in addition to investigating the existence and importance of any crosstalk that may occur between or among them. The ability to substantiate a contributing role of adipokines in the pathogenesis of fructose-fed rats requires the use of pharmacological tools, in particular, the development of specific leptin antagonists. There are several unresolved issues that exist regarding the role of TNF-α in fructosefed rats. The exact role of TNF-α is unclear. Whether TNF-α is involved in the regulation of blood pressure and if elevations in TNF-α are necessary to contribute to the development of hypertension requires additional investigation. The mechanism in which etanercept exerts beneficial effects on blood pressure, vascular reactivity and eNOS expression remains to be determined. Whether etanercept can elicit these effects through a mechanism independent of TNF-α inhibition, possibly through reducing oxidative stress, requires further study. To elucidate the role of calcium sensitization and the RhoA/Rho-kinase pathway in the development of hypertension in fructose-fed rats also requires further investigation. Lastly, as fructose-fed rats exhibit numerous symptoms of the metabolic syndrome, it may be of interest to determine whether this animal model will eventually result in Type 2 diabetes. 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