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Mechanisms of hypertension in hyperinsulinemic and insulin resistant fructose hypertensive rats Verma, Subodh 1996

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MECHANISMS OF HYPERTENSION IN HYPERINSULINEMIC AND INSULIN RESISTANT FRUCTOSE HYPERTENSIVE RATS by SUBODH V E R M A M.Sc. (Pharm.), The University of British Columbia, 1993  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF GRADUATE STUDIES Division of Pharmacology and Toxicology Faculty of Pharmaceutical Sciences  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA July 1997 ©Subodh Verma, 1997  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his or  her  representatives.  It  is understood that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ii  ABSTRACT  The fructose-induced hypertensive (FH) rat is a diet-induced model of mild hypertension. In this model, feeding normal Sprague Dawley rats a fructose-enriched diet (66% fructose) results in hyperinsulinemia, insulin resistance and hypertension (independent of obesity). Several lines of evidence suggest that insulin resistance and hyperinsulinemia may play a pathogenic role in the development of high blood pressure in this model. This view is supported by observations which indicate that drugs that specifically counter insulin resistance (and attenuate hyperinsulinemia) exhibit antihypertensive effects.  Despite the  increasing use of this model, very little information is available regarding the mechanism(s) that link hyperinsulinemia/insulin resistance to high blood pressure in FH rats. A growing body of recent evidence suggests, that insulin, in addition to its well known effects on carbohydrate, protein and lipid metabolism, exerts important hemodynamic effects by modulating both vascular tone and sympathetic activity. This has led to the hypothesis that in states of insulin resistance/hyperinsulinemia, alterations in the hemodynamic actions of insulin may be important in the development and/or reinforcement of hypertension. The experiments outlined in this thesis were designed to examine this proposition in insulin resistant and hyperinsulinemic FH rats. In the first series of experiments, we studied the direct effects of insulin on the reactivity of aortae and perfused mesenteric arteries (MVB) from control (C) and FH rats to norepinephrine and angiotensin JJ. The key observation from this study was that the vascular effects of insulin were vessel-specific and dose-dependent.  In the C rat aortae,  pharmacological insulin concentrations (100 mU/ml) attenuated the contractile responses,  iii  while in the  MVB, physiological concentrations (100 uU/ml) potentiated the pressor  responses to NE. In contrast to the effects noted in C rats, the vascular effects of insulin were altered in arteries from FH rats. In aortae from FH rats, insulin-induced attenuation of contractile responses was blunted. Conversely, in the MVB, insulin-induced potentiation was further enhanced. To determine if these changes preceded the development of hypertension, we examined the effects of insulin (100 pU/ml) on MVB reactivity in pre-hypertensive FH rats (7 days post-fructose feeding).  Strikingly, insulin-induced exaggeration of MVB  contractile responses was present even at this time-point. These data indicate that in states of insulin resistance/hyperinsulinemia, the vascular effects of insulin are altered in a fashion consistent with increases in peripheral vascular resistance. The observation that the effects of insulin in the MVB occurred at physiological insulin concentrations (vs. the aortae) suggests that changes in vascular responsiveness of this bed may be of greater relevance to overall hemodynamics in FH rats. Evidence suggesting that insulin, at physiological concentrations, can increase the synthesis, release and gene expression of endothelin-1, led us to hypothesize that hyperinsulinemia in FH rats may serve as a continual stimulus for endothelin-1 release. To examine the contribution of endothelin-1 in FH rats we examined (a) the effects of in-vitro blockade of endothelin receptors (with bosentan, the specific E T and ET^ receptor a  antagonist) on insulin-induced MVB hyper-reactivity, (b) the effects of chronic bosentan treatment on plasma insulin levels and blood pressure in FH rats, and (c) reactivity of mesenteric arteries from C and FH rats to endothelin-1. Analysis of endothelin-1 levels revealed that the FH rats exhibited a two-fold higher endothelin-1 content in the MVB when compared to C, normotensive rats. Furthermore, in-vitro endothelin receptor blockade  iv  prevented the component of insulin-induced hyper-reactivity in the MVB. More importantly, chronic bosentan treatment prevented the development of hypertension in FH rats. These data suggest that hyperinsulinemia in FH rats may serve to increase blood pressure through alterations in ET-1 production. To determine if vasodilation per se is a determinant of insulin sensitivity in FH rats, we studied the effects of mibefradil (a calcium channel blocker) in this model. Chronic mibefradil treatment both prevented and reversed the development of fructose-induced hyperinsulinemia and hypertension and improved insulin sensitivity (estimated by 5 hour insulin/glucose ratio). Lastly, we studied the role of the sympathetic nervous system in FH rats. This was accomplished by studying the effects of chemical sympathectomy (adrenal medullectomy, followed by weekly 6-hydroxydopamine injections) on plasma insulin levels, blood pressure and the MVB effects of insulin. Sympathectomy abrogated the development of both hyperinsulinemia and hypertension, suggesting that a functional sympathetic nervous system is required for the expression of both hyperinsulinemia/insulin resistance and hypertension in FH rats. Sympathectomy also obliterated the vascular effects of insulin in FH rats without affecting these responses in controls. Based on our observations, we propose that a close interplay between the vascular actions of insulin and the sympathetic nervous system is important in the development and maintenance of hyperinsulinemia, insulin resistance and hypertension in rats fed a high fructose diet. /John H. McNeill, PhD Supervisor  TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  v  LIST OF TABLES  vii  LIST OF FIGURES  viii  LIST OF ABBREVIATIONS PREFACE  x '. •  xi  ACKNOWLEDGEMENTS  xiii  DEDICATION  xiv  INTRODUCTION  1  I.  INSULIN RESISTANCE, HYPERINSULINEMIA AND HYPERTENSION - A N OVERVIEW  1  n.  INSULIN AND HYPERTENSION-MECHANISMS  5  Insulin and the Cardiovascular System 1. Cardiac Effects 2. Vascular Effects (a) Insulin-mediated vasodilation in humans (b) Mechanisms of insulin-mediated vasodilation (c) Effects of insulin on vascular tone and reactivity in rats (d) Insulin regulation of endothelin-1 release 3. Insulin and the SNS 4. Cardiovascular actions of insulin: are they important in long term BP regulation  7 7 8 8 9 14 16 18 21  HI.  SPECIFIC RESEARCH QUESTION, RATIONALE AND APPROACH  24  IV.  WORKING HYPOTHESES  25  VI  MATERIALS AND METHODS  27  GENERAL METHODOLOGY  27  1. BP Measurement 2. Biochemical Analysis 3. Reagents ;  27 27 29  EXPERIMENTAL PROTOCOLS  29  I.  n.  Study A. Study B. Study C. Study D. Study E. m.  Direct Effects of Insulin on Reactivity of Aortae and MVB from Control and Fructose Rats. Direct Effects of Insulin on MVB Reactivity in Pre-Hypertensive Fructose Rats Studies Examining the Role of ET-1 in Fructose-Induced Hypertension Vasodilators and the Insulin Hypothesis of Hypertension The Role of the SNS in Fructose-Hypertension  STATISTICAL ANALYSES  RESULTS Study A. Study B. Study C. Study D. Study E.  29 33 33 38 40 41  43 Direct Effects of Insulin on Reactivity of Aortae and MVB from Control and Fructose Rats Insulin Modulation of Vascular Reactivity in Pre-Hypertensive Fructose Rats Studies Examining the role of ET-1 in Fructose-Induced Hypertension Vasodilators and the Insulin Hypothesis of Hypertension Effects of Sympathectomy on Fructose-Induced Hyperinsulinemia and Hypertension  DISCUSSION Direct Effects of Insulin on Aortic and MVB Reactivity in C and F Rats Role of ET-1 in the Development of Fructose-Induced Hypertension Vasodilators and the Insulin Hypothesis of Hypertension Role of the SNS in Fructose-Induced Hypertension Pathogenesis of Fructose-Induced Hypertension: in Perspective Limitations and Other Mechanisms  43 48 55 68 76 85 86 90 95 100 104 108  CONCLUSIONS  110  BIBLIOGRAPHY  112  vii  LIST OF TABLES Table  Page  1.  General characteristics of the rats in the pre-hypertensive study  56  2.  General characteristics of the rats in the four experimental groups before and after bosentan treatment  64  3. 4. 5.  Functional characteristics of superior mesenteric arteries in response toNEandET-1  66  General characteristics of the rats in the four experimental groups in the mibefradil prevention study  75  General characteristics of the rats in the four experimental groups in the mibefradil reversal study  77  viii  LIST OF FIGURES Figure  Page  1.  Anatomic sites of BP control and potential sites of insulin action  6  2.  Effect of insulin (100 mU/ml for 2 hours) on aortic contraction to NE in C and F rats with intact endothelium  44  Effect of insulin (100 mU/ml for 2 hours) on aortic contraction to AII in C and F rats with intact endothelium  46  Effect of insulin (100 uU/ml for 2 hours) on aortic contraction to NE in C with intact endothelium  49  Effect of insulin (100 mU/ml for 2 hours) on aortic contraction to AII in C with endothelium denuded  51  Effect of insulin (100 uU/ml for 2 hours) on MVB contraction to NE in C and F rats  53  3. 4. 5. 6. 7.  8. 9. 10. 11.  Percent maximum potentiation of NE responses by insulin (lOOpU/ml) in C and F rats at lO'^M NE in the pre-hypertensive study  57  Effect of bosentan and/or indomethacin on insulin-induced MVB reactivity in C and F rats  59  Effect of chronic endothelin blockade with bosentan on plasma insulin levels and systolic BP in C and F rats  62  Wall thickness of mesenteric arteries measured over a range of transmural pressures  69  -log [NE] versus percent constriction at transmural pressures of 120 and 160 mmHg  71  12.  Insulin levels and systolic BP in the mibefradil prevention study  73  13.  Effect of tyramine on tail artery responses from sympathectomized C and F rats Effect of sympathectomy on plasma insulin levels and systolic BP  78 80  14.  15. a) 15. b) 15. c) 16. 17.  Effect of sympathectomy on pressor responses to NE in Control (C) and control-sympathectomized (CS) rats  82  Effect of sympathectomy on pressor responses to NE in fructose (F) and fructose-sympathectomized (FS) rats  82  Insulin-induced potentiation of NE pressor responses in CS and FS groups  82  Hypothetical model linking insulin resistance/hyperinsulinemia, vascular tone and elevated blood pressure Pathogenesis of fructose-induced hypertension: a schematic representation  98 107  LIST OF ABBREVIATIONS BP  Blood Pressure  SHR  Spontaneously Hypertensive Rats  FH  Fructose-induced hypertension/hypertensive  RAS  Renin-angiotensin system  NE  Norepinephrine  AII  Angiotensin II  ACh  Acetylcholine  SNP  Sodium nitroprusside  VSMC  Vascular smooth muscle cell(s)  NO  nitric oxide  MVB  Mesenteric vascular bed  ET  Endothelin  ECE  Endothelin converting enzyme  RIA  Radioimmunoassay  L-NMMA  N^-monomethyl-L-arginine  C  Control rats/group  F  Fructose-fed rats/group  CT  Control-treated group  FT  Fructose-treated group  CS  Control-sympathectomized group  FS  Fructose-sympathectomized group  PREFACE  Portions of the data have been published as: Papers:  Verma S, Bhanot S and McNeill JH. Effects of chronic endothelin blockade in hyperinsulinemic hypertensive rats. Amer. J. Physiol. 269:H2017H2021, 1995. Verma S, Bhanot S, Yao L and McNeill JH. Defective endothelium dependent relaxation in fructose-hypertensive rats. Amer. J. Hypertens. 9:370-376, 1996. Verma S, Bhanot S and McNeill JH. Decreased vascular reactivity in metformin treated fructose hypertensive rats. Metabolism 45:10531055, 1996. Verma S, Skarsgard P, Bhanot S, Laher I, Yao L and McNeill JH. Reactivity of mesenteric arteries from fructose hypertensive rats to endothelin-1. Amer. J. Hypertens. (in press) 1997.  Verma S, Hicke A and McNeill JH. Chronic T-type calcium channel blockade in hyperinsulinemic, insulin resistant and hypertensive rats. Cardiovasc Res (in press) Verma S, and McNeill JH. Vascular insulin resistance in fructose hypertensive rats. Eur. J. Pharmacol, (in press).  Portions of the data have been published as: (cont'd) Abstracts:  Verma, S., Chua, D. and McNeill, J.H. Sympathectomy prevents fructose induced hyperinsulinemia and hypertension in rats. ISHR Conference, Chicago, IL. June 13-16, 1996. J Mol Cell Cardiol 28(6) M57, 1996. Verma, S., Bhanot, S. and McNeill, J.H. Antihypertensive effects of bosentan in fructose hypertensive (FH) rats. ISHR Conference, Chicago, IL. June 13-16, 1996. J Mol Cell Cardiol 28(6) M56, 1996. Verma, S., Bhanot, S., Yao, L.F. and McNeill, J.H. Reactivity of mesenteric arteries (M) from fructose hypertensive (FH) rats to ET-1. ISHR Conference, Chicago, IL. June 13-16, 1996, J Mol Cell Cardiol, 28(6), M55, 1996. Verma, S., Bhanot, S. and McNeill, J.H. Decreased vascular reactivity in metformin treated fructose hypertensive rats. ISHR Conference, Chicago, IL. June 13-16, 1996, J Mol Cell Cardiol 28(6) M54, 1996. Verma, S., Laher I and McNeill JH. Effect of insulin on reactivity of angiotensin II in control and insulin resistant arteries. Angiotensin II receptors and their blockade, Winnepeg, Canada Oct. 16-19, 1996 Verma S. Insulin and Hypertension: is it time to restructure the hypertension paradigm ? Key Note Address 1996 John H. McCreary Health Sciences Week, Vancouver.  xiii  ACKNOWLEDGEMENTS First and foremost, my profound gratitude to my supervisor, Dr. John H. McNeill for his encouragement and support throughout my graduate program. It is quite a coincidence that he trained both my Dad and me. Over and above my thesis work, I thank him for providing me with multiple opportunities within and outside the Faculty. Working for Dr. McNeill has been one of the most enriching experiences of my life. To him, I owe a tremendous intellectual debt. I would like to thank the members of my research committee: Dr. Kath MacLeod, Dr. Jack Diamond, Dr. K. Kwok and Dr. Frank Abbott for their constructive criticism. My special thanks to Dr. MacLeod for her time and valuable suggestions over the past 4 years. I would also like to thank her for providing me with the opportunity to teach a few lectures in undergraduate and graduate courses. We are extremely thankful for the kind help of Dr. Kwok in setting up the ET assay and providing valuable experimental input throughout my Ph.D. program. My special thanks to three important people. Dr. Ismail Laher, Dr. Sanjay Bhanot and Dr. Linfu Yao. Not only have they provided experimental input (as collaborators) but they have taken genuine interest in my work. I would like to acknowledge the support of the following summer students and directed studies students during the period of my degree. Aspasia Michoulas, Alan Hicke and Emi Arikawa. My special thanks to Ms. Mary Battell (laboratory manager) and Ms. Violet Yuen for their help at various stages (technical and otherwise) over the past 4 years. My heartfelt and most sincere thanks to Sylvia Chan for her expert secretarial assistance. I am indebted to the Medical Research Council of Canada for Fellowship Support throughout my degree. I would like to thank every member of the Faculty for making my Ph.D. a memorable experience. Last, but not least, I thank a special friend, Salma, for always being there for me.  DEDICATION  In the memory of my Dad & To Mom and Sujata always placing me ahead of themselves  1  INTRODUCTION  I.  INSULIN RESISTANCE, HYPERINSULINEMIA AND HYPERTENSION: A N OVERVIEW  In the past several years, there has been growing interest in the hypothesis that resistance to the metabolic effects of insulin (insulin resistance) and compensatory hyperinsulinemia may contribute to increased blood pressure (BP) and essential hypertension, especially when associated with obesity (Reaven and Hoffman, 1987; Modan et al, 1985; Manicardi et al, 1986; Ferrari et al, 1990; Ferrannini et al, 1987a; Ferrannini et al, 1987b; Lucas et al, 1985; Christlieb et al, 1985). The observation that insulin resistance is present in lean hypertensive patients (Ferrannini et al, 1987a) has fueled speculation that these metabolic abnormalities may play a more pervasive role in hypertension than previously believed. Interest in the link between insulin and high BP was fueled by two distinct observations: (i) the lack of effective antihypertensive drugs to reduce the increased risk of coronary artery disease in hypertensive subjects (Collins et al, 1990; Korner et al, 1982; Wikstrand et al, 1988, MacMohan et al, 1989, Ferrannini and Natali 1991; Reaven, 1990) and (ii) the realization that essential hypertension per se is frequently associated with insulin resistance and hyperinsulinemia (Ferrannini and Natali 1991; Reaven, 1990). These two observations led to the so-called 'insulin hypothesis' of hypertension where it was postulated that insulin resistance and/or hyperinsulinemia may be causally related to the development of hypertension (Reaven, 1988; DeFronzo, 1992, Bhanot and McNeill, 1996). This was an attractive proposition which could help explain the apparent inability of conventional antihypertensive drugs to decrease the incidence of coronary ischemic events, since these  2  drugs did not improve but rather worsened insulin action (Lind et al, 1994, Pollare et al, 1989; Skarfors et al, 1989). Further support for this hypothesis stemmed from studies demonstrating that insulin resistance and hyperinsulinemia are present in normotensive offspring of hypertensive patients as early as the second decade of life and that these changes antedate the rise in BP (Allemann et al, 1995; Grunfeld et al, 1994). The observation that insulin resistance and hyperinsulinemia occur not only in untreated human hypertensives, but also in several rodent models of hypertension (DeFronzo, 1992; Reaven, 1991ab; Mondon and Reaven 1988; Kotchen et al, 1991; Dall-Aglio et al, 1991; Hwang et al, 1987) strengthened the contention that these abnormalities may be intrinsically linked with hypertension and are not mere coincidental findings. Studies have clearly indicated that hyperinsulinemia is an independent risk factor for coronary artery disease and that even a small degree of glucose intolerance significantly increases the risk for developing coronary artery disease (Ducimetiere et al, 1980; Pyorala, 1979; Fuller et al, 1980; Bhanot and McNeill, 1996). In addition to hypertension, insulin resistance and hyperinsulinemia have been implicated as a common pathophysiologic mechanism in obesity, non-insulin dependent diabetes mellitus (NTJDDM) and dyslipidemia. Epidemiological studies have shown clustering of obesity, hypertension and NIDDM associated with a high risk of coronary heart disease (Reaven, 1991; DeFronzo and Ferrannini, 1991; Kannell et al, 1991; Multiple Risk Factor Trial Research Group, 1986; Sowers et al, 1991; Reaven, 1994, Weidmann et al, 1993; Haffner et al, 1992). The relevance of insulin resistance and hyperinsulinemia as a causal factor in hypertension has been the subject of much current discussion and debate. Although several reports indicate a positive correlation among BP, the degree of insulin resistance and plasma  3  insulin concentrations, the association appears to be especially prominent in obese subjects. Studies have, however, demonstrated that insulin resistance may be present in lean hypertensive subjects. Ferrannini et al, 1987ab have reported that whole body glucose uptake during a hyperinsulinemic euglycemic clamp was reduced by ~ 30-40% in lean hypertensive patients compared to normotensive controls; also, the severity of hypertension in these patients correlated with the degree of insulin resistance. Several studies, however, have not found a correlation between insulin and BP. Others have found that the association between plasma insulin concentrations and BP is weak or nonexistent (Asch et al, 1991; Collins et al, 1990; Saad etal, 1990; Mbanya et al, 1988).. Despite the dispute that exists due to disparate results from several studies, a few consistencies regarding the relationship have emerged. First, as many as 50% of essential hypertensive patients appear to be insulin resistant and hyperinsulinemic (Denker and Polock, 1992; Zavaroni et al, 1992). There is ethnic variation in this relationship; Pima Indians and Mexican Americans often have insulin resistance, but rarely have hypertension (Saad et al, 1990; Miura et al, 1995; Saad et al, 1991). To the proponents of the insulin hypothesis of hypertension, this lack of association between insulin and BP in certain populations has suggested that a genetic predisposition towards insulin-mediated increases in BP must be present before hypertension occurs. However, another interpretation is that there is no direct cause and effect relationship between insulin and hypertension. In other words, the reported correlations between insulin resistance, hyperinsulinemia and hypertension may occur through parallel but unlinked mechanisms (Hall et al, 1993). The association between insulin resistance and hyperinsulinemia in animal models of high BP merits a brief discussion.  In favor of the hypothesis are multiple studies  4  demonstrating an association between insulin resistance, hyperinsulinemia and hypertension in rodent models of high BP. These include the Dahl rat (Kotchen et al, 1991), the spontaneously hypertensive rat (SHR, DeFronzo, 1992; Reaven, 1991; Mondon and Reaven, 1988), the Milan hypertensive rat (Dall-Aglio et al, 1991) and the fructose-induced hypertensive (FH) rat (Hwang et al, 1987). All of these hypertensive models, although etiologically distinct, exhibit common defects in glucose metabolism.  In a series of  experiments, we have recently examined the proposition that insulin resistance and hyperinsulinemia contribute causally to the development of high BP. Essentially, if these defects were pathogenic in the development of hypertension, then drugs that counter these defects should decrease BP. We, therefore, examined the effects of multiple agents (that were known to improve insulin sensitivity) on BP in rodent models of hypertension. We found that chemically diverse drugs that had the common property of attenuating hyperinsulinemia also lowered BP in both the SHR and the F-hypertensive rat (Bhanot and McNeill, 1994; Bhanot et al, 1994abcde; Verma et al, 1994a;Verma et al, 1994b). All of these drugs not only caused sustained reductions in plasma insulin concentrations and BP, but the antihypertensive effects of these drugs could be reversed by simply restoring the plasma insulin levels in the drug-treated rats to those that existed before drug treatment. Similar results have been reported by other laboratories and indicate that there is a strong and close association between hyperinsulinemia and hypertension in rodent models of hypertension (Morgan and Mark, 1993; Meehan et al, 1993; Pershadsingh et al, 1993). In contrast to the reports in rats, hyperinsulinemia (induced by chronic insulin infusion) does not appear to cause increases in BP in dogs (Hall et al, 1990b; Hall et al, 1995a). Intriguingly, when dogs are fed a high fat or fructose diet, they become insulin resistant, hyperinsulinemic and  5  hypertensive (Martinez et al, 1994). The differences between the effects of insulin in dogs vs. rats have been ascribed to species variation in the effects of insulin on cardiovascular and sympathetic responses (Hall et al, 1995ab), an issue that we will address later. Notwithstanding the above discussion, the current evidence strongly suggests that the association between insulin and hypertension is not merely coincidental and that insulin resistance and hyperinsulinemia may be important in the development and course of hypertension in a subset of patients and in certain animal models.  II.  INSULIN AND HYPERTENSION - MECHANISMS  Normal regulation of BP is governed by the basic hydraulic equation: arterial BP is directly proportional to the product of the blood flow (cardiac output) and the resistance to the passage of blood through precapillary arterioles (peripheral vascular resistance). Physiologically, in both normal and hypertensive states, BP is maintained by moment-tomoment regulation of cardiac output and vascular resistance exerted at three anatomic sites: arterioles, postcapillary venules (capacitance vessels) and the heart (Benowitz, 1995). A fourth anatomic control site, the kidney, contributes to the maintenance of BP by regulating the volume of intravascular fluid. Baroreflexes, mediated by sympathetic nerves, act in combination with humoral mechanisms, including the renin-angiotensin system (RAS) to coordinate function at these four control sites and to maintain normal BP. The observation that insulin has the ability to modulate the function of each of these anatomical control sites (Figure 1) has led to the hypothesis that in states of insulin resistance and hyperinsulinemia, alterations in insulin's cardiovascular and renal actions may be important in the development and/or reinforcement of high BP. In this section we shall address two key issues: (a) the  6  FIGURE 1 Anatomic Sites of BP Control and Potential Sites of Insulin Action  Modified from Benowitz, 1995  INSULIN  INSULIN  INSULIN  INSULIN  INSULIN  cardiovascular effects of insulin as they relate to hemodynamics and insulin regulation of glucose uptake, and (b) the potential of alterations in insulin's cardiovascular actions as being contributing factors towards hypertension.  Insulin and the Cardiovascular System 1.  Cardiac Effects:  Experimental data indicate that insulin can alter cardiac  function (Lucchesi et al, 1972; Merin, 1970; Sheldon et al, 1969; Weissler, 1979; Liang et al, 1982). A positive inotropic effect of insulin was first documented in isolated cardiac muscle preparations as well as in newborn lamb (Lee and Downing, 1976) and piglet myocardium (Downing et al.,, 1977). Studies by Liang et al. (1982) in conscious dogs demonstrated that supraphysiological insulin doses increased myocardial contractility and coronary blood flow in the absence of changes in blood glucose concentrations. In contrast, no increment in myocardial blood flow was observed by Barrett et al. (1984) in awake dogs at insulin concentrations within the physiological range. Studies in the human heart have documented an increase in heart rate and cardiac output after insulin administration (Rowe et al, 1981; Anderson et al, 1991; Fisher et al, 1987) or following the ingestion of a mixed meal (Avasthi et al, 1983).  However, most of these data were obtained with  supraphysiological insulin doses or under conditions of concomitant changes in plasma glucose concentrations. By employing the euglycemic hyperinsulinemic clamp technique (which allows evaluation of the effects independent of changes in glucose) studies have demonstrated that insulin in the physiological range caused = 25% increase in cardiac output, secondary to an increase in both stroke volume and heart rate (Baron and Brechtel, 1993). In contrast, Ferrannini et al. (1993) failed to observe any change in cardiac and systemic  8  hemodynamics during physiological hyperinsulinemia. Studies in rats have revealed that hypertension secondary to chronic hyperinsulinemia is not associated with changes in cardiac output or heart rate (Brands et al, 1996).  2.  Vascular Effects: The effects of insulin on vascular tone and reactivity are both  complex and confusing.  The confusion, is, in part, due to the variations in insulin  concentrations employed in various studies and the apparent species variations in the effects of insulin on blood vessels.  (a)  Insulin-mediated vasodilation in humans  The current literature supports a vasodilatory role of insulin in humans (Brands et al, 1991b; Dela et al, 1995; Egan and Stepnaikowski, 1994; Laakso et al, 1990). Insulin has specific and physiologically relevant effects to increase skeletal muscle blood flow. In recent years, results have repeatedly shown that intravenous insulin, independent of glucose changes, increases blood flow in the leg (Baron, 1993; Baron and Brechtel, 1993; Baron, 1994, Baron et al, 1995, Baron et al, 1991). By combining the euglycemic clamp with the leg balance technique, these investigators also demonstrated that insulin-induced vasodilation was specific for skeletal muscle.  This effect, was dose-dependent and occurred at  physiological insulin concentrations with an apparent E D 5 0 of 35-40 pU/ml in lean insulin sensitive subjects (Laakso et al, 1990). The vasodilating action of insulin has been confirmed by several groups over a range of physiological insulin concentrations and by using different techniques (Richter et al, 1989; Bennett et al, 1990; Edelman et al, 1990; Vollenweider et al, 1993; Boden et al, 1993, Anderson et al, 1991). Insulin infused locally, directly into the  9  brachial (Ueda et al, 1995) or femoral artery (Steinberg et al, 1994), leads to similar increases in blood flow in humans, suggesting that the vasodilating properties may be mediated via local factors (discussed later). Additional studies in humans, utilizing the intraarterial infusion model have confirmed a direct effect of insulin in reducing vascular responsiveness to pressor agents (Sakai et al, 1993; Lembo et al, 1993b; Smits et al, 1993). Sakai et al. (1993) demonstrated that local hyperinsulinemia (= 130uU/ml) attenuates the increase in forearm vascular resistance induced by norepinephrine (NE) and angiotensin-U (A U).  Likewise Lembo et al (1993b) observed that intrabrachial infusion reduced the  vasoconstrictive response to reflex sympathetic activation evoked by lower body negative pressure. Although the majority of studies indicate a role of insulin as a vasodilator, one cannot ignore previous studies in which such an effect was not detected (DeFronzo et al, 1985; Jackson et al, 1986; Yki-Jarvinen et al, 1987; Kelley et al, 1990). The reasons for the discrepancies have been linked to differences in (a) blood flow measurement techniques and (b) subject variability among different studies (with regard to age, physical fitness and basal vascular tone) (Capaldo and Sacca, 1995). However, the current data, taken together, clearly support a vasodepressor effect of physiological insulin concentrations in humans.  (b)  Mechanisms of insulin-mediated vasodilation  The mechanisms by which insulin causes vasodilation are still not completely known, but new knowledge has shed light on this issue. Insulin-mediated vasodilation has been suggested to be mediated through (i) a direct effect of insulin on vascular smooth muscle cells (VSMC), (ii) an indirect effect via the release of endothelium-derived vasoactive  10  factors, (iii) an indirect effect coupled to metabolic activity such as oxygen consumption, and (iv) a combination of the above (Baron, 1994). Insulin is known to stimulate the Na K ATPase activity (Prakash et al, 1992). +  +  Thus, insulin could vasodilate by hyperpolarizing VSMC and reducing calcium influx. Kahn et al (1993) have provided evidence for this mechanism. Sowers and coworkers have shown that insulin enhances the gene expression and activity of the VSMC Na K ATPase and +  +  Ca+^ATPase (Tirupattur et al, 1993; Sowers and Eptstein, 1995), which are the main cellular pumps regulating VSMC [Ca+2]j. Several studies have reported that insulin reduces agonist-induced [Ca ]i in cultured VSMC (Kahn et al, 1993; Kahn et al, 1994; Standley +2  et al, 1991; Saito et al, 1993; Touyz et a/t'1994). Pharmacological doses of insulin have been shown to inhibit serotonin-induced Ca+2 influx, (Ca 2)j and contraction in cultured +  VSMC (Kahn et al, 1994; Kahn et al, 1993). Moreover, it was shown that insulin did not inhibit Ca+2 release from internal stores or stimulate Ca 2 efflux from the cell (Kahn et al, +  1994).  Others have reported that insulin hyperpolarizes by increasing Ca 2 activated K +  +  channel activity (Berweck et al, 1993). This would be expected to decrease Ca+2 influx via voltage operated Ca 2 channels. An attenuated ll^-induced Ca+2 mobilization from internal +  stores has also been reported in response to insulin in high doses (Saito et al, 1993). Although the balance of published information indicates that insulin, at physiological concentrations, influences endothelial function (discussed below), the aforementioned systems may be important at pharmacological insulin concentrations and in the long-term regulation of vascular tone (Baron and Steinberg, 1996).  11  Much current attention has focused on the interaction between insulin and the endothelium-derived nitric oxide (NO) system in mediating vasodilation. There is now compelling evidence that insulin-mediated vasodilation in humans is NO dependent. Studies by Steinberg et al. (1994) have provided evidence for this mechanism. In their studies, intrafemoral artery infusion of the specific inhibitor of endothelium-derived NO synthesis, N ^-monomefhyl-L-arginine (L-NMMA), were performed under basal conditions in healthy volunteers and leg blood flow was measured by thermodilution. In a separate group, LNMMA infusions were performed after 3 h of hyperinsulinemia during a euglycemic clamp designed to increase leg blood flow approximately two fold. At baseline, L-NMMA caused ~ 25% fall in leg blood flow.  During hyperinsulinemia, leg blood flow increased  approximately two fold, and in contrast to baseline, L-NMMA caused a =50% fall in leg blood flow indicating that insulin-mediated vasodilation was NO dependent. Additional studies have also indicated that blockade of NO synthesis (with L-NMMA) abrogates insulinmediated vasodilation (Steinberg et al., 1994). Although the exact mechanism/s through which insulin interacts with the NO pathway in humans are unclear, studies indicate that this may involve synthesis/release of NO, but not NO action on VSMC (Steinberg et al., 1994). It is not clear how insulin could attenuate the agonist-induced [Ca 2]j and contraction in +  various preparations which are devoid of endothelial cells. Although studies have attributed this to differences in preparations and insulin concentrations, evidence suggests that insulin may also stimulate nitric oxide synthase (NOS) activity in the VSMC (Han et al., 1995). Another possibility is that the insulin-induced increase in blood flow may be related to the metabolic effect of the hormone. It has been hypothesized that the increase in oxygen  12  consumption and/or lactate release consequent to insulin stimulation might be responsible for vasodilation (Vollenweider et al, 1992). In this context, it is interesting to recall previous studies that indicate that metabolic demand (induced by hypoxia and exercise) is a signal for blood flow (Hudlicka 1985; Johnson, 1986). In this paradigm, insulin's hemodynamic effects would be a consequence, not a determinant of the metabolic effects. Although a previous report indicated that insulin-induced vasodilation may be mediated via stimulation of 8-adrenergic receptors, recent evidence suggests that insulin increases leg blood flow even during a and 6 adrenergic receptor blockade (Randin et al, 1992). The vasodilating action of insulin raises a number of questions regarding the physiological significance of this effect.  Two areas of intense research are (a) the  contribution of insulin-mediated vasodilation towards glucose metabolism, and (b) the role of the insulin-mediated vasodilation in the maintenance of vascular tone. The first question, as to whether the hemodynamic actions of insulin are linked to its metabolic actions (on glucose uptake), has been recently investigated. Analysis of the data available appears to support the observation. One of the key points in favor of this view is the demonstration that muscle perfusion per se is an independent determinant of insulin-mediated glucose uptake in skeletal muscle (Baron, 1996; Schultz et al, 1977; Ebeling and Koivisto, 1993; Wiernsperger et al, 1994; Baron et al, 1991, Baron et al, 1995, Baron and Steinberg, 1996). Studies have indicated that infusion of the vasodilating agent methacholine into the femoral artery leads to an increase in glucose uptake significantly above that obtained with insulin alone (Steinberg et al, 1994). This indicates that an increase in muscle perfusion per se is a factor increasing muscle glucose uptake. In another study, Baron et al. (1995) examined the contribution of  13  insulin-mediated vasodilation to insulin's overall effect to stimulate glucose uptake in skeletal muscle. Steady state euglycemia alone caused =15% increase in leg glucose extraction and a two fold increase in leg blood flow. When insulin-mediated vasodilation was inhibited, leg blood flow returned to baseline rates and the glucose extraction increased by 50% with a net effect to reduce leg glucose uptake by =25%. Thus, it has been suggested that vasodilation per se may account for approximately one-fourth of insulin's overall effect to stimulate glucose uptake (Baron et al, 1991; Baron et al, 1995, Baron, 1994). Further support of a potential role of insulin-mediated vasodilation in modulating metabolic responses comes from studies in insulin-resistant patients. In conditions of insulin resistance (diabetes and obesity), insulin-mediated vasodilation is markedly blunted. In fact, the dose-response curve for insulin-induced-vasodilation is markedly shifted to the right in obese patients and is virtually flat in obese diabetic subjects (Laakso et al, 1990; Laakso et al, 1992). Furthermore, the dose-response characteristics of glucose uptake closely parallel the pattern of vascular response, suggesting that the impaired insulin-mediated vasodilation could partly account for the reduced rates of insulin-mediated glucose uptake (insulin resistance) observed in these patients (Lind and Litherll, 1993). The exact mechanism/s through which insulin-mediated vasodilation is coupled to glucose metabolism remains unknown; however, evidence suggests a role of increased capillary perfusion in this effect (Bonadonna et al, 1992). Insulin has been demonstrated to induce the recruitment of additional capillaries and consequently provide greater surface area for plasma-tissue glucose exchange.  In addition, the insulin gradient from the vascular  compartment to the interstitial space is narrowed, so that insulin delivery to cells is enhanced.  14  In support of this view are observations suggesting that capillary density correlates closely with insulin-mediated glucose uptake (Yang et al, 1989; Lillioja et al, 1987). The role of insulin in the maintenance of vascular tone in humans has been a subject of much discussion and debate. Studies demonstrating that the vasodilatory actions of insulin are blunted in insulin resistant states of obesity and diabetes (Laakso et al, 1990; Laakso et al, 1992) have been extrapolated to imply that insulin-mediated vasodilation is an important determinant of vascular tone and BP. However, acute systemic or local infusions of insulin produce no change or a small change in mean arterial pressure. Studies by Anderson et al. (1991) have elegantly demonstrated that although insulin causes marked reductions in forearm vascular resistance (due to vasodilation), the reason why insulin does not alter blood pressure is because it simultaneously activates the SNS (discussed later). Thus, the effects of vasodilation are offset by an increase in SNS activity. Notwithstanding the above discussion, it is possible that in states of insulin resistance, a loss of vasodilation associated with insulin resistance could diminish vasodilatory reserve and thus "sensitize" the vasculature to the pressor forces (Baron, 1994).  Thus, the available data raise the compelling question as to  whether resistance to the vasodilator effects of insulin is important in the development of  hypertension in states of insulin resistance. This finding could provide new insight into our understanding of the mechanisms underlying the increased prevalence of hypertension observed in states of insulin resistance.  (c)  Effects of insulin on vascular tone and reactivity in rats  In contrast to humans, the effects of insulin on vascular tone and reactivity in rats have been inconsistent. Although a large number of studies have demonstrated that insulin  15  reduces agonist-induced intracellular (Ca+2)j in cultured rat VSMCs, attenuates contraction and affects various sarcolemmal and sarcoplasmic reticular Ca+2 transport systems (Saito et al, 1993; Hori et al, 1992; Kim Y-C et al, 1993; Standley et al, 1991; Standley et al, 1992; Zemel et al, 1992, Wambach and Liu, 1992; Han et al, 1995), the physiological relevance of these findings remains uncertain because of the pharmacological insulin concentrations employed in these studies (approximately 1000 fold higher than physiological concentrations) (Arnquist, 1983). Studies in intact blood vessels in-vitro have reported both constrictory (Juncos and Ito, 1993; Yanigasawa-Miwa et al, 1990; Wu et al, 1994; Townsend et al, 1992) and dilatory effects (Walker et al, 1996; Alexander and Oake, 1977) of insulin. Insulin (in pharmacological concentrations) has been shown to attenuate pressor responses to NE and A II in the tail veins and aortae from control rats (Alexander and Oake, 1977, Hori et al, 1992, Han et al, 1995). Similar observations were made in rabbit femoral arteries and veins, where supraphysiological concentrations of insulin inhibited the vasoconstrictor effect of A U (Yagi et al, 1988). Studies examining the effects of insulin on the mesenteric vasculature have reported conflicting results.  Studies have shown that  physiological insulin concentrations attenuate vasoconstriction by NE, serotonin and potassium chloride in mesenteric arterioles (Wambach and Liu, 1992; Walker et al, 1997). However, when the effects of similar concentrations of insulin are examined in the entire perfused MVB, insulin consistently exaggerates the pressor responses to vasoactive agents (Wu et al, 1994; Townsend et al, 1992). In the rat mesenteric artery-gut preparation insulin has been demonstrated to markedly potentiate the pressor responses to NE (Townsend et al, 1992). Similarly, insulin-mediated potentiation of AVP responses has been observed in normotensive control Sprague Dawley rats (Wu et al, 1994). Although the mechanism/s  16  through which insulin affects vascular tone in rats remains unknown, a variety of endothelium-dependent and -independent effects have been suggested (Han et al, 1995). It is important to point out that studies examining the effects of insulin on blood vessel reactivity in-vitro provide limited information regarding the effects of insulin on global vascular tone and reactivity in-vivo. Studies examining the effects of acute and chronic insulin infusion in rats project a clearer picture. These studies demonstrate that unlike humans, insulin infusion in rats is associated with no vasodilatory responses. In fact, total peripheral resistance and mean arterial pressure are elevated in response to chronic insulin infusions (Brands et al, 1996; Brands et al, 1991a). As the changes in peripheral vascular resistance in response to insulin have been shown to be independent of changes in cardiac output and heart rate (Brands et al, 1996), it is safe to conclude that the net hemodynamic effect of insulin is vasoconstriction in control rats.  (d)  Insulin regulation of endothelin-1 release  To add to the diversity of the biological actions of insulin are reports indicating that physiological concentrations of insulin stimulate the synthesis, secretion and gene expression of the potent vasoconstrictor agent endothelin (ET)-l (Oliver et al, 1991; Frank et al, 1993; Hu et al, 1993; Hattori et al, 1991). ET-1 is a newly isolated vasoactive peptide that is synthesized and secreted from vascular endothelial cells (Yanagisawa et al. 1988). ET-1 is by far the most potent endogenous vasoconstrictor yet identified (Yanagisawa and Masaki, 1989). ET-1 is primarily released by endothelial cells in response to several mediators such as shear stress, thrombin, transforming growth factor-61, angiotensin TJ, arginine-vasopressin, calcium ionophores and more recently insulin.  The hormone is produced as  17  preproendothelin, cleaved into big ET and finally converted into ET-1 by the endothelin converting enzyme (ECE). More than 2/3rd of the produced ET-1 is released preferentially towards the vascular smooth muscle where it causes vasoconstriction by interacting with chiefly E T and ET5 receptors in some vascular beds. As very small amounts of ET-1 are a  released towards the lumen, it has been postulated that ET-1 may play a vital role as a local modulator of vascular smooth muscle tone and reactivity. Interestingly, insulin has been demonstrated to increase immunoreactive ET release from cultured VSMC and enhance arginine-vasopressin and A II induced immunoreactive ET release (Anfossi et al, 1993). In conjunction with these observations are studies demonstrating elevated ET-1 levels in insulin-treated diabetic patients (Takahashi et al, 1990) and in experimental models of diabetes during insulin treatment (Hu et al, 1993; Takeda et al, 1991). In addition to stimulating ET-1 release, studies have demonstrated a two fold increase in ET receptor number in normal VSMC treated with insulin in-vitro (Frank et al, 1993). The combination of insulin and ET has been shown to result in a 10-fold increase in cell proliferation. Although the exact mechanism through which insulin increases ET-1 production is not known, reports indicate that this effect may be mediated through the tyrosine kinase action of insulin and probably results from the stimulation of a nuclear protein which acts in trans on cis elements of the ET-1 promotor (Hu et al, 1992; Hu et al, 1993). In summary, accumulating evidence from experimental and clinical studies indicate that insulin, in addition to it's well known effects on carbohydrate, lipid and protein metabolism, exhibits important hemodynamic effects.  At the level of the vasculature,  physiological insulin concentrations appear to cause vasodilation in humans. This, in turn, may be part of insulin's overall action to enhance glucose uptake. Studies examining the  18  effects of insulin on reactivity of blood vessels in rats have yielded inconsistent results. This is, in part, due to the pharmacological concentrations of insulin employed and the apparent species differences in the effects of insulin in rats vs. humans.  Insulin may activate  completely divergent pathways, for example insulin may stimulate the L-arginine-NO system while activating the synthesis and release of the potent vasoconstrictor ET-1. Although the question as to what role insulin plays in regulating vascular tone in-vivo remains far from being resolved, the observation that insulin exhibits vascular effects poses the intriguing question: are changes in insulin's vascular effects important in the development and/or reinforcement of hypertension ?  3.  Insulin and the SNS. Insulin-induced stimulation of the SNS has been long proposed  to represent a link between hyperinsulinemia and hypertension. For detailed reviews the reader is referred to some excellent articles (Anderson, 1993; Mediratta et al, 1995; Landsberg and Krieger, 1989; Moreau et al, 1995; Muntzel et al, 1995). Infusion of insulin during euglycemic clamps increases plasma NE levels in experimental animals (Tomiyama et al, 1992; Liang et al, 1982) and in humans (Rowe et al, 1981; Anderson et al, 1991; Berne et al, 1992; Lembo et al, 1992). Sympathoexcitatory effects of insulin are also well demonstrated through direct recordings of muscle sympathetic nerve activity in response to insulin. Insulin has been demonstrated to augment sympathetic outflow to skeletal muscle in humans (Anderson et al, 1991; Berne et al, 1992; Vollenweider et al, 1993; Muntzel et al, 1995) and to the hundlimb in normotensive rats (Morgan et al, 1993; Muntzel et al, 1995). This effect appears to be specific as no alterations in SNS activity were reported in the skin sympathetic fibers in humans or in renal or adrenal sympathetic activity in normotensive rats  19  (Morgan et al, 1993). In rats, a pressor role for insulin-induced sympathetic activation has been suggested by several studies. For example, insulin-induced elevations in heart rate and arterial pressure were prevented by ganglionic blockade (Edwards et al, 1989). In addition, Tomiyama et al (1992) chronically infused insulin into Dahl salt-sensitive and salt-resistant rats. Insulin increased plasma NE levels and arterial pressure in Dahl salt-sensitive rats without affecting either parameter in Dahl salt-resistant animals. Finally, Kaufman et al (1991) reported that a high fat diet elevated insulin levels, urinary NE levels and arterial pressure in Sprague Dawley rats. The increase in SNS activity would be expected to cause increases in cardiac output, peripheral vasoconstriction and a consequent increase in BP. However, studies in humans have shown that although physiological increases in insulin concentrations cause an increase in muscle sympathetic activity and nerve firing rate, it results in no change in BP (Anderson et al, 1991; Berne et al, 1992). The reason why insulin does not increase BP, despite stimulation of the SNS, is that insulin causes preferential vasodilation (discussed above) in the skeletal muscle vasculature and thereby leads to a redistribution of cardiac output to skeletal muscle (Baron, 1993; Baron, 1994). Thus the vasodilatory effects of insulin offset the increase in cardiac output. The mechanism/s through which insulin evokes sympathetic overactivity are not well defined. Three mechanism/s have been suggested: (i) a direct action on the CNS; (ii) a baroreflex action secondary to hemodynamic changes, and (iii) a direct action on NE metabolism. In support of the first hypothesis are studies demonstrating the presence of insulin-specific binding sites in the hypothalamus. Additionally, intraventricular injection of insulin has been shown to increase catecholamine turnover in the brain (Sauter et al, 1983). Although the possibility that insulin stimulates the SNS secondary to vasodilation and  20  activation of the baroreflex mechanism has been suggested (Anderson et al, 1991), this has not been confirmed (Hall et al, 1990a). The third mechanism relates to the direct effects of insulin on NE flux. In support of this hypothesis are studies that demonstrate that insulin affects NE uptake by atrial strips (Bhagat et al, 1981). Recently, Lembo et al (1993a) examined the contribution of changes in NE metabolism during intrabrachial insulin infusion. Under these conditions, no change in NE release could be detected, indicating that insulin did not affect muscle NE metabolism. In the 'insulin hypothesis' of hypertension, hyperinsulinemia (in states of insulin resistance) has been suggested to increase SNS activity and cause hypertension (discussed above). However, an equally compelling case has been made for elevated SNS activity as the primary defect, modulating both insulin resistance (and consequently hyperinsulinemia) and elevations in BP. In this section, we will discuss the possibility that enhanced SNS drive may contribute mechanistically to the insulin resistance syndrome in patients with hypertension. Three main mechanisms have been proposed to link SNS overactivity to the development of insulin resistance. First, studies by Diebert and DeFronzo (1980) have demonstrated that stimulation of B-adrenergic receptors can cause acute insulin resistance. In their experiments in humans, infusion of epinephrine caused an acute decrease in insulin-stimulated glucose uptake. Secondly, chronic B-adrenergic stimulation appears to favor an increased proportion of insulin-resistant fast-twitch fibers in experimental rats (Zeman et al, 1988). Since the skeletal muscle is the major site of insulin resistance, a change in fiber composition could have a major impact on total body insulin sensitivity.  The third mechanism by which  excessive SNS activity can lead to insulin resistance is through vasoconstriction (Jamerson et al, 1993). Vasoconstriction may decrease capillary blood flow and thus decrease the  21  delivery of glucose to skeletal muscle. As skeletal muscle accounts for a significant portion of insulin-mediated glucose uptake, it follows that decreases in perfusion may be important in the development/reinforcement of insulin resistance.  4.  Cardiovascular Actions of Insulin: Are They Important in Lone Term BP  Regulation ? Given the above preamble, it is clear that insulin exhibits important effects on the cardiovascular system.  These effects (especially insulin-mediated vasodilation) have  been viewed as being an essential component of overall insulin action in humans. As insulin has the ability to affect a variety of BP regulatory mechanism/s, it is not surprising that it has been suggested that abnormalities in the cardiovascular actions of insulin (in states of insulin resistance and hyperinsulinemia) may be important in the development of hypertension. As hyperinsulinemia and insulin resistance are often present together, it has been difficult to distinguish between the effects of hyperinsulinemia or insulin resistance per se in cardiovascular disease. The question as to the exact interaction between insulin resistance, hyperinsulinemia and SNS has also been the subject of much debate. Although, it has been postulated that hyperinsulinemia may serve as a continual stimulus for SNS activation, while on the other hand resistance to the vasodilator actions of insulin may result in increased vasoconstriction and BP, direct evidence for this mechanism in humans is not available. Clinical data indicate that intensive insulin therapy (that creates chronic hyperinsulinemia) is not associated with a higher incidence of hypertension (Tsutsu et al, 1990; Pontiroli et al, 1992). Furthermore, acute systemic or local infusions of insulin produce either little or no change in BP in humans (discussed above) (Brands et al, 1991b; Hall et al, 1989; Hall et al, 1990ab; Hall et al, 1991; Hilderbrandt et al, 1994; Hall et al, 1995ab).  Systemic  22  hyperinsulinemia in humans has been demonstrated to produce both SNS activation with simultaneous (and paradoxical) reductions in vascular resistance. Thus, the actions of insulin to cause vasodilation and SNS activation are distinct from one another and the lack of BP increase in response to hyperinsulinemia is due to the effects of one being offset by the other. Taken together, evidence in humans suggests that hyperinsulinemia per se is not associated with increased BP. Notwithstanding the above points, it is possible that insulin resistance when superimposed on hyperinsulinemia could predispose to increased vascular tone (via a loss of insulin-mediated vasodilation). This could sensitize the vasculature to the pressor effects of vasoactive agents and tilt the balance in favor of increased systemic vascular resistance. Thus, while insulin resistance per se may not be causal in hypertension, it may make the vascular wall more sensitive to pressor agents and predispose to hypertension. In contrast to studies in humans, studies indicate that sustained hyperinsulinemia increases BP in conscious rats (Brands et al, 1991a; Brands et al, 1992; Brands et al, 1996, Mehan et al, 1994). Although the mechanism/s that link hyperinsulinemia to increases in BP in rats remain unknown, the observations that chronic hyperinsulinemia can elevate BP are of interest. As alluded to earlier, there are apparent differences in the vascular and BP responses of rats vs. humans, to insulin. However, whether these contribute to differences in the BP response remains to be determined. A crucial point to highlight is that observations in the rat may not reflect the vascular actions of insulin in humans. This, however, should not preclude studies that have examined these effects, since they provide critical information regarding potential hypertensinogenic actions of insulin that may be uncovered in humans under some pathophysiological conditions.  23  The discussion of the insulin hypothesis of hypertension would not be complete without a brief discussion regarding the potential of insulin to cause renal sodium retention. Studies have demonstrated that increases in plasma insulin concentrations elicit sodium retention by increasing sodium reabsorption at a site beyond the proximal tubule. Studies employing the euglycemic hyperinsulinemic clamp have demonstrated that insulin has minimal effect on proximal tubular reabsorption; however, it markedly stimulates chloride reabsorption in the loop of Henle (Kirchner, 1988). Studies in humans are consistent with the notion that insulin may increase sodium reabsorption at a site beyond the proximal tubule, probably the diluting segment (Friedberg et al, 1991). There is some evidence that low, basal levels of insulin are necessary for sodium reabsorption by the kidney. For example, Baum (1987) found that addition of physiological insulin concentrations raised fluid reabsoprtion in isolated rabbit proximal tubules bathed in an insulin-deficient solution. In contrast to these observations, recent studies demonstrate that intrarenal infusion of insulin (in the physiological range) caused no change in sodium excretion (Briffeuil et al, 1992). Studies by O'Hare et al, (1988) have found no reduction in urinary sodium excretion in normal men when plasma insulin concentrations were raised to 50 pU/ml. It has been suggested that the reported anti-natriuretic effects of insulin may be secondary to systemic changes such as a decrease in arterial pressure or hypokalemia. Elegant studies in humans (using the euglycemic hyperinsulinemic clamp) have demonstrated that sodium retention observed during the procedure resulted mainly from an insulin-induced fall in extracellular potassium concentration. When plasma potassium was held constant, no changes in sodium excretion were observed (Friedberg et al, 1991).  Other studies have confirmed that  24  intravenous insulin infusion (which causes hypokalemia) causes much greater sodium and water retention than intrarenal insulin infusion (Hall et al, 1991).  III.  SPECIFIC RESEARCH QUESTION, RATIONALE AND APPROACH  The main objective of the present thesis was to examine the mechanisms of hypertension in hyperinsulinemic and hypertensive FH. The F-hypertensive rat is an acquired form of hypertension, where feeding normal Sprague Dawley rats a fructose-enriched diet (60% fructose) results in hyperinsulinemia, insulin resistance and hypertension without changes in body weight (Hwang et al, 1987; Verma et al, 1994a; Bhanot et al, 1994a). By employing the euglycemic hyperinsulinemic clamp technique in conscious rats, we recently demonstrated that these rats are extremely insulin resistant and hyperinsulinemic as compared to their controls (Bhanot et al, 1994a). We also found that chemically diverse drugs that have the common property of improving insulin resistance and attenuating hyperinsulinemia also prevent the development of fructose-induced hypertension (Verma et al, 1994a; Bhanot et al, 1994a). Furthermore, the antihypertensive effects of the drugs could be reversed simply by increasing insulin levels in the drug-treated rats to those that existed before treatment.  These studies, in conjunction with other reports (Morgan and Mark, 1993;  Meehan et al, 1993; Pershadsingh et al, 1993), strongly suggest that insulin resistance and hyperinsulinemia are very closely related to the development of hypertension in Fhypertensive rats. The model allows examining the inter-relationship between insulin and BP independent of obesity. Despite the increasing use of the F-hypertensive rat, the mechanism/s that contribute towards increased BP in this model remain unknown. In an attempt to elucidate the  25  mechanism/s of hypertension and potential inter-relationships with insulin, our experiments were designed with the following specific aims:  (1)  To examine the direct effects of insulin on the reactivity of arteries [(aortae and mesenteric vascular bed (MVB)] from control (C) and F-hypertensive rats.  (2)  To determine whether changes in insulin's vascular effects precede the development of hypertension in this model. To this aim, the vascular effects of insulin were examined in pre-hypertensive F hypertensive rats.  (3)  To examine the role of ET-1 as a mediator of high BP in F hypertensive rats. To this aim, both in-vitro and in-vivo studies were performed in F rats, chronically treated with the ET receptor blocker bosentan.  (4)  To evaluate the role of a vasodilator-antihypertensive agent on the development of hyperinsulinemia and hypertension in F-hypertensive rats. This was accomplished by studying the chronic effects of a calcium channel blocker on fructose-induced hyperinsulinemia and hypertension.  (5)  To examine the contribution of the SNS in the development of fructose-induced hypertension. This was studied by examining the effects of chemical sympathectomy on fructose-induced changes in plasma insulin levels and BP. The effects of sympathectomy on the direct vascular effects of insulin were also evaluated.  IV.  WORKING HYPOTHESES  1.  Insulin resistance and hyperinsulinemia play a role in the development and regulation of high BP in F-hypertensive rats.  2.  The mechanisrn/s underlying the development of insulin resistance, hyperinsulinemia and hypertension in F rats involve a close interaction between the vascular actions of insulin and the SNS.  3.  At the vascular level, alterations in the effects of insulin are important in the development and reinforcement of hypertension. Thus, arteries isolated from Fhypertensive rats will exhibit changes in vascular reactivity in a fashion that is consistent with increased peripheral vascular resistance.  4.  Chronic hyperinsulinemia in F-hypertensive rats may serve to exaggerate pressor responsiveness through increases in ET-1 production. Thus, chronic treatment of Fhypertensive rats with a specific ET receptor blocker will attenuate the development of hypertension.  5.  The antihypertensive effects of vasodilator-antihypertensive associated  with improvement in insulin sensitivity  compounds are  and amelioration of  hyperinsulinemia. This effect is mediated largely via an improvement in perfusion to insulin-sensitive tissues. Thus, chronic treatment with a calcium channel blocker (mibefradil) will attenuate the development of hyperinsulinmeia and hypertension in F-hypertensive rats. 6.  The SNS plays an important role in the development of F induced hypertension. Therefore, sympathectomy will prevent the development of fructose-induced hypertension.  27  MATERIALS AND METHODS  I.  GENERAL METHODOLOGY  1.  BP Measurement  In all the studies described, systolic BP was measured in conscious rats using the indirect tail-cuff method without external preheating (Bunag, 1973). The animals were preconditioned to the experimental procedure before the actual measurements were conducted. In this method, the reappearance of pulsations on gradual deflation of the BP cuff are detected by a photoelectric sensor and are amplified and recorded digitally as the systolic BP. An. average of five such readings was taken to obtain the individual systolic BP. We have validated readings obtained by this method by comparison with those obtained by direct intra-arterial cannulation.  Recorded pressures were similar (within 5 mmHg) to those  obtained by direct cannulation; similar results have been reported by other laboratories (Bunag, 1973; Hwang et al, 1987; Reaven et al, 1989).  2.  Biochemical Analysis  For all the studies, biochemical measurements were performed in an identical fashion, as described below: (i)  Plasma glucose was determined by the glucose oxidase method using kits  from Boehringer Mannheim, Laval, Quebec, Canada. (ii)  Plasma insulin levels were determined  using a double antibody  radioimmunoassay using a kit from Linco Diagnostics Inc., USA.  Samples from all the  28  weeks in each study were analyzed together at the end of the study to avoid inter-assay variations. (iii)  Measurement of ET from plasma and blood vessels was done using the  method of Lariviere et al. (1993) with a few modifications. Briefly, the frozen tissues were homogenized in 2 ml extraction buffer (composition: IN HC1, 1% formic acid, 1% trifluoroacetic acid, TFA and 1% NaCl) using a polytron for 30 seconds (2X15 seconds; with an interval of 30 seconds between each homogenization). The homogenate was centrifuged at 2000g for 30 minutes . The supernatant was passed through a nylon mesh and then centrifuged at 1500g for 15 minutes. The supernatant was passed through Amprep C2 minicolumns (Amersham International, Amersham UK) as described below. Prior to passing the supernatant through the columns, the columns were equilibrated by washing with 4ml methanol (2X2ml) followed by 2ml water. For this and subsequent washes and elutions, a flow rate of ~ 3ml/minute was maintained. The entire supernatant was passed through the Amprep 500 mg C2 columns, washed with 5ml of water (containing 0.1% TFA) and finally washed with 2ml of 80% acetonitrile (containing 0.1% TFA) to elute ET. The eluent was collected in polypropylene tubes. The eluent was dried in a Speed-Vac for ~ 5 hours and then reconstituted in 250ul assay buffer (Amersham International pic, Amersham UK), lmmunoreactive ET was measured by a radioimmunoassay (RIA) using a specific anti-serum against ET obtained from Amersham laboratories. The antiserum had the following crossreactivity: 100% to ET-1, 1305% to ET-2, <0.001% to ET-3, 189% to big ET (human), <0.00625% to atrial natriuretic peptide (Amersham International).  Cross reactivity is  expressed as a percent of ET-1. The RIA kit from Amersham utilizes a high specific activity [125I]THX-1  (synthetic) tracer, together with a highly specific and sensitive antiserum as  29  described above. The assay allows measurement of ET-1 in the range of 0.25-32 fmol per tube(0.623-79.74 pg/tube). For determination of ET from plasma, 1ml of plasma was acidified with 0.25m 2M HC1, centrifuged at 8000g for 10 minutes and passed through the columns as described above.  3.  Reagents  All chemicals and reagents used in this study were of reagent grade and were obtained from Sigma. Bosentan and mibefradil were kindly supplied by Hoffman La Roche, Basil, Switzerland. The fructose diet was obtained from Teklad Laboratory Diets Inc, USA. Regular Insulin {Humulin N) was purchased from a community pharmacy.  II.  EXPERIMENTAL PROTOCOLS  Study A.  Direct Effects of Insulin on Reactivity of Aortae and MVB from Control and Fructose rats  Animals and Experimental Design.  Prior to the commencement of the experiments, all  protocols were approved by the University of British Columbia Use of Animals in Research  Committee. Male Sprague Dawley rats were procured at 5 weeks of age {University of British Columbia Animal Care Facility) and were assigned to two experimental groups (n=10  per study): control (C) and fructose (F). At week 6 (weeks signify the age of the rats) systolic BP, plasma glucose and plasma insulin (5 hour fasted) were measured in all groups. Starting at week 6, the rats in the F group were started on a 66% fructose diet for 11 weeks. The  30  fructose diet (66% fructose, 12% fat and 22% protein) had an electrolyte, protein and fat content comparable to the standard rat chow. Weekly measurements of plasma insulin, glucose (5 hour fasted values) and systolic BP were conducted. At week 17, the MVB and aortae from C and F rats were removed and pressor responses to NE and All were examined in the presence and absence of insulin, as described below.  Aortic Ring Preparation and Protocol. At week 17, rats were sacrificed with an overdose of pentobarbital, and the thoracic aortae were carefully dissected out, cleaned of adherent connective tissue and cut into ring segments. For each rat, four rings from the thoracic aorta (each ring =3mm in length) were used. The endothelium was removed in some ring segments by gently rotating a stainless steel rod through the lumen of the vessel. Successful removal of the endothelium was assessed by the loss of the vasodilator effect to 10~5 M ACh. The tissues were suspended on wire hooks (passed through the lumen of the vessel) in isolated tissue baths containing modified Krebs-Ringer bicarbonate solution containing 0.1% bovine serum albumin with the following composition (in mM): NaCl (118), KC1 (4.7), CaCl2 (2.5), KH P0 2  4  (1.2), MgS0 (1.2), NaHC0 (25), dextrose (11.1) edetate calcium disodium 4  3  (0.026), and indomethacin (20 pM) maintained at 37°C, and oxygenated with 95% 0 and 2  5% CO2. Each aortic ring was placed under a resting tension of 3.0 g. The tissues were allowed to equilibrate for 60-90 minutes before the responses to various agonists were tested. Isometric responses were recorded on a Grass 79D polygraph. After an initial equilibration period, the tissues were contracted twice with a high K  +  solution (KC1 40-60mM;  concentration expressed as final molar concentration in the tissue bath) before the  31  experiments were conducted. After each contraction, the buffer was replaced several times to wash the tissues until the resting tension of each tissue was reached. The above procedure was carried out to stretch the tissues so as to ensure an optimal and reproducible response to a given concentration of agonist. After these initial steps, cumulative dose-response curves were constructed to NE (10"9 to lO^M) and All (1012 to lO^M) (separate studies) in the _  absence and presence of insulin (Humulin N, 100 pU/ml and 100 mU/ml for 2 hours, diluted in buffer containing 0.1% albumin). The concentrations of insulin chosen were based on previously published studies (Wu et al., 1994, Han et al., 1995). The plasma insulin levels of F-hypertensive in the post-prandial state are =65-80 Mj_J/ml. Therefore, throughout the thesis, we have used the term "physiological insulin concentration" to represent 100 p,U/ml while "pharmacological" to represent lOOmU/ml.  Contractile responses to each agonist were  expressed as a percentage of maximum contraction in the absence of insulin. The percent maximum inhibition or potentiation by insulin was calculated by using the following formula: (maximum contractile response without insulin - maximum contractile response with insulin) / (maximum contractile response without insulin) x 100%. In addition, agonist pD2 values (-log ED50) were calculated by nonlinear regression analysis of the individual dose-response curves and were used as an index of sensitivity.  Mesenteric Vascular Bed Preparation and Protocol. MVB perfusion was performed by the  method of McGreggor (1965) and modified by Wu et al. (1994). Rats from both groups were anesthetized (pentobarbital 65 mg/kg, i.p) and the superior mesenteric artery was cannulated at its aortic origin and flushed with approximately 5-10 ml of modified Krebs-Ringer bicarbonate buffer containing 0.1% bovine serum albumin [composition in mM: NaCl (118),  32  KC1 (4.7), CaCl (2.5), K H P 0 (1.2), MgS0 (1.2), NaHC0 (25), dextrose (11.1) and 2  2  4  4  3  calcium disodium edetate (0.026)]. An incision was made to separate the intestine from the stomach and a second incision was made to separate the sigmoid colon.  The entire  mesenteric bed was dissected out away from the intestine and rinsed with 30-40 ml of buffer and suspended in a 100-ml jacketed organ bath. The superior mesenteric artery was perfused at a constant flow rate (using a peristaltic pump) of approximately 7-10 ml/min with buffer maintained at 37°C, and oxygenated with 95% 0 /5% C 0 2  2  The flow rate was chosen based  on the minimum reproducible flow rate that the peristaltic pump could provide. The MVB was covered with a wet gauze-pad to prevent tissue drying. The tissues were allowed to equilibrate for at least 30 minutes and responses were measured after a stable resting pressure was obtained. Changes in perfusion pressure were measured by a Stratham P23 AA pressure transducer at a point proximal to the perfusion cannula. Cumulative concentration-response curves to NE were first determined in the absence of insulin. Following a 30 minute washout and 30 minute equilibration period, the MVB was perfused with buffer containing insulin {Humulin N, 100 pU/ml) for 2 hour, following which cumulative concentration-response curves to NE were repeated. Perfusion pressure data were calculated as a percent of the maximum contraction in the absence of insulin. Agonist pD values (-log 2  ED50)  were  calculated by nonlinear regression analysis of the dose-response curves and were used as an index of sensitivity. Although a time-control was run in each experiment, our data was not controlled for this factor.  As the concentration-response curves were conducted after maximum KC1  contraction, we feel that correction for time-related increases (if any) is not warranted.  33  Study B.  Direct effects of Insulin of MVB Reactivity in Pre-Hypertensive Fructose Rats.  This study was conducted to determine whether defects in insulin's vascular effects precede the development of hypertension in F rats. Thus, the direct vascular effects of insulin were examined in rats that had been fed a fructose-enriched diet for a period of 7 days. We have previously demonstrated that hypertension in F rats occurs after about 2 weeks of fructose feeding (Verma et al, 1994a). Thus, we selected the one week post-fructose time-point to represent the pre-hypertensive state.  Animals and Experimental Design. Male Sprague Dawley rats (6 weeks of age) were divided into C and F groups (n=10 in each group). The rats in the F group were started on a 66% fructose diet at week 6. At week 7, plasma insulin, glucose (5 hour fasted values) and systolic BP were measured and the MVB's were removed to examine the effects of insulin on the reactivity to NE as described above.  Study C.  Studies Examining the Role of ET-1 in Fructose-Induced Hypertension.  These series of studies were based on reports suggesting that hyperinsulinemia may increase ET-1 production in-vitro and in-vivo (Oliver et al, 1991; Frank et al, 1993; Hu et al, 1993; Hattori et al, 1991). Thus, we hypothesized that elevated plasma insulin levels in F rats may serve as a continual stimulus for ET-1 production, which in turn could lead to increases in BP  34  through altering either circulating and/or local ET-1 concentrations. To examine the potential contribution of ET-1 in fructose-induced hypertension, we examined: (i) the in-vitro effects of ET receptor blockade (with bosentan) on MVB responses to insulin, and (ii) the chronic effects of bosentan on plasma insulin levels, systolic BP, MVB ET-1 levels and reactivity of mesenteric arteries from control and F rats.  Bosentan. Bosentan (Ro 46-2005, F. Hoffmann-La Roche Ltd., Switzerland) is a nonpeptide ET antagonist that blocks both the ET and ET^ receptors (Clozel et al, 1993; Clozel et al, a  1994). The drug is orally effective and at doses of 100 mg/kg/day has been demonstrated to exhibit antihypertensive properties and anti-ischemic effects (Clozel et al, 1993; Clozel et al, 1994). Bosentan has been demonstrated to competitively antagonize the specific binding of labeled ET-1 with a Ki of 4.7 nM for the ET receptor and 95 nM for the ETj, receptor. It a  selectively inhibits the binding of ET-1 in isolated rat aortae and blocks contractions elicited by ET-1 and sarafotoxin S6C with pA2 values of 7.2 and 6.0, respectively. The binding of approximately 40 other peptides including prostaglandins, ions and neurotransmitters is not affected by bosentan. In vivo, bosentan has been demonstrated to block the pressor responses to ET-1 (acutely) and exhibit antihypertensive/antihypertrophic effects (chronically) (Clozel et al, 1993, Clozel et al, 1994). Bosentan is one of the most potent orally active antagonists of ET receptors described todate. Its profile makes it extremely useful as a pharmacological tool and potential therapeutic agent (Clozel et al, 1994).  In-vitro Effects of Bosentan on Insulin-induced Exaggeration of MVB Responses. Studies  in section (a) revealed that insulin at concentrations of 100 uU/ml (which may be considered  35  as being physiological in hyperinsulinemic F rats) exaggerated the pressor responses of MVB to NE. To examine the potential contribution of ET-1 towards this effect, we studied the effects of bosentan treatment (in-vitro) on insulin-mediated MVB hyper-reactivity in control and F rats. 16 week old C and F rats (n=10) were set up for MVB perfusion as described above. The tissues were allowed to equilibrate for at least 30 minutes and responses were measured after a stable resting pressure was obtained. Following a 30 minute washout and 30 minute equilibration period, the MVB was perfused according to the following protocol: (i) a cumulative contraction response curve to NE, (ii) a cumulative concentration-response curve to NE after perfusion with insulin (100 uU/ml for.2 hours), and (iii) a cumulative concentration-response curve to NE after perfusion with bosentan (3X10"6 M for 15 minutes) followed by insulin (100 pU/ml for 2 hours). In a separate study, the protocol above was repeated in presence of buffer containing indomethacin (20 pM) with/without bosentan. Perfusion pressure data were calculated as a percent of the maximum contraction.  Effect of Chronic Bosentan Treatment in Fructose Rats. Male Sprague Dawley rats were procured at 5 weeks of age and were assigned to four experimental groups: control (C, n=9), control bosentan-treated (CT, n=10), fructose (F, n=10) and fructose bosentan-treated (FT, n=10). At week 6 (weeks signify the age of the rats), BP, plasma glucose and plasma insulin (5 hour fasted) were measured. Starting at week 6, chronic bosentan treatment was initiated in the CT and FT groups. Bosentan was administered at a dose of 100 mg/kg/day by a single daily oral gavage. The dose was selected based previous studies reporting a BP lowering effect at this dose (Clozel et al., 1993; Clozel et al., 1994). One week after initiating bosentan treatment, the rats in the F and FT groups were started on a 66% fructose diet. Starting at  36  week 8, BP, plasma insulin and plasma glucose were measured weekly for the next 3 weeks. At week 15, the entire MVB from all groups was removed, dissected free of fat and connective tissue and frozen for the measurement of immunoreactive ET-1 content as described above.  Reactivity of Mesenteric Arteries from Fructose Rats to ET-1. In a separate study, male  Sprague Dawley rats were procured at 5 weeks of age were assigned to four groups as described above (n=15 per group): C, control-bosentan treated (CT), fructose (F) and fructose-bosentan treated (FT). At week 16, superior mesenteric arteries and mesenteric resistance arteries were removed for study. Superior mesenteric arteries were carefully dissected, cleaned of adherent connective tissue and cut into ring segments. Two rings, approximately 2.5 to 3mm in length, were dissected from each rat. One ring of each pair was left intact, while the other was gently rotated on a stainless steel rod to remove the endothelium. The lack of a dilator response to 10~5 M ACh was used as an index of an endothelium-denuded vessel. The tissues were suspended on wire hooks in an isolated tissue bath containing modified Krebs-Ringer bicarbonate buffer maintained at 37°C, and gassed with 95% 02/5% CO2. Each ring was placed under a resting tension of l.Og which was determined as the ideal tension in preliminary experiments. The tissues were then allowed to equilibrate for 90-120 minutes before the experiments were conducted. The vessels were stimulated according to the following protocol: (a) a cumulative concentration-response curve to NE and (b) a cumulative concentration-response curve to ET-1 (10 minutes for each concentration). The ET-1 curve was done last as the sustained nature of the contraction is resistant to washout. The arteries were allowed to equilibrate for approximately 90 minutes  37  between the steps (a) and (b). Hence, the effects of NE and ET-1 were tested in the same tissue. In a separate study, we examined the effects of chronic endothelin receptor blockade (with bosentan) on endothelium-dependent and independent responses of isolated mesenteric arteries to acetylcholine (ACh) and the endothelium-independent vasodilator sodium nitroprusside (SNP) in arteries pre-constricted with the ED90 of NE (for the respective groups). At the completion of each experiment, the length of the tissues was measured, the tissues placed on a filter paper, lightly blotted and weighed. The cross-sectional area of each tissue was calculated using the formula: cross sectional area (CSA, mm^) = weight (mg)/length(mm)Xdensity (mg/mm^). The density of the arteries was assumed to be 1.05 mg/mm^. Tension responses of each preparation were then corrected for both cross-sectional area and length of the tissue and expressed as (g/mm^). Agonist pD2 values (-log ED50) were calculated by nonlinear regression analysis of the dose-response curves and were used as an index of sensitivity. Pressurized Artery Experiments. To study the reactivity of resistance vessels from C and F rats, we examined the reactivity of pressurized mesenteric resistance vessels to NE from C and F rats. A tertiary branch of the superior mesenteric artery (length 0.6-1.0 mm, diameter 180-250 pm) was carefully dissected from surrounding connective tissues and transferred to the experimental chamber of a Mulvany-Halpern (Halpern et al., 1984) pressure myograph. Each vessel was tied onto proximal and distal glass microcannulae (tip diameter 80-90 pm) using single strands (20 pm) of braided 3-0 nylon surgical suture. The lumen of the vessel was gently flushed with oxygenated Krebs buffer, and the intraluminal pressure was set to  38  values between 20 and 180 mm Hg using an electronic pressure servo system. The buffer in the vessel chamber was continuously recirculated at a flow rate of 25 ml/min through an external reservoir that was bubbled with a gas mixture of 95% 0 +5% C 0 . The pH was 2  2  measured by a micro-pH probe positioned in the tissue chamber, and was maintained at 7.4±0.04 by adjustment of the gassing rate. A heating pump connected to a heat exchanger maintained bath temperature at 37°C. The arteriograph containing the pressurized artery was then placed on the stage of an inverted microscope, and equilibrated for sixty minutes at a transmural pressure of 75 mmHg. Arterial dimensions were measured using a monochrome video camera mounted to the microscope viewing tube and a video dimension analyzer (Living Systems, Burlington, VT) which provides continuous digital readout of internal luminal diameter and right and left wall thickness. The information is updated every 17 ms, and the precision of the diameter measurement is within 1%. Wall thickness (expressed as percent of internal diameter) was measured over a range of transmural pressures (10-180 mm Hg) in mesenteric arterioles from F and C rats.  Subsequently, constriction responses  (expressed as percent of initial internal diameter) to increasing concentrations of NE (10~8 to 10" M) were measured at a transmural pressure of 120 mmHg, then after 30 minute 5  equilibration, at a transmural pressure of 160 mmHg.  Study D.  Vasodilators and the Insulin Hypothesis of Hypertension.  Background. As alluded to earlier, accumulating data suggests that blood flow (or conversely the resistance to blood flow) are important determinants of insulin-sensitivity and glucose uptake. The rate of glucose uptake is determined by two components, i.e. glucose  39  extraction and blood flow. Glucose uptake (GU) is commonly measured by using the Fick principle, based on knowledge of the arterial-venous blood glucose difference (AV) and blood flow (F), glucose uptake=AV X F. From this formula, it becomes immediately evident that an increment in glucose uptake may result from an increase in glucose extraction, blood flow or both. The data from our studies (above) and others (Brands et al, 1991a; Brands et al, 1996) have demonstrated that hypertension in hyperinsulinemic and insulin resistant rats is associated with increases in total peripheral resistance. Thus, it was of interest to examine the effects of a vasodilator antihypertensive agent (which has no intrinsic effect on insulinsensitivity) on the development of hyperinsulinemia, insulin resistance and hypertension in F rats. To this aim, we examined the chronic effects of a calcium antagonist (mibefradil) on plasma insulin levels, plasma triglyceride levels and systolic BP in insulin resistant and hyperinsulinemic F rats. Mibefradil (Ro 40-5967) is a new calcium channel blocker that has been demonstrated to have potent antihypertensive and anti-ischemic effects and is currently in phase HI clinical trials. Mibefradil appears to be the only calcium antagonist that completely blocks the T-type calcium channels. Although the available calcium blockers exert their effects on the L-type (long-lasting, high-voltage-activated) calcium channels, the T-type (transient, low-voltage activated) calcium channel is found in relatively high density in spontaneously active vascular muscle and may play a role in vascular hypertrophy and remodeling (Osterrieder and Hoick, 1989; Billman, 1992).  Prevention Protocol.  Male Sprague Dawley rats were procured locally (180-200g body  weight) at 6 weeks of age. The animals were divided into four experimental groups: control-  40  untreated (C, n=6), control mibefradil-treated (CT, n=5), fructose-untreated (F, n=7) and fructose mibefradil-treated (FT, n=6). After recording basal values of plasma insulin, glucose, triglycerides and systolic BP, chronic mibefradil treatment was initiated in the CT and FT groups. Treatment was initiated at a concentration of 30 mg/kg/day (p.o. via daily oral gavage) as previously described. One week after initiating mibefradil treatment, the animals in the F and FT groups were started on a 66% fructose diet. The fructose diet (66% fructose, 12% fat and 22% protein) had an electrolyte, protein and fat content very comparable to the standard rat chow. Beginning at week 9, BP, plasma insulin, glucose and triglycerides were measured each week for the next four weeks. Insulin sensitivity (IS) was estimated by comparing the ratios of plasma insulin to glucose (5 hour fasted) in the experimental groups at week 12.  Reversal Protocol: In this study the effects of mibefradil were examined after the development of hyperinsulinemia and hypertension in F rats. Male Sprague Dawley rats that had been treated with fructose-diet for 8 weeks (and their age and weight matched control rats) were divided into: C (n=6), CT (n=6), F (n=6) and FT (n=6). Mibefradil treatment was started in the CT and FT groups (30 mg/kg/day p.o. via daily oral gavage) for 2 weeks. Plasma glucose, insulin, triglycerides and BP were determined before and after treatment.  Study E.  The Role of the SNS in Fructose-Induced Hypertension.  Effects of Chemical Sympathectomy on Fructose Induced Hyperinsulinemia and  Hypertension. Male Sprague Dawley rats were procured at 5 weeks of age and were divided into the following experimental groups: control (C, n=16), control-sympathectomized (CS,  41  n=7), fructose (F, n=16) and fructose-sympathectomized (FS, n=8).  After baseline  measurements (glucose, insulin and BP), adrenal medullectomy (under pentobarbital anesthesia) was performed in the CS and FS groups, following which the rats in CS and FS groups received weekly intraperitoneal injections of 6-hydroxy dopamine (50 mg/kg dissolved in 0.5 ml of 0.9% sodium chloride saline containing 0.5 mg ascorbic acid) (Finch and Leach, 1970). Starting at week 9, the rats in the F and FS groups were started on the fructose diet.  Weekly measurements of plasma insulin, glucose and systolic BP were  conducted in the four groups. At week 17, tail artery responses to 10'^M tyramine were examined in the four experimental groups. The lack of tyramine responses was used to indicate successful sympathectomy.  Effects of Sympathectomy on the Vascular Actions of Insulin in Control and F Rats. To  examine the interaction between the SNS and the vascular actions of insulin, we examined the direct effects of insulin on MVB reactivity in control and sympathectomized rats. To this aim, MVB from C (n=8), CS (n=7), F (n=10) and FS (n=8) rats were removed and pressor responses to NE were recorded in the presence and absence of insulin as described above.  III.  STATISTICAL ANALYSES  All data presented are as mean±SE.  Statistical evaluation was done by repeated  measures ANOVA (two way) with grouping factors for the evaluation of the interaction between agonist and insulin. Mean values were considered significantly different at a value of P<0.05. For the studies examining the chronic effects of an intervention (bosentan or  42  mibefradil) on plasma insulin levels, plasma glucose levels and systolic BP, differences among groups were examined by MANOVA. When a significant difference in the mean vector was noted, the individual variables were analyzed by employing the Newman-Keuls test for multiple comparisons.  43  RESULTS  Study A.  Direct Effects of Insulin on Reactivity of Aortae and MVB from Control and Fructose Rats  The F group exhibited hyperinsulinemia and elevated BP when compared to the C group (plasma insulin: 4.7±0.5 vs. C 2.6±0.3 ng/ml, P<0.001; systolic BP: 153±2 vs. C 127±3 mmHg, P<0.001). The presence of hyperinsulinemia in the presence of similar glucose levels (5.8±0.5 vs. C 5.3±0.4 mM) is indicative of insulin resistance; we have previously demonstrated that these rats are markedly insulin-resistant when assessed using the euglycemic hyperinsulinemic clamp (Bhanot et al, 1994a). Figure 2 depicts the effects of 2 hour insulin incubation (100 mU/ml) on the reactivity of aortic rings (with intact endothelium) from C (left panel) and F (right panel) to NE. Please note that the absolute tension values corresponding to 100% were not different between C and F aortae (2.3±0.1 vs. F: 2.4±0.2 g/ram^ P>0.05). Insulin caused vasodepressor effects in C rat aortae; in the presence of insulin, both the percent maximum contraction and the sensitivity to NE were attenuated (percent maximum attenuation by insulin: -25±3; pD  2  values: 7.49±0.04 vs. C+I 6.65±0.15, P<0.05). Strikingly, insulin-induced attenuation of NE responses was absent in aortae from F rats. Neither a rightward, nor downward shift of the NE curve was observed in F rats in the presence of insulin (Figure 2, right panel). Similarly, in the presence of insulin, AH- induced responses were attenuated in C aortae (Figure 3, left panel). This effect was absent in aortae from F rats (percent maximum attenuation by insulin (I): F+I: -9±4 vs. C+I: -33±3, P<0.05).  It is important to note that although insulin  44  FIGURE 2  Effect of insulin (•, 100 mU/ml for 2 hours) on contraction induced by cumulative addition of NE in isolated aortic rings from control (C, n=10, left panel) and (F, n=10, right panel) rats with intact endothelium. Data are shown as mean±SE. *P<0.05, different from C without insulin.  o  CM  T—  o  O  T—  o  00  o  CD  o  TJ-  o  CM  uoipejjuoo LunoiixeiAl %  o  46  FIGURE 3  Cumulative concentration-response curves of isolated aortae from control (n=8, left panel) and fructose (n=8, right panel) rats to angiotensin TJ (A H) in the absence (O) and presence (•) of insulin (100 mU/ml for 2 hours). Data are shown as mean±se. *P<0.05, different from control without insulin.  47  UOipCUJUOQ  LUnujIXDiAj  %  48  attenuated reactivity in C rats at insulin concentrations of 100 mU/ml, this effect was absent at more physiological insulin concentrations (100 pU/ml, Figure 4). Additionally, removal of the endothelium was associated with a complete loss of insulin's vasodepressor effect in C rat aortae (Figure 5). Figure 6 depicts the effects of insulin incubation (100 pU/ml, for 2 hours) on MVB reactivity to NE in C (left panel) and F rats (right panel). There was no difference between the reactivity of MVB between the C and F group to NE (absolute change in pressure corresponding to 100%: C 63±5 vs. F 72±4 mm Hg P>0.05). By marked contrast to its action in the aortae, physiological insulin concentrations (100 pU/ml vs. 100 mU/ml in the aortae), potentiated the pressor responses to NE in MVB from C rats. Strikingly, insulin-induced enhancement of NE pressor responses was further exaggerated in MVB from F rats (% maximum potentiation of NE responses by insulin at 10"4 M [NE]: 48±4% vs. C 28±3%, p<0.05). Analysis of the pD2 values calculated on the absolute responses indicated no change in NE sensitivity was produced by insulin in either the C or F rats (pD2 values: C: 6.04+0.25, C+I: 6.98±0.4, F: 6.2±0.3 and F+I: 6.73±0.2, P>0.05).  Study B.  Insulin Modulation of Vascular Reactivity in Pre-Hypertensive Fructose Rats.  To examine whether insulin modulation of vascular reactivity precedes the development of hypertension in F rats, we examined the direct effects of insulin incubation on MVB reactivity in rats fed a fructose-enriched diet for a one-week period. As shown in  49  FIGURE 4  Effect of insulin (100 jiU/ml for 2 hours) on the contraction induced by the cumulative addition of NE in isolated aortic rings from control rats with intact endothelium. Data are shown as mean ±SE.  51  FIGURE 5  Effect of insulin (100 mU/ml for 2 hours) on the contraction induced by the cumulative addition of angiotensin II (A IT) in isolated aortic rings from control rats (n=8) with endothelium denuded. Data are shown as mean ±SE. P=NS between groups.  120  100  h  o •  —Insulin +Insulin  O  • i—i  o cd  50  ^—i  o 60  X  40  CO  20  0  13  — I  11  10  - 9  - 7  - 6  A n g i o t e n s i n II ( —logM)  •4  53  FIGURE 6  Effect of insulin (100 uU/ml for 2 hours) on the percent maximum contraction induced by the cumulative addition of NE in mesenteric vascular beds (MVB) from control (C, n=10, left panel) and fructose (F, n=9, right panel).  Please note that the actual pressor response  corresponding to 100% was not different between C and F groups (see text for details). Data are shown as mean ±SE. * P<0.05, different from respective C and F groups with insulin.  uoipejjuoQ lunoiixew %  55  Table 1, after one week of fructose feeding the rats exhibited elevated plasma insulin levels without an increase in systolic BP. Analysis of the reactivity of MVB revealed the pre-hypertensive F group exhibited a greater degree of insulin-induced potentiation when compared to the C group (Figure 7, %  maximum potentiation of MVB responses at 10~ M NE: F+I: 43±4 vs. C+I: 24±4, p<0.05). 4  Study C.  Studies Examining the Role of ET-1 in Fructose-Induced Hypertension.  Figure 8 depicts the acute effects of bosentan (3X10"6M for 15 minutes) and indomethacin (20 pM) incubation on insulin-induced exaggeration of MVB responses in C (left panel) and F (right panel). Results are presented as percent maximum potentiation. The absolute pressure responses corresponding to 100% were not different between C and F groups (as noted earlier). In MVB from C rats, indomethacin completely abolished the ability of insulin to potentiate NE pressor responses. Endothelin receptor blockade (with bosentan) did not affect the insulin response. The combination of indomethacin and bosentan did not produce an additional attenuation of that observed with indomethacin alone. As observed previously, MVB from F rats exhibited a greater potentiation of NE pressor responses in the presence of insulin (% maximum potentiation: 62±7 vs. C+I: 32±4, p<0.05.  In contrast to the effects observed above (in C rats), in MVB from F rats,  indomethacin only partially attenuated insulin's effect (% maximum potentiation 39±5 vs. C+I, P<0.05).  A similar observation was made with bosentan alone.  Strikingly, the  combination of indomethacin and bosentan completely obliterated insulin-induced potentiation of NE pressor responses (% maximum potentiation: 8±5 vs. C, P>0.05).  56  TABLE 1 General Characteristics of the Rats in the Pre-Hypertensive Study  Control (n=10)  Fructose (n=10)  126±3  124±5  Plasma insulin (ng/ml)  2.1±0.3  3.1+0.1*  Plasma glucose (mM)  4.7±0.6  5.3±0.5  Body weight (g)  205±4  210±9  Systolic BP(rnmHg)  P<0.05, different from Control  57  FIGURE 7  Insulin-induced potentiation of NE responses in pre-hypertensive FH rats. MVB were isolated from control (C) and pre-hypertensive fructose rats (F) as described in the Materials and Methods section. Note that "pre-hypertensive" is defined as one-week post-fructose feeding when the rats are hyperinsulinemic yet normotensive. The bar graph depicted here represents the percent maximum potentiation by insulin (100 pU/ml for 2 hours) in C and F rats. 100% corresponds to the % maximum contraction in the absence of insulin. *P<0.05, different from F+Insulin.  160  150  C + Insulin F+Insulin 140  130  59  FIGURE 8  Effect of bosentan (3X10~6M for 15 minutes) and indomethacin (20uM) on insulin (100 uU/ml for 2 hours) -induced MVB responsiveness in control (C, left panel) and fructose (F, right panel) rats. Data are mean±SE and presented as maximum potentiation (% of control).. Left panel: *P<0.05, different  from control, control+indomethacin+insulin and  control+indomethacin+bosentan+insulin. Right panel: P<0.05, different from fructose+insulin, fructose+indomethacin+insulin, a  fructose+bosentan+insulin and fructose+indomethacin+bosentan+insulin. P<0.05, different D  from control, fructose+insulin and fructose+indomethacin+bosentan+insulin.  (|o..n'.ioo jo  uoiv:M)i.ia io i t  UIIUUIX>-.!|-\'  .61  To examine the potential in-vivo contribution of ET-1 in F rats, we examined the effects of chronic bosentan treatment on MVB ET-1 levels, plasma insulin and glucose levels and systolic BP in F rats. The F group exhibited hyperinsulinemia and elevated BP when compared to the C group (average of weeks 8-10: plasma insulin: 4.9±0.6 vs. C 3.2±0.6 ng/ml, P<0.001; systolic BP: 149±2 vs. C 125+3 mmHg, P<0.001; Figure 9). Treatment of the F rats with bosentan (FT) prevented the rise in BP in this group (average of weeks 8-10: 130±4 mmHg, P<0.001 vs. F 149±2; Figure 9). Bosentan treatment did not affect the BP of the C group (average of weeks 8-10: 128±4 mmHg, P>0.05 vs. C 125±3; Figure 9). The treatment did not alter the plasma insulin levels in FT (average of weeks 8-10: 5.2±0.5 ng/ml P>0.05 vs. F4.9±0.6; Figure 9) and CT groups (average of weeks 8-10: 3.5±0.7 ng/ml P>0.05, vs. untreated C 3.2±0.6; Figure 9). Chronic bosentan treatment did not affect the plasma glucose levels, plasma ET levels, weight gain, food or fluid intake in any group (Table 2). The mesenteric vascular tissue from the F group weighed significantly more than the C group (103.2±6 vs. C 69.4±9 mg, P<0.05).  Although treatment of the F rats with  bosentan, showed a trend towards a lower mesenteric weight, this was not statistically significant (88.2±10 mg vs. untreated F, P>0.05). ET content (expressed as total ET content per vascular tissue assayed) was higher in the F rats when compared to the C group (21.5+3 vs. C 14.1±2 fmol, P<0.05.). Bosentan treatment did not affect the tissue ET levels in either the FT (18.8+5 fmol, P>0.05 vs. untreated F) or CT (10.3±3 fmol, P>0.05 vs. untreated C.)  We have expressed the ET content as total ET-1 content per mesenteric tissue assayed and have not corrected for the wet weight of the tissue. If the results are expressed as ET-1  62  F I G U R E  9  Effect of chronic endothelin receptor blockade with bosentan (100 mg/kg/day) on systolic BP (mmHg) and plasma insulin levels (ng/ml). (C O, n=9), (CT • , n=10), (F V, n=10) and (FT T , n=10).C and F rats. Left panel: * P<0.05, different from C, CT and F. Right panel: * P<0.05, different from C and CT.  63  64  TABLE 2 General Characteristics of Rats in the Four Experimental Groups Before and After Bosentan Treatment.  CT  F  FT  Weight (g) week 6 avg. weeks 8-10  213±5.0 397±8.0  203±6.0 407±7.0  201±6.0 400±7.0  223±5.0 415±9.0  Plasma glucose (mM) week 6 avg. weeks 8-10  5.7±0.4 6.2±0.3  6.3±0.5 5.8±0.5  5.8±0.3 5.5+0.8  6.0±0.5 6.3±0.6  Plasma ET-1 (fmol/ml) week 15  3.4±0.5  3.6±0.9  3.8±0.4  4.3±0.7  Food Intake (g/day) week 6 avg. weeks 8-10  14+1.0 30±1.0  15±1.0 33±1.0  14±1.0 28±1.0  15±1.0 30±2.0  Fluid Intake (ml/day) week 6 avg. weeks 8-10  24±1.0 42±2.0  26±2.0 46±3.0  21+1.0 44±3.0  21±1.0 40±3.0  Values are means±SE. control, C (n=9); control bosentan-treated, CT (n=10); fructose, F (n=10); fructose bosentan-treated, FT (n=10). P=NS between any groups. n=5 in each group for ET-1 determination.  65  content per wet weight of mesenteric tissue, the content is not different between the C and F group, because the F group has a higher mesenteric weight compared with the C group. Similar results have been obtained in the DOCA-salt hypertensive model. As discussed by Lariviere et al., the increased weight of the mesenteric arteries from hypertensive animals is chiefly attributable to hypertrophy of the intima and media rather than hypertrophy of the endothelial cells. Because the primary source of ET-1 is the endothelium, correcting for the weight of the entire mesenteric tissue would represent a diluted value rather than the actual total ET-1 content.  With this factor taken into consideration, we have expressed the  mesenteric. ET-1 levels as total ET-1 content per entire mesenteric tissue assayed. However, caution is warranted in interpreting the data on mesenteric ET-1 content, because a microscopic evaluation of the mesenteric arteries was not done to confirm the degree of hypertrophy of the intima and/or media vs. The endothelium. Because the weight of the entire mesenteric bed could be influenced by numerous factors, such as number of vessels and amount of adhering nonvascular tissue, in addition to hypertrophy, we cannot reach an unequivocal answer at this point.  However, the observation that chronic ET receptor  blockade prevents the development of high BP clearly suggests a role of this peptide in FH.  Responses of Superior Mesenteric Arteries to ET-1: Comparison with NE. The maximum  contractile response of mesenteric arteries to NE did not differ between the four groups either with or without endothelium [with endothelium (+):. C 0.89±0.05, CT 0.89±0.04, F 0.82±0.05 and FT 0.84±0.03 g/mm , P>0.05; without endothelium (-): C 0.95±0.03, CT 1.01±0.04, F 3  0.99±0.05 and FT 1.03±0.03 g/mm , P>0.05, Table 3]. 3  Removal of the endothelium  increased the developed tension in all groups (Table 3). Chronic bosentan treatment did not  66  cn co  O d o +1 d+i oo m d o  PQ  X> >n O d d +l +l CN  CO IT)  00 IT) 00 ON  o o +1 +i o NO o CO  o  NO  d O +1 +i CN  oo 00 ON  _>  o  X)  .3H  >/-> m  o o  CD  d d +l +l P  CN ON oo ON »  co  CS oo  m  W  crj  3  OO  CQ  < u  a I 1  o o  +1 +1  m ON  m NO  CO  NO  d  x>  ' CN  i O O d +1 +1 NO  o  co oo od oo  (U 1/3  C  3  "S  XS  c  O  o -o a o  a  s  •<*  o o d d +l +l ON  NO NO CN 00  O O +1 +1 O 00  1  OH  -4—I  d  03  I—I  oo O d ^  3  O o +1 +1 CN NO  S4,  1)  m u  <u p  r-H i—I  IT) i—I  d d +i +i  o o  ON r-H r-H CN  ON IT)  O O O O +1 +1  o  1  *H  U  D  13 c o o c  X<  rS  U  00 CN ON r - < od ON  C P  U  o c y ra CH  T3  "S ^ uo  p  T3  •.—I  c  o o v CH  X>  3  IT) CO  o o d d +i +i ON >n 00 ON  d  +  d  CN 00  o  ? l +1 oo in 00 O *-H  — < o  d d +1 +1 NO NO CN  CN  + £  r- CN o o CO Q 00 O 00 OS  + 2  pq m  67  change the sensitivity (as assessed by pD values) of the arteries to NE in any group (Table 2  3). The maximum contractile response of mesenteric arteries to ET-1 was depressed in the F group both (+) and (-) the endothelium [(+): 1.50+0.11 vs. C 1.88+0.1 g/mm , P<0.05, 3  (-): 1.68+0.11 vs. C 2.05±0.1 g/mm , P<0.05, Table 3]. Chronic bosentan treatment in the F 3  group (FT) restored the maximum responses of arteries to ET-1 [(+): 1.88±0.12 g/mm vs. C, 3  P>0.05, (-): 1.95±0.05 g/mm vs. C, P>0.05, Table 3] but had no effect on the responses of 3  the CT group [(+): 1.92±0.1 g/mm vs. C, P>0.05, (-): 2.16±0.1 g/mm vs. C, Table 3]. 3  3  Analysis of the pD values indicated that only F arteries (with intact endothelium) had a 2  reduced sensitivity to ET-1 which was restored by bosentan treatment (Table 3). In arteries pre-contracted with NE, ACh produced a concentration-dependent relaxation in arteries with intact endothelium, while it failed to cause dilation in arteries wherein the endothelium was removed (not shown).  The percent maximum relaxation  produced by ACh in F arteries was significantly lower than that in the C arteries (68±5 vs. C 92±5, P<0.05). The dilator responses to ACh were improved by bosentan treatment (FT: 88±5 vs. C, P>0.05). The sensitivity of arteries to ACh was not affected in any group (pD  2  values: C: 7.12±0.43, CT 7.24±0.50, F: 6.53±0.16 and FT 6.86±0.17, P>0.05). In arteries pre-contracted with NE, the endothelium-independent vasodilator SNP produced a concentration-dependent relaxation in all groups both with and without the endothelium [maximum decrease in tension as a percent of the contraction to NE was 100% in all groups]. The sensitivity of arteries to SNP was similar in all groups (removal of the endothelium caused a similar increase in sensitivity in all groups). [pD values (with endothelium) C: 2  68  7.44±0.08, CT 7.26±0.2, F: 7.50±0.1 and FT 7.20±0.5. (without endothelium) C: 8.12±0.06, CT 8.26±0.4, F: 8.17±0.17 and FT 8.53±0.3).  Reactivity of Small Mesenteric Resistance Arteries to NE. The diameters of unstretched  tertiary branches of the superior mesenteric arteries were not different between C (204.8±9.8 pm) and F rats (234.3±9.3 pm) p>0.05. Wall thickness, expressed as percent of internal diameter, did not differ between arteries from C and F rats when measured over a range of transmural pressure (Figure 10). As noted in the superior mesenteric arteries, constrictory responses to NE were similar for mesenteric resistance arteries from C and F rats when studied at transmural pressures of either 120 mmHg (mean systolic pressure of C rats is 130) or 160 mmHg (mean systolic pressure of F rats is 157 mm Hg) [Figure 11].  Study D.  Vasodilators and the Insulin-Hypothesis of Hypertension  Effects of Mibefradil Treatment Before the Development of Hwerinsulinemia and  Hypertension in FH Rats (Prevention Study): Chronic mibefradil treatment of the F group caused marked and sustained decreases in both plasma insulin levels and systolic BP when compared to the untreated F group (Figure 12). Although the drug decreased insulin levels in the CT group it did not affect BP. Additionally, chronic mibefradil treatment attenuated the development of hypertriglyceridemia in the FT group (Table 4).  69  F I G U R E 10  Wall thickness of mesenteric resistance arteries (expressed as percent of internal diameter) measured over a range of transmural pressures (10-180 rnmHg). C (•, n=5), F(B, n=5)  TRANSMURAL  PRESSURE  71  F I G U R E 11  Left Panel: -log [NE] versus percent constriction at a transmural pressure of 120 mmHg in C (•, n=5) and F (•, n=5) mesenteric resistance arteries. Right Panel: -log [NE] versus percent constriction at a transmural pressure of 160 mmHg in C (•, n=5) and F (•, n=5) mesenteric resistance arteries.  72  73  F I G U R E 12  Effects of chronic mibefradil treatment (30mg/kg/day) on plasma insulin levels (ng/ml) and systolic BP (mmHg) in the experimental groups (mibefradil prevention study). Control (C O, n=6), control-mibefradil treated (CT • , n=5), fructose (F A, n=7) and fructosemibefradil-treated (FT A, n=6). Values are mean±SE.  74  TIME (WEEKS)  75  T A B L E  4  General Characteristics of the Rats in the Experimental Groups in the Mibefradil Prevention Study  CT  F  FT  Plasma glucose (mM) week 6 avg. weeks 9-12  8.1±0.5 7.4±0.1  7.8±0.2 7.6±0.1  7.5±0.1 7.4±0.1  7.5±0.1 7.1±0.2  Plasma insulin (ng/ml) week 6 avg. weeks 9-12  2.8±0.1 2.3±0.07  2.5±0.08 1.4±0.3  2.4±0.1 3.2±0.1  2.3±0.2 1.6±0.8  1.4±0.3 1.6±0.3  1.3+0.1 1.0±0.2  1.2±0.1 5.4±0.8  1.4±0.2 2.6±0.5  Plasma triglycerides (mM) week 6 avg. weeks 9-12  a b c  P<0.05, different from C, CT and FT P<0.05, different from C and CT P<0.05, different from C and F  C  a  a  b  76  Effects of Mibefradil Treatment After the Development of Hwerinsulinemia and Hypertension in FH Rats (Reversal Study): The effects of mibefradil treatment on various parameters determined in this study are described in Table 5. Treatment of F rats (after 8 week of fructose feeding) with mibefradil completely reversed the elevated BP, insulin and triglyceride levels in the FT group. Treatment had no affect on the glucose levels in either studies.  Study E .  Effects of Sympathectomy on Fructose-Induced Hyperinsulinemia and Hypertension.  Adrenal medullectomy followed by weekly 6-hydroxy dopamine injections resulted in successful sympathectomy as shown by a lack of tail arterial responses to lO'^M tyramine (Figure 13).  Figure 14 depicts the effects of sympathectomy on fructose-induced  hyperinsulinemia and hypertension. Sympathectomy prevented the development of fructoseinduced hyperinsulinemia and hypertension without an effect in the control group. To assess the modulatory role (if any) of the SNS on the direct vascular effects of insulin, we examined the effects of insulin (100 pU/ml for 2 hours) on reactivity of MVB to NE in sympathectomized control (CS) and fructose rats (FS). Sympathectomy per se enhanced the responses (Figures 15a and 15b) and sensitivity to NE possibly due to supersensitivity (pD2 values C: 7.34±0.4, CS: 8.85±0.5*, F: 7.26±0.5, FS: 8.55±0.6*, *P<0.05, different from respective C and F groups). Please note that the change in pressure corresponding to 100% was not different between CS and FS groups (CS 118±5 vs. FS 129±6 mmHg, P>0.05). Figure 15c depicts the effects of insulin on percent potentiation of  77  T A B L E  5  General Characteristics of the Rats in the Mibefradil Reversal Study  C  CT  F  FT  Plasma glucose (mM) before after  7.3±0.4 7.4±0.1  7.1±0.4 7.6±0.1  7.9±0.4 7.4±0.1  Plasma insulin (ng/ml) before after  2.4±0.3 2.2±0.1  2.1±0.2 1.9±0.2  3.8±0.3 3.6±0.3  Plasma triglycerides (mM) before after  1.8±0.3 1.6±0.3  1.6+0.1 1.0+0.2  4.2±0.3 5.4±0.8  Systolic BP before after  132±2 130±2  128±2 128±2  7.5±0.1 7.1±0.2 3.6±0.2 2.6±0.3  b  b  a  3.4±0.2 2.6±0.5  b a  153±4 149±4  a a  b b  148±3 121+3 b  Fructose hypertensive rats (8 weeks fructose-fed) and their age and weight-matched control rats were divided into the 4 experimental groups as shown above. Plasma glucose, insulin, triglycerides (5 hour fasted values) and systolic BP were determined after (2 weeks) mibefradil treatment. Values are mean±SE. «P<0.05, different from C, CT and FT ^P<0.05, different from C and CT  78  F I G U R E 13  Effect of tyramine (lO'^M) on tail artery responses. Control (C, n=5), fructose (F, n=5), control-sympathectomized (CS, n=5) and fructose-sympathectomized (FS, n=6). * P<0.05 different from C and F.  79  80  f i g u r e 14  Effect of chemical sympathectomy on plasma insulin levels (left panel) and systolic BP (right panel) in the experimental groups at termination.  Control (C, n=16), control-  sympathectomized (CS, n=7), fructose (F, n=16) and fructose-sympathectomized (FS, n=8). Sympathectomy was performed by adrenal medullectomy followed by weekly i.p. injections of  6-hydroxydopamine.  *P<0.05,  different  from  C,  CS  and  FS.  (|uu/6u)  ui|nsui  OLUSOIJ  82  FIGURE 15 15a). Effect of sympathectomy on pressor responses of MVB to NE in control (C, n=8) and control-sympathectomized (CS, n=7) rats.  Data are expressed as percent maximum  contraction in C rats. *P<0.05, different from C.  15b). Effect of sympathectomy on pressor responses of MVB to NE in fructose (F, n=10) and fructose-sympathectomized (FS, n=8) rats. Data are expressed as percent maximum contraction in F rats. *P<0.05, different from F.  (Note: Absolute pressure values corresponding to 100% were not different in any group. See text for details).  15c). Insulin-induced potentiation of NE pressor responses of MVB in CS and FS groups. Data are expressed as percent maximum potentiation at 10"^M [NE]. 100% corresponds to the percent maximum response in the absence of insulin and at 10'^M NE concentrations. * P<0.05, different from CS and FS groups.  83  o m  LO  c\i  o o  ssuodssy  m r^-  o  ui  un CM  ixinuuixD^ %  o  84  MVB responses in sympathectomized control (CS) and fructose rats (FS) at 10'^M NE. Although insulin potentiated the responses in both the CS and FS groups, the exaggeration of MVB responses in F rats (noted earlier) was abolished in FS rats (Figure 15c).  85  DISCUSSION  The main objective of the work presented in this thesis was to examine the link between hyperinsulinemia, insulin resistance and hypertension from a mechanistic standpoint. To examine this relationship, we employed the fructose-induced hypertensive rat model. In this model, feeding normal Sprague Dawley rats a fructose-enriched diet results in marked insulin resistance, hyperinsulinemia and hypertension. The model is widely used to explore the inter-relationship between metabolic defects and hypertension independent of obesity or genetic propensity. The main questions that this thesis addressed were: (a) are the direct vascular effects of insulin altered in F rats in favor of increased peripheral vascular resistance? (b) do alterations in the vascular effects of insulin precede the development of hypertension in F rats? (c) what is the role of vasodilators in the development of fructose-induced hyperinsulinemia and hypertension? and (d) does the SNS play a role in the development of fructose-induced hypertension? It is important to realize that BP is the measurable endproduct of an exceedingly complex series of factors, including those which control blood vessel caliber and responsiveness, those which control fluid-volume within and outside the vascular bed, and those which control cardiac output. None of these factors is independent; they interact with each other and respond to changes in BP. It is not easy, therefore, to dissect out cause and effect. However, throughout this section we will attempt to integrate the complex inter-relationships between insulin action, insulin resistance, hyperinsulinemia, vascular reactivity and the SNS into logical hypotheses.  86  Direct Effects of Insulin on Aortic and MVB Reactivity in C and F Rats  Examination of the direct effects of insulin on vascular reactivity revealed two main observations. First, insulin's vascular effects are vessel-specific and dose-dependent. In control rat aortae, insulin, at concentrations of 100 mU/ml attenuated the contractile responses to NE and A II (Figures 1,2), while in the MVB, insulin at concentrations of 100 pU/ml exaggerated the pressor responses to NE (Figure 6). Similar vessel-specific effects of insulin have been previously reported (see Introduction section; Wu et al, 1994; Townsend et  al, 1992). Second, in arteries from F rats, insulin's vascular effects were altered in both arterial beds studied. In aortae from F rats, insulin (100 mU/ml) failed to attenuate NE- or A U-induced contractile responses (Figures 1, 2) (Verma et al, 1997b). By contrast, insulininduced potentiation of MVB responses was further augmented in arteries from F rats (Figure 6). Thus, the direct vascular effects of insulin are altered in hyperinsulinemic, insulinresistant F rats in favor of increased peripheral vascular resistance. It is important to note that the effects of insulin on aortic reactivity were observed at pharmacological insulin concentrations (100 mU/ml vs. 100 pU/ml in the MVB). As the plasma insulin levels of hyperinsulinemic rats in the post-prandial state are = 80-100 u'U/ml, it is reasonable to conclude that the effects of insulin on MVB reactivity are more relevant to altered hemodynamics in F-hypertensive rats than a loss of insulin-mediated vasodilation in aortae. This factor assumes greater significance given the contribution of the MVB towards global systemic vascular resistance. Thus, based on these observations, we suggest that in Fhypertensive rats, chronic hyperinsulinemia may serve to increase peripheral vascular resistance (via exaggeration of MVB responses) which, in turn, may play an important role in  87  the development or maintenance of elevated BP in these rats. Although the loss of the vasodilatory effects of insulin in the aortae is consistent with clinical reports indicating the presence of "vascular insulin resistance" (Laakso et al., 1990; Laakso et al., 1992), the contribution of this effect to hypertension in F rats may be minimal given the pharmacological concentrations that are required to elicit this effect. It is important to point out that both the peripheral vascular actions of insulin and their relative role towards hypertension differ between humans/dogs and rats (Hall et al., 1995a). Although acute insulin infusions (in the physiological range) have been repeatedly shown to elicit vasodilatory effects in both humans and dogs (Brands et al., 1991b; Dela et al., 1995), it appears that a loss of insulin-mediated vasodilation is not associated with hypertension in these cases (Hall et al., 1995ab). Thus, although insulin resistance modifies the peripheral vascular actions of insulin in favor of increased tone, this effect may be largely confined to the effect of insulin to stimulate skeletal muscle glucose uptake. By contrast, results from our studies and others (Brands et al., 1991a; Brands et al., 1992, Brands et al., 1996) have demonstrated that in rats, physiological concentrations of insulin are associated with no vasodilatory responses. In fact, total peripheral resistance measured during chronic insulin infusions in rats has been shown to be elevated (independent of changes in cardiac output and heart rate) (Brands et al., 1996). These observations corroborate our data indicating an exaggeration of pressor responses of the MVB in response to physiological insulin concentrations in rats. A critical point to clarify is whether these changes in MVB reactivity precede the development of high BP. To this aim, we examined the effects of physiological insulin concentrations (100 pU/ml) on MVB reactivity in F rats after one week of fructose feeding.  88  We chose the one week post-fructose time point to represent the pre-hypertensive state. After one week of fructose feeding the rats in the F group were hyperinsulinemic yet normotensive (Table 1). We have previously demonstrated that fructose-induced hypertension develops after ~ 2 weeks of fructose feeding (Verma et al, 1994a, Bhanot et al, 1994a). Results from our study in pre-hypertensive F rats demonstrate that altered MVB responses to insulin are evident prior to the development of hypertension in these rats. MVB from pre-hypertensive F rats exhibited a greater potentiation of NE reactivity when compared to C rats (Figure 7). Thus, it is reasonable to suggest that hyperinsulinemia in FH rats may serve to exaggerate MVB responses and increase BP through increasing systemic vascular resistance. Although the blunted attenuation of NE responses by insulin in F rat aortae may be of minimal importance in modulating increased vascular resistance in-vivo (owing to the pharmacological concentrations required to elicit this effect), it is important to note that insulin-mediated vasodilation in this bed was completely obliterated by removal of the endothelium. As discussed in the introduction section, evidence suggests that NO release may account for insulin's' vascular effects in humans (Steinberg et al, 1994; Baron and Steinberg, 1997). Studies examining the role of NO in insulin-sensitive subjects suggest that insulin may modulate the endothelial synthesis/release of NO, but not NO action on VSMC (Steinberg et al, 1994). Although a variety of endothelium-dependent and -independent mechanism/s have been suggested to contribute towards insulin's effect on aortic tone (in pharmacological doses) (Wu et al, 1994; Han et al, 1995), preliminary observations from our laboratory indicate that this effect is blocked by inhibitors of NO synthesis (Yao, Arikawa and McNeill, unpublished observations).  As the buffer in our studies contained  indomethacin, the role of cyclooxygenase factors (vasodilatory prostaglandins E2 and I2) in  89  mediating this response can be excluded. Although the exact mechanism through which insulin interact with the L-arginine-NO pathway in rat aortae remain unknown, of interest are recent studies demonstrating that insulin may act on the Na K ATPase located in endothelial +  +  cells (Tack et al, 1996). Hyperpolarization (secondary to activation of Na K ATPase) will +  +  cause an influx of calcium into the endothelial cell because of the increased electrogenic driving force (calcium channels in endothelial cells are voltage-independent). The increase in cytosolic calcium will in turn stimulate endothelial synthesis and release of NO (via activation of cNOS).  Recent observations from our laboratory suggest that the interaction  between insulin and the NO pathway may involve stabilization of tetrahydrobiopterin (a cofactor for NOS) in the endothelium (Yao, Arikawa and McNeill, unpublished observations). Whatever the exact mechanism of action of insulin, what is perhaps more important are the observation that: (a) in rats insulin-mediated aortic vasodilation occurs at pharmacological insulin concentrations, (b) this effect is endothelium-dependent, and (c) in insulin-resistant and hyperinsulinemic rats, the ability of insulin to attenuate aortic contraction is blunted. It may be argued that the effects of insulin on MVB hyper-reactivity were due to a time-dependent increase in NE reactivity. Although the is a valid concern if this was the only reason we would: (a) not be able to block the effect with indomethacin, and (b) observe hyper-reactivity in the aortae at 100 |iU/ml. Furthermore, our data is in agreement with previously published reports indicating MVB hyper-reactivity to insulin in control rats (Wu et al., 1994).  90  Role of ET-1 in the Development of Fructose Induced Hypertension  In search of a mechanism underlying insulin-induced increases in MVB reactivity, we became interested in the interaction between insulin and ET-1.  Reports indicating that  plasma insulin levels may modulate ET-1 release (both in-vivo and in-vitro) (Oliver et al, 1991; Frank et al, 1993; Hu et al, 1993; Hattori et al, 1991), led us to hypothesize that hyperinsulinemia in F rats may serve as a continual stimulus for ET-1 release, which could increase BP via increases in vascular tone and reactivity. To this aim, we examined (a) the effects of acute ET receptor blockade (with bosentan) on insulin-induced changes in MVB reactivity, and (b) effects of chronic ET receptor blockade on plasma insulin levels, systolic BP and total MVB ET-1 content (Verma et al, 1995). Two main observations emanate from our in-vitro studies: first, ET receptor blockade (with bosentan) did not affect insulin-mediated MVB potentiation of NE responses in C rats. By contrast, indomethacin completely prevented the insulin response in MVB from C rats (Figure 8). The marked inhibition by indomethacin of insulin potentiation of NE responses in the MVB suggests that a cyclooxygenase metabolite from the endothelium is involved in this response. Whether the effect of insulin was reduction or inactivation of a relaxation metabolite or increased production of a contractile factor for the cyclooxygenase pathway remains to be determined (Wu et al, 1994). It is possible that insulin stimulates synthesis of cyclooxygenase, which would increase vascular levels of its products. In BALB/c 3T3 cells primed with epidermal growth factor or platelet-derived growth factor, insulin-like growth factor-I has been shown to elicit an influx of calcium that was slow in onset but was sustained for 2 hours (Kojima et al, 1988). It is also possible that in the endothelial cells of the MVB, insulin initiated a gradual increase in calcium entry that activated phospholipase  91  A2 leading to an increased flux of arachadonic acid through the cyclooxygenase pathway (Wu et al, 1994). The identity of the cyclooxygenase product(s) that mediate the effect of insulin in control MVB remains to be determined. As insulin and insulin-like growth factor-I have been demonstrated to not affect cGMP levels in the mesenteric bed (Wu et al, 1994), it is reasonable to suggest that vasodilators that enhance this pathway were not altered in this vessel. As observed earlier, in MVB from F rats, insulin's effects to potentiate MVB reactivity were potentiated (percent maximum potentiation: F+I 62±5 vs. C+I 32±5 %, P<0.05, Figure 8). In marked contrast to the observations in MVB from C rats, data in F rats revealed the involvement of both cyclooxygenase products and ET-1. Although indomethacin attenuated the pressor responses in F rats (to a similar degree to that seen in C rats), in the presence of both indomethacin and bosentan, the effects of insulin were completely abrogated (Figure 8). As bosentan is a potent inhibitor of E T and ET^ receptors a  (Clozel et al, 1993, Clozel et al, 1994), it is reasonable to speculate that the component of hyper-reactivity observed in MVB's from F rats in response to insulin may be mediated via ET-1. In conjunction with the in-vitro data, analysis of the total ET-1 content in MVB's revealed that F rats had an almost two fold higher mesenteric ET content compared to C rats (21.5±3 vs. 14.1±2 fmol, P<0.05). More importantly, chronic bosentan treatment completely prevented the development of hypertension in F rats (Figure 9). These data clearly indicate a role of ET in FH. We realize that the responses to ET-1 were conducted in superior mesenteric arteries while total ET-1 content was measured in the entire MVB. Although it  92  would be ideal to have measured both in the same vessel, this was not possible in our experimental setting. The plasma levels of ET-1 were not different in any group (Table 2), which supports the notion that ET-1 acts primarily as a local vascular modulator rather than a circulating homeostatic hormone (Rubanyi and Polokoff, 1994). Similar results have been documented in the aorta and mesenteric arteries of the DOCA-salt hypertensive rat (Lariviere et al, 1993). Although the finding that the bosentan treatment did not alter the total mesenteric ET-1 content in FT rats may, at first, seem contradictory, it does not contradict our observations. As mentioned earlier, ET-1 is released preferentially towards the vascular smooth muscle, where it is believed to mediate contraction through interaction with specific ET and ET^ a  receptors. Chronic treatment with an ET antagonist would make the receptors unavailable for ET-1 binding, and hence a lower total mesenteric ET-1 content would not be expected after bosentan treatment. Taken together, these data indicate that hyperinsulinemia in FH rats may serve to exaggerate MVB responses (possible through increases in ET-1 production) which in turn may lead to increases in vascular tone, reactivity and BP. The next question we addressed is whether the reactivity of arteries to ET-1 is altered in arteries from F rats (Verma et al, 1997a). To this aim, we compared the reactivity of superior mesenteric arteries from C and F rats (with and without chronic bosentan treatment) to ET-1 and NE. The main finding of this study was that mesenteric arteries from F rats exhibit a decreased responsiveness to the constricting effects of ET-1. Tissues that responded normally to NE (Table 3) exhibited reduced contraction to ET-1, indicating a specific alteration in the responsiveness to ET-1 in F rats. Similar results have been documented in  93  the DOCA salt hypertensive rat model where a diminished responsiveness of resistance arteries to ET-1 is associated with down regulation of the ET receptors and second messenger systems due to an increased endothelial cell ET-1 production and ET-1 gene expression (Lariviere et al, 1993; Nguyen et al, 1992, Deng and Schiffrin, 1992). Thus, it is plausible that a reduced responsiveness of mesenteric arteries to ET-1 may be secondary to increased ET-1 production in F rats. Although a recent study by Navarro-Cid et al reported that constrictor responses of isolated perfused MVB to ET-1 were similar between F rats and C rats, in that study rats were fed a fructose diet for a shorter period (3-4 weeks) than that used in our study (10 weeks). When resistance arteries from the mesenteric bed were studied directly using a pressure myograph, we were unable to detect any structural differences in small mesenteric arterioles from F and C rats, as determined by identical pressure-diameter curves and diameter (% constriction) changes over a range of NE concentrations (Figures 10, 11). The diameter changes would not indicate absolute levels of force; structural changes would be represented by pressure-diameter curves. This method has consistently detected changes in reactivity of vessels due to structural changes (Mulvany and Korsgaard, 1993; Schiffrin, 1992). As the constrictory responses to NE were similar for C and F rats when studied at transmural pressures of either 120 mmHg or 160 mmHg, respectively, it is reasonable to suggest that the smooth muscle function in mesenteric resistance arteries was not intrinsically different between C and F rats. Chronic bosentan treatment led to sustained decreases in BP in the F rats and restored the responsiveness of mesenteric arteries to ET-1 (Table 3).  Additionally, treatment  improved endothelium-dependent relaxation in FH rats. As a critical balance exists between  94  endothelium derived constricting and relaxing factors, it is possible that the improvement in relaxation was due to a reduction in ET-1 mediated constriction secondary to chronic bosentan treatment. The possibility that the responses were normalized secondary to a decrease in BP by bosentan cannot be ruled out. There is increasing evidence from studies using endothelin converting enzyme (ECE) inhibitors and ET receptor antagonists that basal generation of ET-1 may contribute to the maintenance of basal vascular tone and to the regulation of BP (for review see Rubanyi and Polokoff, 1994). Carefully designed studies that have followed hemodynamic responses for several hours after administration of an ET blocker do provide evidence for a role of ET-1 in regulation of basal vascular tone. Such studies are able to take into account the slow reversal of ET-1-induced vasoconstriction by anti-ET agents.  Inhibition of ET generation by  phosphoramidon (an ECE inhibitor) slowly decreases mean arterial pressure in normotensive and SHR rats over several hours. Studies suggest that ET-1 may contribute to maintenance of BP under physiological conditions, at least in some species, probably through its actions as a potent vasoconstrictor (for review see Rubanyi and Polokoff, 1994).  Another well  documented effect of ET-1 is its ability to potentiate vasoconstriction induced by a variety of vasoactive agents including serotonin and NE possibly through increasing Ca entry into the +2  VSM (Rubanyi & Polokoff 1994). This may represent one mechanism responsible for the enhanced insulin-mediated pressor responses of MVB to NE observed in F rats. It is also important to mention that ET-1 may increase the activity of the RAS system. ET-1 stimulates the tissue renin-angiotensin system of the rat mesenteric bed, increasing the generation of renin and A II. On the contrary, evidence suggests that A II may induce the synthesis and release of ET-1 (Emori et al, 1989, Scott-Burden et al, 1991). Furthermore, ET receptor  95  blockade inhibits A H-induced ET-1 production in rat VSM cells (Sung et al, 1994). Of interest, are recent observations demonstrating that blockade of the angiotensin-1 receptor with lorsartan prevents the development of hypertension in F rats (Navaro-Cid et al, 1995), suggesting a role of A II in FH. These observations, coupled with our studies documenting the antihypertensive effects of bosentan in F rats suggests an intriguing possibility of "crosstalk" between AII and ET-1 at the level of VSM in this rodent model of hypertension. However, we were interested in the relationship between hyperinsulinemia and elevated MVB responses in F rats. Based on the observations of study A, B and C, it may be hypothesized that chronic hyperinsulinemia in F rats serves as a stimulus for ET-1 release, which in turn may lead to an increased local ET-1 production, followed by a down regulation of the ET-1 receptors and an altered responsiveness to ET-1. Our observations demonstrating an increased total mesenteric ET-1 content in F rats make this a plausible hypothesis; however, whether such a mechanism is unequivocal in F rats remains to be determined.  Vasodilators and the Insulin-Hwothesis of Hypertension  Thus far, we have presented evidence that the vascular effects of insulin are altered in F-hypertensive rats. We propose a pathogenic model wherein hyperinsulinemia (possibly via ET-1 production) enhances reactivity in peripheral vasculature and that this effect precedes the development of hypertension. Our next objective was to examine the role of vasodilation per se on fructose-induced insulin resistance/hyperinsulinemia and hypertension.  There is  strong data to support a role for perfusion per se as an independent determinant of glucose uptake into skeletal muscle (Baron, 1996; Schultz et al, 1977; Ebeling and Koivisto, 1993; Wiernsperger et al, 1994; Baron et al, 1991, Baron et al, 1995). The role of blood flow in  96  modulating insulin's action to stimulate glucose uptake is further strengthened by studies demonstrating a loss of insulin-mediated vasodilation in states of insulin resistance such as obesity and diabetes (Laakso et al, 1990; Laakso et al, 1992). Although insulin's effects vary between insulin-sensitive humans and rats, in states of insulin resistance and hyperinsulinemia the vascular effects of insulin are altered in favor of increased peripheral vascular resistance. Fundamentally, if increased peripheral vascular tone plays a role in the development and/or maintenance of the insulin-resistant state, then a vasodilatorantihypertensive agent should attenuate these defects in F-hypertensive rats. To this aim, we studied the long term effects of mibefradil (a calcium channel blocker) on the development of hyperinsulinemia and hypertension in F rats. Data from this study indicate that the antihypertensive effects of chronic mibefradil treatment are associated with sustained and marked reductions in plasma insulin levels (Verma et al, 1997c). Although insulin sensitivity was not measured in this study, analysis of the 5-hour insulin/glucose ratios (an index of insulin sensitivity) revealed an improvement in insulin sensitivity after mibefradil treatment (insulin/glucose ratios in the four groups: C: 0.3±0.05, CT: 0.2±0.1, F:0.48±0.06 and FT:0.25±0.1. P<0.05 F vs. C, CT and FT). The treatment was effective in lowering the triglyceride levels in FT rats in both the prevention and reversal protocols employed (Table 4, 5). These data indicate that mibefradil exhibits beneficial effects on carbohydrate and lipid metabolism in addition to its antihypertensive effects. Additionally, these data suggest that vasodilation may play a role in modulating insulin resistance/hyperinsulinemia in this model. As discussed earlier, interventions with drugs that possess insulin sensitizing properties, lead to concurrent decreases in plasma insulin levels and BP in F rats (for review  97  see Bhanot and McNeill, 1996). By contrast, diverse antihypertensive-vasodilator agents (angiotensin-receptor blockers, peripheral alpha antagonists, angiotensin converting enzyme inhibitors) have also been shown to improve insulin sensitivity and decrease plasma insulin levels (Lithell, 1991; Navaro-Cid et al, 1995). In the former case, the antihypertensive effects of insulin sensitizers have been attributed to their ability to counter hyperinsulinemia and thereby correct the hypertensinogenic mediator/s linking insulin to BP (discussed above). On the other hand, the ability of vasodilator-antihypertensive agents to increase insulin sensitivity may be an indirect consequence of drug-induced vasodilation and hence an increased blood flow to insulin-sensitive tissues. On the basis of these observations, it has been speculated that some common mechanism (such as increases in VSM tone) may underlie both the expression of insulin resistance/hyperinsulinemia and hypertension. Thus, on one hand, insulin resistance/hyperinsulinemia results in vasoconstriction while on the other hand vasoconstriction results in decreases in blood flow to insulin target tissues, which further worsens insulin resistance. As discussed elegantly by Kotchen (1996), if the cycle is interrupted by agents that directly improve insulin sensitivity or by vasodilators that improve blood flow, the final outcome is an improvement in insulin sensitivity, a decrease in plasma insulin levels and a decrease in BP. A diagrammatic representation of this hypothesis is presented (Figure 16). An issue that merits attention are studies that have associated calcium channel antagonists with the development of diabetes, impaired glucose tolerance and insulin resistance, presumably through blocking calcium-induced insulin secretion (Semple et al, 1988; Iversen et al, 1990; Bhatnagar et al, 1984). However, studies suggest that at hemodynamically active doses, calcium antagonists do not normally interfere with insulin  FIGURE 16 Hypothetical Model Linking Insulin Resistance/Hyperinsulinemia, Vascular Tone and Elevated BP  Environmental Factors , , Smoking etc.)  Genetic Predisposition  r  D i e t  Insulin R e s i s t a n c e " ' ^ ^ Hyperinsulinemia  Resistance to the Vasodilatory Actions of Insulin and/or T MVB Reactivity  » / V  Insulin Sensitizers ^Metformin, Vanadium)  \  Decreased Blood Flow to Insulin Sensitive Tissues  J  Increase in VSM Tone |  V  Vasodilators (Mibefradil)  Increased Peripheral Vascular Activity  I  Increase in Blood Plessure  99  release nor impair glucose tolerance in man. This is further supported by animal studies that report that calcium channel blockers inhibit insulin secretion in-vitro but not in-vivo (Semple etal, 1988). An obvious and central question is how much overall insulin-mediated glucose uptake can be accounted for by insulin's hemodynamic action and the corollary, namely, how much insulin resistance can be accounted for by the impaired insulin-mediated vasodilation ? As stated earlier, the answer to the former questions requires physiological manipulations that abrogate insulin-mediated vasodilation in control situations, and the answer to the latter requires physiological manipulations that normalize skeletal muscle perfusion in insulinresistant situations. Although definitive answers to these questions are not available yet, studies in humans suggest that approximately 20-30% of insulin responsiveness is dependent upon vasodilatation (Baron et al, 1995, Baron and Steinberg, 1996; Baron, 1994). Whether the decrease in insulin levels and improvement in the insulin sensitivity index (i.e. 5 hour fasted insulin/glucose ratios) is due to a direct effect of mibefradil to improve insulin sensitivity or is secondary to an improvement of blood flow in insulin target tissues, is an important question that needs to be further examined. Although it is tempting to speculate that the attenuation of insulin resistance/hyperinsulinemia in FT was due to an improved blood flow to insulin sensitive tissues, no direct evidence for that is provided in this study. Clearly, studies aimed at examining both blood flow and glucose extraction (in skeletal muscle) in response to chronic mibefradil treatment will provide a clearer picture. We acknowledge that this study is clearly phenomenological and the hypothetical model proposed is one possible logical hypothesis, but in no way is unequivocal.  100  Role of the Sympathetic Nervous System in Fructose-Induced Hypertension  As discussed in the introduction section, multiple lines of evidence indicate that in rats, hyperinsulinemia may increase BP via activation of the SNS. On the other hand, evidence implicates a role of the SNS in the development of insulin resistance and hyperinsulinemia. To clarify the role of the SNS in the development of fructose-induced hypertension we examined the effects of chemical sympathectomy (via adrenal medullectomy, followed by weekly 6-hydroxy-dopamine injections) on fructose-induced increases in plasma insulin levels and BP. Results from this study demonstrate that chemical sympathectomy completely prevents the development of hyperinsulinemia and hypertension in F rats without affecting these parameters in C rats (Figure 14). These data indicate that the presence of a functional SNS is required for the development of both elevated plasma insulin levels and BP in F rats. Although previous studies have demonstrated the obligatory role of the SNS in mediating the acute effects of insulin infusion on BP in rats (Mozaffari et al, 1996), this is the first study to examine the role of the SNS in the development of high BP in a model of chronic insulin resistance, hyperinsulinemia and hypertension. The key observation from this study relates to the primacy of the SNS in the development of both hyperinsulinemia and hypertension in these rats. As alluded to earlier, the exact relationship between the SNS, insulin resistance and hypertension remains unclear. Although there appears to be general agreement in the literature regarding the dependence of insulin-induced hypertension (in rats) on a functional SNS (Mozaffari et al, 1996; Moreau et al, 1995; Kitamura et al, 1994; Brands et al, 1994), the question as to which comes first (SNS overactivity or insulin resistance) has been debated (Julius and Jamerson, 1994). The balance of published work (in  101  rats) favors a paradigm in which elevated SNS activity leads to insulin resistance (Julius and Jamerson, 1994, Moreau et al, 1995, Kaufman et al, 1991, Etchegoyen et al, 1997). Compensatory hyperinsulinemia that ensues in the face of insulin resistance, serves as a continual stimulus for SNS activation, further reinforcing the insulin resistant state. Thus, it is plausible that SNS hyperactivity is an early and integral part of fructose-induced hypertension and that the metabolic consequences are secondary to SNS-induced insulin resistance (The mechanism/s through which SNS overactivity can lead to insulin resistance are discussed in the introduction section). In support of this view are data demonstrating that chronic moxonidine treatment attenuates the development of both hyperinsulinemia and hypertension in F rats (Rosen et al, 1997). As moxondine is a centrally acting imidazoline-1 receptor agonist (which reduces sympathetic discharge), (Ernsberger et al, 1990; Ernsberger et al, 1992), the authors conclude that the improvement in insulin sensitivity and attenuation of hyperinsulinemia in F-treated rats was secondary to a reduction in sympathetic outflow, a view that is supported by our sympathectomy data. In this respect, however, it is intriguing to note that the central sympatholytic agent clonidine did not show beneficial effects on hyperinsulinemia in the F rat model, despite a marked reduction in BP (Hwang et al, 1987). It is important to note that both clonidine and 6-hydroxy-dopamine are sympatholytic agents, however, only sympathectomy abrogates F-induced hyperinsulinemia. Although unclear at this point, this may be related to the difference in site of action of the two agents. While clonidine reduces central sympathetic outflow, the action of 6-hydroxy-dopamine is limited to peripheral sympathetic post-ganglionic fibres. This may contribute towards the differences observed. However, the observation that moxonidine (a central sympatholytic) decreases plasma insulin levels and BP in F-hypertensive rats suggests that at the level of the  102  CNS immadozoline receptors may play a role in the development of hyperinsulinemia and insulin resistance secondary to SNS activation. Previous studies have demonstrated that adrenal medullectomy followed by weekly injections of 6-hydroxy-dopamine is an effective method of sympathectomy (Karlson and Ahren, 1991; Kostrzewa and Jacobowitz, 1974; Fench and Leach, 1970). Although NE levels were not measured in this study (owing to technical difficulties), sympathectomy was confirmed by a loss of tail artery responses to tyramine. The lack of tyramine response (which releases NE from endogenous stores) has been previously documented to be a sensitive index of functional dennervation (sympathectomy) of the cardiovascular system (Barres et al, 1992; Julien et al, 1989; Julien et al, 1990). The SNS is highly responsive to dietary intake. Food restriction or fasting suppresses SNS activity (Rappoport et al, 1982; Young and Landsberg, 1977). Food restriction also lowers insulin levels in obese humans and reduces BP in hypertensive obese humans as well as in SHR (Resin et al, 1978; Tuck et al, 1981). It is well known that the SNS function is sensitive to the macronutrient composition of the diet as well as to the total calorie content (Fournier et al, 1986; Kaufman et al, 1986; Schwartz et al, 1983; Walgren et al, 1987). Specifically, dietary fat and carbohydrate can enhance SNS activity. In addition, dietary sucrose has been associated with increased BP (via activation of the SNS) in both SHR and normotensive rats (Fournier et al, 1986; Michaelis et al, 1981; Preuss and Preuss, 1980; Young and Landsberg, 1981). Although enhanced SNS activity has not been previously demonstrated in FH rats, the available data (on sucrose-fed and other diet fed models) strongly suggests a similar diet-induced SNS activation mechanism.  103  The next question we addressed is whether the SNS modulates the vascular effects of insulin. To this end, we examined the direct effects of insulin on reactivity of CS and FS groups to the main sympathetic transmitter NE.  Sympathectomy per se resulted in an  increased sensitivity of MVB from both CS and FS groups. Supersensivity, secondary to sympathectomy, has been well established (Julien et al, 1989; Julien et al, 1990). The central observation, from this study however, was that sympathectomy abolished the component of hyper-reactivity in FS rats; MVB from CS and FS groups exhibited the same degree of insulin-induced exaggeration (Figure 15c). Although we are unable to provide an explanation for the lack of insulin-mediated exaggeration of MVB responses in FS rats, we have considered two possibilities. First, the SNS may modulate the MVB effects of insulin via blocking the release of ET-1. Our previous data suggest that the component of hyper-reactivity to insulin in MVB from F rats can be ascribed to ET-1 (Figure 8). Thus, it is possible that sympathectomy (through mechanism/s unknown) blocks ET release. In support of this view are recent observations which suggest that long-term guanethidine sympathectomy suppresses ET release from endothelial cells of the rat vascular bed (Milner et al, 1996). On the contrary, it is plausible that this effect was unrelated to an interaction between the SNS and ET, and was merely a consequence  of alleviation of hyperinsulinemia and hypertension  sympathectomy.  secondary to  Clearly, further studies are needed to define the exact mechanism/s  underlying this effect.  104  Pathogenesis of Fructose Induced Hypertension: in Perspective  One of the biggest hurdles in hypertension research is ascribing cause and effect. The pressure required to move blood through the circulatory bed is provided by the pumping action of the heart (cardiac output) and the tone of the arteries (peripheral resistance). Each of these primary determinants of BP is, in turn, determined by the interaction of an exceedingly complex series of events involving an interplay between genes and the environment. Hypertension has been ascribed to abnormalities in virtually every one of these factors and multiple hypotheses may prove to be correct, since the hemodynamic hallmark of hypertension, a persistently elevated vascular resistance, may be reached through a number of different paths. Although the search for a single underlying abnormality that begins the hemodynamic cascade towards sustained hypertension continues to attract the imagination and energies of numerous investigators, there may be no such single defect. In view of the multiple factors involved in the control of BP, the concept of a multifaceted mosaic, introduced by Irvine Page (1963) may be more appropriate. Given the above preamble, our research effort was directed at examining the potential links between insulin and BP. Our studies were based on an exceedingly large amount of experimental and clinical data that suggested that essential hypertension per se is often associated with insulin resistance and hyperinsulinemia. Additionally, drugs that specifically counter insulin resistance (and decrease plasma insulin levels) exhibit antihypertensive effects in rodent models of hypertension (including the F rat).  The observation that  hyperinsulinemia and insulin resistance correlate well with hypertension, piqued interest into understanding the mechanisms and links underlying this association. Much current interest has focused on the cardiovascular actions of insulin, chiefly, (a) the ability of insulin to alter  105  vascular smooth muscle tone and reactivity, and (b) the distinct sympathoexcitatory actions of insulin. This, in turn, has led to the proposal that alterations in insulin's effects on these two systems may play an important role in hypertension. Although, at first glance, the proposal seems attractive, studies in humans and dogs consistently demonstrate that hyperinsulinemia per se does not elevate BP. By marked contrast, chronic hyperinsulinemia (via insulin infusion or through fructose-feeding) is associated with hypertension in rats. The question as to why hyperinsulinemia causes hypertension in rats vs. dogs/humans has been linked to the species variation in the vascular responses of insulin. Although in both cases, physiological insulin concentrations stimulate SNS activity, in humans these concentrations of insulin are associated with vasodilation. In rats, however, physiological insulin concentrations are associated with no vasodilatory responses; total peripheral resistance is in fact markedly increased. Thus, a question that must be considered is whether or not the observations in rats are relevant to human hypertension?.  Hyperinsulinemia may activate pressor mechanism/s in rats that are  completely absent in humans and dogs (for example increases in mesenteric ET-1 content). Alternatively, dogs and humans may be protected from the hypertensive effects of insulin. The fact that insulin can elevate BP in some species, such as the rat, is intriguing and keeps alive the possibility that there may be pathologic conditions in which BP regulatory systems are altered in a manner that allows insulin to express its hypertensive effects in humans and dogs (Halloa/., 1995ab). Notwithstanding the above discussion, it is, however, plausible that in humans, insulin resistance rather than hyperinsulinemia with its attendant impaired insulin-mediated vasodilation could predispose to increased vascular tone and hypertension. Indeed, studies in  106  dogs suggest that although hyperinsulinemia per se may not be enough to induce increases in BP, when insulin resistance is superimposed, hypertension develops (Martinez et al, 1994). Studies by Anderson et al. (1991) and subsequently others (Berne et al, 1992) have elegantly demonstrated that although hyperinsulinemia causes marked SNS activation, the lack of an increase in BP is related to insulin-mediated vasodilation and a redistribution of cardiac output to skeletal muscle. Therefore, a combination of both hyperinsulinemia and insulin resistance may be necessary to increase BP in humans and dogs. It is important to stress that the pathogenesis of hypertension in the FH rat appears to involve a close inter-relationship between alterations in insulin, vascular reactivity and the SNS. Since the actual pathogenesis remains unknown, all one can do is take separate pieces of data from the various studies and try to construct reasonable hypotheses. Enough is known to provide a logical scheme that offers, at least, a better integration of the reams of seemingly disparate experimental data and, at best, models with rather broad explanatory powers. In the scheme presented (Figure 17), we suggest that the SNS may be one of the primary systems activated in response to fructose-feeding.  Activation of the SNS may lead to the  development of insulin resistance. Compensatory hyperinsulinemia (that ensues in the face of insulin resistance) may serve as a continual stimulus for SNS activation, thereby completing  the  vicious  cycle  of  TSNS-insulin resistance-hyperinsulinemia-TSNS.  Concurrently, hyperinsulinemia may serve to increase ET-1 levels and enhance the pressor responses of resistance vasculature to sympathetic stimulation. Increases in vascular tone appear to be important in the development of hypertension; our data indicate that these changes precede high BP in this model. The fact that the vascular effects of insulin are altered after 7-10 days of fructose feeding while BP elevations are observed after 2-3 weeks,  107  FIGURE 17 Pathogenesis of Fructose-Induced Hypertension: A Schematic Representation  FRUCTOSE FEEDING t S  I  N  N  S  U  *  S A  L  C  I  T  N R  I  E  V  S  I  I  T  S  T  E I N C R E A S E  Y  T  A  -  P E R I P H E R A L  V A S C U L A R  T O N E  HYERTENSION  I N  1 P  R  C  O  E  D  —  U  C  T  •  I  O  108  suggests that homeostatic mechanisms compensate for the increases in vascular resistance for this period. The observation that vasodilator antihypertensive agents (mibefradil) improve insulin sensitivity and decrease plasma insulin levels suggest that increases in vascular tone may be linked to insulin resistance and hyperinsulinemia. Thus, if the cycle is broken at any site, by drugs that (a) improve insulin sensitivity, (b) block the effects of ET, (c) cause vasodilation, or (d) antagonize the SNS, the net result is a decrease in BP. We would like to emphasize that we are not claiming primacy of any one system in FH. The model proposed represents one possible explanation. We appreciate that ET-1, the SNS, insulin resistance/hyperinsulinemia may all play independent roles in the development of high BP and may not be inter-related. Thus at best the proposed model should be used to highlight the various systems that may be involved in the development of FH.  Limitations and Other Mechanisms  Although recent studies have excluded the involvement of altered renal function in F rats (Iyer and Katovich, 1996), our studies would have greatly benefited from data on (a) cardiac output, (b) heart rate, (c) NE levels and (d) MVB ET-1 levels in the pre-hypertensive experiment and after sympathectomy. The involvement of the RAS in FH (using ACE inhibitors) has been suggested (Navarado-Cid et al, 1996). However, given the effects of ACE inhibitors on insulin sensitivity, it is difficult to distinguish whether a functional RAS is necessary to induce hyperinsulinemia and hypertension or whether the effects on plasma insulin levels are secondary to an improvement in insulin sensitivity. Although the exact contribution of hyperinsulinemia and insulin resistance towards the pathogenesis and clinical course of essential hypertension is still debatable, the evidence  109  that these defects are associated with an atherogenic risk profile and other cardiovascular diseases stands strong. Whether insulin resistance and hyperinsulinemia are causally related to hypertension or are linked through parallel yet distinct mechanism/s is still a topic of considerable discussion and debate.  The F-hypertensive rat is a diet-induced model of  hypertension that is now being used extensively to examine the interaction between insulin resistance, hyperinsulinemia and hypertension (independent of obesity). Although we have provided some mechanistic information regarding the development of hypertension in this model, it is important to realize that the cardiovascular effects of insulin are different in rats vs. humans. Hence, caution is warranted in drawing extrapolative conclusions from these data. We do hope, however, that our effort at identifying a few potential sites in FH rats will add to the field of experimental hypertension research.  no  CONCLUSIONS  1.  The vascular effects of insulin are vessel-specific and concentration dependent. In control aortae, pharmacological insulin concentrations (100 mU/ml) attenuate the contraction to vasoactive agents. In contrast, physiological insulin concentrations (100 pU/ml) exaggerate pressor responses in the perfused MVB. As the effects of insulin in the aortae occur at pharmacological concentrations, we suggest that the effects of insulin on aortic responses are of minimal importance towards overall hemodynamics.  2.  The vascular effects of insulin are altered in hyperinsulinemic, insulin-resistant FH rats. In the aortae, insulin-induced attenuation of pressor responses is blunted, while in the MVB, insulin-mediated potentiation is markedly enhanced.  3.  Hyperinsulinemia in FH rats may exaggerate MVB responses via an increased production of ET-1. This is supported by observations indicating (a) an increased ET1 content in MVB from FH rats, (b) the acute blockade of insulin-mediated MVB potentiation of NE responses, and (c) the antihypertensive effect of chronic endothelin receptor blockade (with bosentan).  4.  Chronic treatment of FH rats with a vasodilator calcium-channel blocker (mibefradil) both prevented and reversed the development of hyperinsulinemia, insulin resistance (estimated by 5 hour fasted insulin/glucose ratios) and hypertension. As mibefradil is  Ill  devoid of any direct effects on insulin-sensitivity, we suggest that vasodilation may be coupled to improvement of insulin action in this model.  A  functional  SNS is  necessary for the  development  of both insulin  resistance/hyperinsulinemia and elevated BP in FH rats. This is supported by studies demonstrating the effects of chemical sympathectomy in FH rats. Additionally, the SNS may modulate the vascular effects of insulin in the MVB.  112  BIBLIOGRAPHY  Alexander WE and Oake RJ. 1977. The effect of insulin on vascular reactivity to norepinephrine. Diabetes. 26:611-614. Alleman Y, Weidman P. 1995. Cardiovascular, metabolic and hormonal dysregulations in normotensive offspring of essential hypertensive parents. J. 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