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Insulin resistance and hypertension: the hemodynamic and metabolic effects of deuterium oxide, enalapril,… Maxwell, Sharon 1994

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Insulin Resistance and Hypertension: The Hemodynamic and Metabolic Effects of Deutenum oxide, Enalapril, and Metformin By Sharon Maxwell B.Sc. (Hons.), Queen’s University, 1991 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Masters of Science in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHARMACOLOGY & THERAPEUTICS FACULTY OF MEDICINE We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1994 © Sharon Maxwell, 1994  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  Tht-\ OcCDOcj  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  I  II  ABSTRACT Elevated blood pressure has been recognized as a marker for disease since the early I 800s.  It is commonly divided into two categories: primary hypertension and  secondary hypertension. inheritable  and/or  Primary hypertension is defined as hypertension where  environmental  factors  are  unknown  whereas,  secondary  hypertension is defined as hypertension caused by a known congenital or acquired disease. Primary hypertension is discussed in this paper. In an attempt to better understand the pathophysiology of hypertension, a common syndrome in patients was described called Syndrome X.  Patients with this  syndrome have resistance to insulin-stimulated glucose uptake, glucose intolerance, hyperinsul inemia, increased very-low-density lipoprotein triglyceride, decreased highdensity lipoprotein cholesterol, and hypertension. In an attempt to better understand the relationship between elevated insulin concentrations (hyperinsulinemia) and elevated  blood  pressure,  experiments  were  designed  using  spontaneously  hypertensive rats (SHR) as a genetic model of hypertension. Agents which lower blood pressure (deuterium oxide and enalapril) and an agent which lowers plasma glucose concentrations were used to try to elucidate the relationship between insulin resistance and hypertension.  If insulin resistance and hypertension are causally related one  would expect that by pharmacologically altering one of the abnormalities a similar direction and magnitude of effect would occur in the other. Two experiments were performed. The first experiment examined the effects of 10% 020 and 50 mg/L enalapril on hemodynamic and metabolic factors in the SHR.  III  The second experiment examined a dose range (10, 30, 100, and 300 mg/kg/day) of metformin in SHR and its effects on hemodynamic and metabolic factors.  In both  experiments body weight, systolic blood pressure, insulin, glucose and triglyceride concentrations in plasma, water intake, and urine volume were recorded weekly. At the end of each experiment direct blood pressures were recorded from the iliac artery. In the D 0 and enalapril experiment, enalapril significantly lowered the systolic 2 pressure compared to the control and 10% D 0 groups. 2  There was no significant  difference in the insulin (mU/L) or glucose (mmol/L) concentrations between the three groups and the insulin:glucose ratio (mU/mmoL) was not significantly different between the groups.  These results suggest that there is no effect on insulin or glucose  concentrations when the blood pressure is lowered in the SHR. In the metformin experiment, metformin did not significantly lower the systolic blood pressure during the treatment period. There was also no significant difference in fasting plasma insulin and glucose concentrations.  The insulin:glucose ratio also  showed no significant difference between the groups. Conclusions: 1. Ten % D 0 decreases fasting plasma glucose concentrations, thus possible 2 causing a decrease in insulin resistance. 2. Despite this, chronic 10% D 0 has no effect on blood pressure or fasting plasma 2 insulin concentration in the SHR. 3. This suggests that insulin resistance does not cause increased blood pressure.  iv 4. Enalapril decreases blood pressure but has no effects on glucose and insulin concentrations in the SHR, confirming that high blood pressure does not cause insulin resistance. 5. Enalapril causes a large increase in urine volume and water in the SHR. 6. Chronic metformin (10 to 300 mg/kg/day) has no effect on insulin and glucose concentrations or blood pressure in SHR. 7. The SHR may not be an appropriate model for studying the link between hypertension and insulin resistance.  V  TABLE OF CONTENTS  Abstract  ii  List of Contents  v  List of Tables List of Figures  viii  Acknowledgements  ix  CHAPTER 1 1. INTRODUCTION 1.1 Hypertension A general background  I  1.2 Hyperinsulinemia and Hypertension  2  -  1.3 Genetic Model of Hypertension The spontaneously hypertensive -  rat (SHR)  6  1.3.1. The Control Strain Wistar-Kyoto Rats (WKY) -  1.4 Pharmacological agents Deuterium oxide, Enalapril, Metformin -  10  10  1.4.1. Deuterium oxide  10  1.4.2. Enalapril  13  1.4.3. Metformin  14  1.5 Objectives  16  CHAPTER-2 2. METHODS 2.1 Deuterium oxide and Enalapril  16  vi 2.2Metformin  18  2.3 Statistics  20  CHAPTER -3 3. RESULTS 3.1 Deuterium oxide and Enalapril  20  3.2 Metformin experiment  22  CHAPTER-4 4. DISCUSSION 4.1 Deuterium oxide and Enalapril  68  4.2 Mefformin experiment  72  4.3 Is the SHR an Effective Model of Insulin Resistance?  73  4.4 Conclusion  75  5. REFERENCES  76  vii  LIST OF TABLES Table 1.  Page Hemodynamic and metabolic data, Metformin experiment  67  VIII  LIST OF FIGURES Figure  Page  1.  Body weights, D 0 and enalapril 2  25  2.  Indirect systolic blood pressure, D 0 and enalapril 2  27  3.  Dircet blood pressure, D 0 and enalapril 2  29  4.  Twelve hour water intake, D 0 and enalapril 2  31  5.  Twelve hour urine volume, 020 and enalapril  33  6.  Urine potassium, D 0 and enalapril 2  35  7.  Urine sodium, D 0 and enalapril 2  37  8.  Percentage of D 0 in urine, D 2 0 and enalapril 2  39  9.  Fasting plasma glucose concentrations, D 0 and enalapril 2 Fasting plasma triglyceride concentrations, D 0 and enalapril 2  41  10. 11.  43 45  12.  Fasting plasma insulin concentrations, D 0 and enalapril 2 lnsulin:Glucose ratio, 020 and enalapril  13.  Body weight, Metformin  49  14.  Indirect systolic blood pressure, Metformin  51  15.  Direct blood pressure, Metformin  53  16.  Twenty-four hour water intake, Metformin  55  17.  Twenty-four hour urine volume, Metformin  57  18.  Fasting plasma glucose concentration, Metformin  59  19.  Fasting plasma triglyceride, Metformin  61  20.  Fasting plasma insulin, Metformin  63  21.  Insulin:Glucose Ratio, Metformin  65  47  ix  ACKNOWLEDGMENTS The author would like to thank Dr. J.M. Wright for all of his guidance and encouragement; Drs. M.C. Sutter, R.A. Wall, and C.C.Y. Pang for their advice and as members of the Supervisory Committee. The author would also like to thank Christina and Kenneth Poon for their help with the surgeries performed in this experiment. A special thanks to the departmental secretaries for their assistance.  1  1.  INTRODUCTION 1.1.  Hypertension A general background -  Elevated blood pressure was recognized as a disease entity in 1827 (1). In 1895, Allbutt called a rise in blood pressure without proteinuria “senile plethora”, which was later revised to “hyperpiesis” (2). The term “hyperpiesis” was modified to “essentille hypertonie” by Frank, in 1911, and was translated to essential hypertension (3). Today, primary hypertension is most commonly used to describe elevated blood pressure. In 1955, Pickering characterized primary hypertension as high blood pressure with hypertensive cardiovascular hypertrophy and proposed that it was dependent on inheritance and environment (4). Thus, primary hypertension was thought to be initiated by a polygenic and multifactorial cause.  Secondary  hypertension was defined as hypertension caused by a known congenital or acquired  disease  such  as  renovascular  hypertension  or  primary  hyperaldosteronism. This is in comparison to primary hypertension where the inheritable and/or environmental factors are unknown (5). More recently, primary hypertension has been postulated to be caused by genetic factors (6). This differs from secondary hypertension caused by environmental factors or disease (6-8).  There is probably an interaction  between environmental and genetic factors such as salt, alcohol, obesity, low exercise, etc. Genetic hypertension is thought to be caused by abnormalities in arterial smooth muscle causing increased peripherial vascular resistance; blood pressure increases steeply at 30 to 50 years of age, without any known environmental factors. In humans, hypertension can be further divided into four groups on the basis of the blood pressure measurements. Borderline hypertension is defined  2 by systolic pressure between 140-159 mmHg and diastolic pressures of 90-94 mmHg.  Mild and moderate hypertension are defined by systolic pressures of  160-219 mmHg and diastolic pressures of 95-114 mmHg.  Lastly, systolic  pressures greater than 220 mmHg and diastolic pressures greater than 115 mmHg are indicative of severe hypertension (9). Hypertension is a particular problem because it is usually asymptomatic and it is the most common cardiovascular disorder in North America, affecting more than I in 10 persons (10).  It is important to control elevated blood  pressure because it can lead to a greater risk of stroke, heart failure, renal disease, peripheral vascular disease, and coronary artery disease (11). There are a number of methods that are used for lowering blood pressure. These include non-pharmacologic therapies such as restricted sodium intake, weight loss, reduced alcohol intake, and exercise as well as antihypertensive drugs (9). The pathophysiology of hypertension is complex and not well understood. Recently, Reaven in an attempt to better understand the pathophysiology of hypertension, described a common syndrome in patients called Syndrome X. Patients with this syndrome have resistance to insulin-stimulated glucose uptake,  glucose intolerance,  hyperinsul inemia,  increased very-low-density  lipoprotein triglyceride, decreased high—density lipoprotein cholesterol, and hypertension (12).  The mechanistic reason for this association between  hypertension and hyperinsulinemia has not been elucidated. A review of the relationship between insulin resistance and hypertension follows. 1.2.  Hyperinsulinemia and Hypertension  In healthy individuals actions of insulin are numerous but in general insulin is secreted from the pancreas after meals and promotes the storage of carbohydrates, protein, and fat (13).  Specific actions of insulin include  3 increased glucose entry in adipose tissue and muscle as well as an increase in triglyceride deposition in adipose tissue. Insulin is synthesized in the 13-cells of the Islet of Langerhans in the pancreas and once it is secreted it has a half-life of approximately 5 minutes. The degradation of insulin mostly occurs in the liver and kidneys but almost all tissues have the ability to metabolize insulin (13). Thus, in normal individuals insulin secretion is triggered by a rise in blood glucose associated with food intake.  The release of insulin then promotes  glucose uptake into specific tissues where the glucose is metabolized. Insulin resistance has been described in a variety of ways, but in general it is a reduction in the response to insulin (14).  Glucose uptake requires a  specific concentration of insulin to promote the transfer of glucose into a cell. Binding of insulin to specific surface receptors triggers unknown intracellular messages which in turn activate glucose transporters that transport glucose across the cell membrane (15). The molecular biology of insulin resistance has recently been described and it has been shown that the impairment of insulin action can be attributed to decreased insulin receptor number and post binding defects of insulin action (16). A malfunction at any stage of this process could result in resistance to insulin-stimulated glucose uptake. Insulin resistance is present in all patients with non-insulin dependent diabetes mellitus (NIDDM) (12) and is frequently combined with a defect in insulin secretion (17).  Obesity is a common precursor of NIDDM and is  commonly associated with insulin resistance (17-19). A relationship also exists between NIDDM and hypertension (Syndrome X) (20-22). A common pathogenic link between diabetes and hypertension may be insulin resistance (20). It has been demonstrated that hypertensive patients, on average, are more insulin resistant than a control population in the absence of obesity or NIDDM (23-25). Bonora, et al. conducted studies involving 247 non-  4 obese and 120 obese non-diabetic subjects (24).  All subjects underwent a  standard oral glucose tolerance test which included the measurement of plasma insulin concentrations. One single blood pressure measurement was obtained I to 2 hours following the glucose load. Results showed a significant relationship between either systolic or diastolic blood pressure and both fasting and postglucose plasma insulin. It was suggested that the post-glucose plasma insulin response was independently associated with blood pressure in the non-obese subjects, while the association between plasma insulin and blood pressure in obesity was mainly mediated by factors such as age and body weight. It was concluded that insulin may play a role in the regulation of blood pressure in the absence of obesity (24). Pollare, carbohydrate  et al.  studied the relationship between abnormalities in  metabolism  and  hypertension  in  143  newly  detected  hypertensives, which were divided into obese and non-obese groups, and 51 normotensive controls (25). The euglycemic clamp technique was used (initially described by Defronzo, et al. 26) to calculate steady state plasma insulin and glucose concentrations.  The non-obese hypertensive group had significantly  increased fasting plasma insulin values compared with the control group. The obese hypertensive group had significantly higher plasma insulin values compared to both control and non-obese groups. These results suggest that the abnormalities of carbohydrate, insulin, and lipid metabolism in primary hypertensive patients may occur independently from obesity (25). In 1985, Mancini, et a). compared obese normotensive and obese hypertensive patients (26).  The patients were subjected to an oral glucose.  tolerance test (OGTT) by giving 75g of glucose as a 33% solution and taking blood samples for glucose and insulin measurements at 0 up to 240 minutes after the glucose load. The results of this study showed a significant increase in  5 serum insulin in the hypertensive group.  The authors also concluded that  impaired glucose tolerance was more common in the obese hypertensive group although this finding was based on one data point at 120 minutes after the oral load.  It was concluded that in obese patients, high blood pressure was  independently associated with impaired glucose tolerance and higher fasting serum insulin levels. The results from this experiment suggest that hypertension and insulin resistance occur independently of obesity, however because of the small number of patients and the other criticisms mentioned above it is not conclusive. As previously mentioned, Reaven defined the relationship between insulin resistance and hypertension as Syndrome X. Various studies have been carried out since this publication that confirm these results. Glucose clamp studies have shown the presence of insulin resistance in elderly patients with hypertension as compared to normotensive controls (27). Oral glucose loads have been given to middle-aged hypertensive patients who showed exaggerated glucose and insulin responses (28), which could be indicative of insulin resistance. The bulk of data confirms a relationship between these two abnormalities.  However, it is not  known whether this is a causal relationship or if these two abnormalities develop individually, possibly from an early genetic defect. The primary site of insulin resistance in hypertensive patients is the skeletal muscle (29,30).  Direct measurements in the forearm of hypertensive  patients has displayed an impairment in insulin action at the muscle tissue level (29).  Julius, et al. postulated that the pressure-induced restriction of the  microcirculation, associated with hypertension, would limit nutritional flow and thereby impair glucose uptake in the skeletal muscle. Thus decreased skeletal muscle blood supply may be a possible link between insulin resistance and hypertension (30).  6 Hwang, et al. conducted an experiment to try to determine if hypertension could be produced by feeding a fructose-enriched diet to normotensive rats (31).  The rats were fed the fructose diet for 2 weeks and blood pressure, steady state plasma insulin and glucose, and biochemical measurements were taken. Systolic blood pressure was significantly higher in the fructose fed group in comparison to controls.  Hyperinsulinemia and hypertriglyceridemia were also  associated with the increase in blood pressure.  However, when clonidine  (antihypertensive agent) was added to the drinking water the fructose induced hypertension was inhibited, but the increases in plasma insulin and triglyceride were not effected. This evidence suggests that elevated blood pressure is not the cause of the metabolic changes seen in these fructose fed rats. Furthermore, in response to an oral glucose tolerance test (OGTT) hypertensive men treated with an antihypertensive agent did not show a significant difference in the steady state plasma insulin (SSPI) levels when compared to normotensive and non-treated hypertensive men.  This indicates  that lowering blood pressure with an antihypertensive agent does not necessarily effect plasma insulin levels (32).  Thus, results from both animal  experiments and human patients suggest that the metabolic abnormalities associated with insulin resistance are not altered when elevated blood pressure is lowered by antihypertensive agents. 1.3.  Genetic Model of Hypertension  -  The spontaneously hypertensive  rat (SHR) The spontaneously hypertensive rat (SHR) is the most commonly used experimental model of inherited hypertension since its development 27 years ago by Okamoto and Aoki (33). The development of this strain of rat began from inbred Wistar rats, who were sent from the United States to Kyoto University.  7 These rats (Wistar-Kyoto, WKY) were then outbred within a closed colony and were subsequently screened for elevated blood pressure (34). A single male rat was found with a systolic blood pressure in the range of 150-175 mmHg. There was a subsequent mating of this male rat with a female rat whose systolic blood pressure ranged between 130-140 mmHg, which was above the average of the colony. There was further inbreeding from three generations of offspring until the rats developed blood pressures of  >  150 mmHg (34). Okamoto and Aoki  defined these rats as arbitrarily hypertensive (33).  Blood pressures rose with  each generation of hypertension and the development of high blood pressure began to occur at a younger age (34).  In 1969, after 20 generations of  inbreeding a line of SHRs had become fixed. However, some of these SHRs were released to other laboratories before the 20th generation.  Therefore,  “genetic variation exists between colonies of SHRs because they either arose from different inbred SHR strains released prior to the strain reaching genetic homogeneity, or because of the genetic drift that is known to occur within and between colonies arising from the same inbred strain” (34). Differences between the colonies are more likely trait difference, which are not related to the development of hypertension (35). Therefore, even though there may be slight differences between colonies, the SHR is an appropriate model for hypertension and may provide clues as to the basis of primary hypertension in humans as explained below. The similarities of genetic hypertension between man and rat has been explored by Trippodo and Frohlich (36).  Certain important differences are  recognized; 1) in the human population of primary hypertension, hypertensive patients tend to be heavier than those without hypertension whereas SHR normally weigh less than their normotensive controls, 2) the SHR may have altered thyroid function which does not occur in most primary hypertensive  8 patients, and 3) the rat has relative resistance to developing significant atherosclerosis, in contrast to primary hypertensive patients whose elevated blood pressure facilitates the onset of atherogenesis (36). The SHR develops increased arterial pressure as early as 3 weeks of age and it continues to increase until approximately 20-28 weeks of age.  The onset and rate of  development of arterial pressure in humans is not clearly defined. Despite these differences, the SHR has a number of similarities to human primary hypertension. Increased total peripheral resistance and normal cardiac output are two hemodynamic factors that are found in established hypertension  in both humans and SHRs. Moreover, both SHR and human patients display either normal or slightly reduced blood volume, an elevated heart rate, and the progressive development of hypertrophy in the left ventricle. The persistence of vascular resistance in both cases may lead to impaired myocardial function which can result in congestive heart failure. Thus, the hemodynamic alterations  in both forms of hypertension appear to follow a very similar course (36). The participation of renal factors and their effect on arterial blood pressure continues to be studied. Studies have shown that renal blood flow is usually normal or decreased with a normal or slightly reduced glomerular filtration rate and increased filtration fraction in both humans with uncomplicated hypertension (37) and SHR (38). Renal vascular resistance is also elevated in both forms of hypertension (36). Other similarities such as increased venoconstriction and increased sympathetic nerve activity have been implicated in both humans with primary hypertension and in the SHR, but the exact mechanism of how these factors effect blood pressure in man and rat is still being investigated (36). Because the similarities outweigh the differences, the SHR is considered to be an appropriate model for studying the mechanism of hypertension in man.  9 However, is it also an appropriate model for studying the relationship between primary hypertension and insulin resistance? A number of studies have been published which support the view that SHRs develop an insulin resistant state similar to that seen in human hypertensive patients (39-42).  In a preliminary  report Mondon and Reaven showed that abnormalities in insulin secretion, action, and catabolism existed in rats with spontaneous hypertension (39). This evidence was supported in a later publication which demonstrated cellular resistance to glucose uptake in adipocytes from SHR (40). In 1989, Mondon, et al. suggested that high plasma insulin concentrations (hyperinsulinemia) associated with insulin resistance may be due to a decreased removal of insulin by skeletal muscle and the kidneys rather than impaired hepatic removal of insulin (41).  The presence of peripheral insulin resistance in the SHR was  observed, specifically in the skeletal muscle (42, 43).  Further study of this  relationship suggests that SHR release insulin normally, but they exhibit reduced tissue sensitivity to insulin (46) and that this reduced sensitivity is a primary rather than a secondary event in hypertension (47). Experiments by Swislocki and Tsuzuki contribute to the previous findings that SHR are suitable models for insulin resistance and primary hypertension through the expression of insulin resistance in terms of glucose and fatty acid metabolism in SHR (48). Thus it is suggested that the SHR, as well as being hypertensive, may also have metablolic abnormalities. However, some evidence exists that contradicts the presence of insulin resistance in SHR (44, 45).  Gaboury, et al. demonstrated that the action of  insulin on glucose metabolism is not impaired in the SHR at a time when their blood pressure is clearly elevated (44). Buchanan, et al. showed no significant difference between SHR and their control strain WKY in the response of skeletal  10 muscle to insulin (45). Therefore, it is not clear whether insulin resistance is a consistent finding in the SHR. Does this potential insulin resistant state in the SHR parallel the one seen in human patients?  A review by Gerald Reaven concludes that the  abnormalities of glucose, insulin, and lipoprotein metabolism that occur in hypertensive patients also occur in the SHR (49). Therefore, it appears that the SHR is the best animal model for studying the relationship between elevated blood pressure and insulin resistance at this time. 1.3.1.  The Control Strain Wisrar-Kyoto Rats (WKY) -  The Wistar-Kyoto rat (WKY) has been used as the normotensive control strain for the SHR.  The WKY differs from the SHR because the increase in  arterial blood pressure occurs at a slower rate in the WKY and reaches its maximum at approximately 6-10 weeks of age (36). The mean arterial pressure of the WKY reaches 115-130 mmHg, while the average mean arterial pressure of the SHR is between 190-200 mmHg depending on the colony.  However,  there is some speculation as to the validity of this normotensive control because it was not developed simultaneously with the SHR (36, 50, 51).  Genetic  “fingerprint” patterns were examined from both SHR and WKY (50).  Results  showed that SHR were genetically quite different from the normotensive WKY; only 50% of the DNA fingerprint bands were common between the two strains. Thus, continued comparison of SHR to WKY may have limited value for investigating the pathogenesis of hypertension.  Therefore, in the following  experiments WKY rats were not studied.  1.4.  Pharmacological agents Deuterium oxide, Enalapril, Mefformin -  1.4.1.  Deuterium Oxide  11 0) is a stable nonradioactive isotope of water, which 2 Deuterium oxide (D has been studied in mammals since the late 1950’s.  In 1958, a study was  conducted in rats to determine the effect of D 0 on glomerular filtration and 2 renal plasma flow (52). The rats were given 50 mole % D 0 as drinking water 2 for 38 days. Results showed a decrease in both filtration rate and renal plasma flow to about 40% of rats on normal water. It was also noted that when the rats were returned to normal tap water the filtration rate and renal plasma flow returned to its normal state. They concluded that the effect of D 0 may have 2 been due to a disturbance of adrenal function.  A couple of years later, the  effects of D 0 were studied in heart, and voluntary muscle at concentrations 2 varying between 99.8 to 25 % in drinking water (53). D 0 decreased the force 2 and velocity of contraction in both the heart and voluntary muscle.  Further  investigations in frog muscle, suggested that the contractile proteins could be affected by deuterium (54). Muscles of the barnacle were used by Kaminer and Kimura to test their hypothesis that calcium release was prevented by D 0, in the coupling of 2 excitation and contraction (55).  Aequorin, a protein which luminesces in the  presence of calcium, was used to determine the amount of calcium present in the muscle tissue after exposure to 99.9 % D 0. The results showed that in the 2 presence of 020 no calcium was released and therefore no contractile response observed.  It has been suggested that D 0 depresses the mobilization of 2  calcium ions by lowering the rate of release of calcium ions, decreasing amount of calcium release, and reducing diffusion of calcium ions (56, 57). 0 in Sprague-Dawley rats have demonstrated that 2 Recent studies with D 0 affects vascular muscle relaxation (58). It was suggested that these results 2 D occurred through action on the sarcoplasmic reticulum calcium mobilization or  12 contractile proteins.  Therefore, D 0 may have multiple sites of action on 2  vascular smooth muscle. Deuterium oxide has been shown to affect both insulin and glucose in experimental rats. Experiments with D 0 in Sprague-Dawley rats have shown 2 that 50% D 0 in the drinking water decreases blood glucose over a period of 35 2 days. It appears that the D 0 treatment slows down gluconeogensis, thus blood 2 sugar cannot be maintained at a normal range (59). It has also been shown that 0 inhibits insulin release, probably through its stabilizing action on the 2 D microtubular system of the 13-cell (60). D 0 may also mimic the action of insulin 2 by increasing glucose metabolism in adipose tissue (61). This specific action of 0 is of interest because D 2 D 0 may also promote glucose uptake in the skeletal 2 muscle by acting like insulin at this site. Vasdev, et at. hypothesized that D 0 may help prevent the development 2 of hypertension, by preventing the abnormal contractile activity of the vascular smooth muscle associated with this abnormality (62). Twenty-five percent D 0 2 was given to male Dahl salt-sensitive rats for four weeks. The D 0 treatment 2 caused a significant decrease in the systolic blood pressure compared to the non treated rats. It was suggested that the antihypertensive effect of D 0 was 2 the result of increased blockage of calcium channels by bound deuterium ions. A further  study  showed  that  0 2 D  (25%)  prevented  hypertension  in  spontaneously hypertensive rats (SHR) compared to their control strain Wistar Kyoto (WKY) (63). It was also demonstrated that 25% D 0 normalized elevated 2 calcium uptake in the aorta.  It was again postulated that the blood pressure  lowering effect of D 0 was the result of bound deuterium ions in the vascular 2 calcium channels. An investigation of the dose-dependent effect of D 0 in drinking water on 2 systolic blood pressure and aortic calcium uptake was conducted in SHR to  13 0 (64). 2 determine the minimum effective dose of D  SHR were treated for 7  0. 10% and 20% D 2 0 prevented the increase 2 weeks with 5%, 10%, and 20% D  in systolic blood pressure. These two groups also displayed normal values of aortic calcium uptake. It was concluded that 10% D 0 was the minimum dose 2 required to completely prevent the development of hypertension and elevated aortic calcium uptake in SHR. Because of its potential effect on both glucose metabolism and blood pressure we decided to use 10% D 0 as an 2 antihypertensive agent to try to determine the relationship between elevated blood pressure and insulin resistance. 1.4.2.  Enalapril  Enalapril is an angiotensin-converting enzyme (ACE) inhibitor and is one of the drugs currently available for the treatment of hypertension (65).  It is a  “prodrug” and is converted into the active compound enalaprilat by the liver. The production of angiotensin II (Ang II) is prevented by enalapril, thus preventing the pressor action of Ang II on the arteriolar smooth muscle (66). There is a decrease in arteriolar resistance and arteriolar pressure. There is also a decreased production of aldosterone because of the lack of Ang II action to increase aldosterone secretion.  The lack of aldosterone prevents sodium  retention in the renal tubule which, along with the lack of arteriolar constriction, causes a decrease in blood pressure (66). Enalaprilat, once converted from enalapril, has a greater affinity for the angiotensin-converting enzyme than its predecessor, captopril (67). It is rapidly absorbed from the gastrointestinal tract and reaches peak serum concentrations in about one hour.  Its half-life is approximately 11 hours, which is more than  twice as long as the half-life of captopril.  Because of the time needed for  hydrolysis by the liver to convert it to its active form, the onset of action of  14 enalapril is slow (two to four hours). The excretion of enalapril and enalaprilat is unchanged in the urine. The efficacy of ACE-inhibitors in hypertensive patients has been well documented (65-67). The antihypertensive effects of this class of drugs are also seen in the spontaneously hypertensive rat (68-70). ACE-inhibitor treatment in young SHR for 4 weeks was sufficient to prevent the full expression of genetic hypertension (68).  As in humans, ACE-inhibitors exert their antihypertensive  effect in SHR by blocking the renin-angiotensin system (69). Enalapril, at a dose of approximately 25 mg/kg/day in the drinking water, significantly reduced mean arterial pressure in the SHR compared to the control group receiving normal tap water (70). Therefore, enalapril was used in our experiments to compare it’s antihypertensive effects to the antihypertensive effects of deuterium oxide.  1.4.3.  Metformin  Metformin is an oral hypoglycemic agent, widely used in Europe and Canada for the treatment of NIDDM. It is composed of two guanidine molecules that are linked together with the elimination of an amino group, thus it is in the drug class of the biguanides with other agents such as buformin and phenformin (79). Metformin is not metabolized. Its absorption is slow (approx. 6 hours) and it is excreted in the urine at a renal clearance rate of about 450 mLlmin. Mefformin has a rapid elimination in humans, with a half-life between 1.7 and 3 hours and plasma concentration at a steady state ranges from about I to 2  i  g/mL (80). Metformin differs from sulfonylureas in a number of various respects. It does not undergo biotransformation and is not bound to plasma proteins. It is eliminated solely by the kidney and rarely cauSes hypoglycemia. Generally, it does not cause weight gain. The doses of metformin given range from 500 to 1000 mg, up to three times a day and it is usually given with meals (80).  15 The mechanism of action of metformin is not completely understood. However, studies show that it does not stimulate the release of insulin (81-85) as do the sulfonylureas.  Metformin has been shown to reduce basal hepatic  glucose production and improve oral glucose tolerance without increasing glucose uptake in patients with NIDDM (80).  It also improves insulin-induced  whole-body glucose uptake in these patients. Metformin causes a decrease in fasting blood glucose, insulin and C-peptide concentrations in plasma (86).  It  also increases insulin action at the cellular level (83) without raising the plasma insulin concentrations (82). A number of studies have attempted to elucidate the mechanism of action of metformin. One suggestion is that metformin’s action is due to a post receptor event that causes glucose lowering and that the effects of metformin on insulin binding are indirect (87). It is also postulated that the basis for the hypoglycemic effect of metformin is at the level of the skeletal muscle, where it increases glucose transport across the cell membrane (88). In muscle cells, mefformin has been shown to stimulate specific glucose transporters; GLUTI and GLUT4 (8890).  Metformin has also been shown to increase insulin stimulated glucose  transport by potentiating GLUTI and GLUT4 transporters in the plasma membrane in rat adipocytes (89).  It has been suggested that the increased  glucose uptake caused by metformin results from an increase in glucose transporter number without the need to invoke a modification of intrinsic transporter activity (90).  These results suggest that metformin stimulates  glucose transport in muscle cells independently of insulin.  Therefore, insulin  and metformin may be exerting their effects through different subcellular pathways (90).  Although the effects of metformin on glucose transporters  enhances our knowledge of its mechanism of action, it is still not fully understood how metformin lowers plasma glucose.  16 Due to its ability to lower plasma glucose levels without increasing insulin levels, metformin has been used to study the relationship between insulin resistance and hypertension. The effect of metformin on blood pressure and metabolism was studied by Landin, et at. in nine non-obese men with hypertension to try to determine the role of insulin resistance (91). They showed that metformin treatment (30 mg/kg/day) significantly lowered blood pressure after six weeks. Two months after the removal of the drug the blood pressure increased, suggesting insulin resistance plays a role in the etiology of hypertension. Spontaneously hypertensive rats were also treated with metformin (200-250 mg/kg/day intraperitoneal) and a significant reduction in MAP (control; 142±6, metformin; 125±4) was observed after seven days (92). Therefore, we decided to examine these effects of metformin in the SHR for an extended treatment period (10 weeks). Given the usual dose of metformin in patients is 30 mg/kg/day, four doses at 10, 30, 100, and 300 mg/kg/day, were used to study the effects of chronic metformin treatment in SHR.  1.5.  Objectives  To try to elucidate the relationship between insulin resistance and hypertension using the spontaneously hypertensive rat by studying agents which primarily lower blood pressure (deuterium oxide, enalaprit) and possibly have an effect on glucose metabolism and an agent which primarily lowers plasma glucose levels (metformin) and possibly has an effect on blood pressure.  If  insulin resistance and hypertension are causally related one would expect that by pharmacologically altering one of the abnormalities a similar direction and magnitude of effect would occur in the other.  2.  METHODS 2.1.  Deuterium oxide and Enalapril  17 Animals:  Twenty-four male spontaneously hypertensive rats (SHR) were obtained from Charles River Canada (200-220g).  These rats were maintained on a  12/1 2h light/dark cycle and food and water were available ad libitum. For one week prior to experimental onset the rats were acclimatized to restraining tubes for subsequent blood pressure measurement via tail cuff (approximately 15 mm ./day). Experimental Setup: At eight weeks of age  ( 200g),  the 24 male SHR were randomly divided  into three groups: control, 10% 2 D 0 , and 50 mg/L enalapril (n=8).  Drug  treatment was given through the drinking water a by single-blind experimental protocol.  Water bottles were filled and coded by an individual who was not  involved in any of the measurements. In addition all plasma analyses were done on coded samples.  During the six week treatment period body weight, urine  volume, and water intake were recorded weekly. Blood samples were taken via the tail and systolic blood pressure was measured by tail cuff weekly. Systolic blood pressure was taken in the morning (09:00  -  12:00) and all blood samples  were also taken in the morning following a 12-14 hour fast. After the six week treatment period direct blood pressure was measured by iliac artery cannulation under pentobarbital anesthesia (0.1 mg/I OOg). An intracardiac blood sample was obtained for plasma biochemistry prior to sacrifice. Measurements/Analysis: Weekly blood samples (O.5mL) were collected by loosely wrapping each rat in a towel, to restrict movement, with their tail exposed. Approximately 1mm of the tail tip was cut-off to allow for bleeding.  Blood was collected in I.OmL  eppindorf tubes which were coated with heparin. The tail tip was subsequently submerged into a 3% hydrogen peroxide solution for antiseptic purposes. Blood  18 samples were then centrifuged for 10-15 mm. and the plasma removed and transferred to a second tube and stored at -20°C for future use. Fasting plasma samples were analyzed for insulin (lmmuncorp Inc.), glucose (Sigma Co.), and triglycerides (Sigma Co.) with diagnostic kits.  The  ratio of insulin to glucose (mU:mmol) was used from each rat as an indicator of insulin resistance. The SHR were housed overnight (12-14h) in metabolic cages for measurement of water intake and urine output. Urine samples were analyzed for 0 content with a single-beam infrared spectrometer (MIRAN I FF, The 2 D Foxboro Co.) and for sodium and potassium levels with a flame photometer. Systolic blood pressure was recorded indirectly from the tail artery using a pneumatic pulse transducer (Narco Bio Systems Inc.).  Blood pressure was  recorded as an average of three measurements. Cannulae were inserted into the iliac artery while rats were under pentobarbital anesthesia. Systolic and diastolic pressure were recorded after a 10 minute wait to allow for pressure stabilization.  2.2.  Metformin  Animals: Twenty-four male spontaneously hypertensive rats (SHR) were obtained from Charles River Canada (200-220g).  These rats were maintained on a  12112h light/dark cycle and food and water were available ad libitum. For one week prior to experimental onset the rats were acclimatized to restraining tubes for subsequent blood pressure measurement via tail cuff (approximately 15 min./day). Experimental Setup: Preliminary experiments with 10 mg/kg/day metformin followed the same experimental protocol as below.  19 At eight weeks of age  ( 200g), the 24 male SHR were randomly divided  into four groups: control, 30 mglkg/day, 100 mglkg/day and 300 mg/kg/day metformin (n=6). Drug treatment was given through the drinking water. During the 10 week treatment period body weight, urine volume, and water intake were recorded weekly.  Blood samples were taken via the tail and systolic blood  pressure was measured by tail cuff weekly. Systolic blood pressure was taken in the morning (09:00  -  12:00) and all blood samples were taken in the morning  following a 12-14 hour fast. After the 10 week treatment period direct blood pressure was measured in concious rats by an iliac artery cannula (see below). This technique was used to eliminate the effect of the anesthetic on blood pressure as was observed in the first experiment. Measurement/Analysis: Weekly blood samples (0.5mL) were collected by loosely wrapping each rat in a towel, to restrict movement, with their tail exposed. Approximately 1 mm of the tail tip was cut-off to allow for bleeding.  Blood was collected in 1.OmL  eppindorf tubes which were coated with heparin. The tail tip was subsequently submerged into a 3% hydrogen peroxide solution for antiseptic purposes. Blood samples were then centrifuged for 10-15 mm. and the plasma removed and transferred to a second tube and stored at -20°C for future use. Fasting plasma samples were analyzed for insulin as described in 2.1. The SHR were housed overnight (12-14h) in metabolic cages for the measurement of water intake and urine output. Systolic blood pressure was recorded indirectly from the tail artery using a pneumatic pulse transducer (Narco Bio Systems Inc.).  Blood pressure was  recorded as an average of three measurements. For direct blood pressure measurements, cannulae were inserted into the iliac artery while rats were under halothane anesthesia.  Two small incisions  20 were made, one in the back of the neck between the ears and the other in the thigh region above the iliac artery. A cannula was then run subcutaneously from the neck to the thigh region. The iliac artery was exposed and cannulated. Both incisions were sutured and the rats were placed back in their cages. After a 24 hour recovery period, direct systolic and diastolic pressures were recorded. The protruding cannula at the neck was attached to a Grass transducer and direct blood pressure was recorded after a 10 minutes wait to allow for pressure stabilization.  After the direct blood pressure was recorded the rats were  reanesthetized and the chest wall retracted to expose the heart for an intracardiac puncture 2.3.  ( 2mL of plasma was collected).  Statistics  A one-way ANOVA and an unpaired two tailed student’s t-test were used to compare direct blood pressure between the groups. A repeated measures ANOVA with Duncan’s multiple range test was used to compare the weekly differences in body weight, urine volume, water intake, insulin, glucose, triglycerides, and indirect systolic blood pressures. P <0.05 was accepted as a significant difference and all results are recorded as mean ± S.E.M.  All  experimental methods were pre approved by the Animal Care Committee of U.B.C.  3.  RESULTS 3.1.  Deuterium oxide and Enalapril  Body weight was not significantly different between the three groups, although there was a significant increase in weight during the six treatment weeks (p <0.05, figure 1). Systolic blood pressure (mmHg) rose in the control group from I 39±2 to I 54±10 and this increase in blood pressure was not significantly prevented by 10% D 0 (132±4 to 161±5) but was prevented by 2  21 enalapril (127±6 to 113±6, p  <  0.05, figure 2).  The direct blood pressure  (mmHg) measurements by iliac artery cannulation also confirm that the systolic and diastolic blood pressure of the enalapril group (133±2/92±4) was significantly lower than both the control (156±11/107±11) and 10% D 0 groups 2 (144±8/97±6) (figure 3). The enalapril group had significantly higher 12 hour water intake (44±3 mL) compared to the control (20±3 mL) and the 10% D 0 (17±4 mL) groups 2 (figure 4).  Urine output (mL) was also significantly higher over the six week  treatment period in the enalapril (34±1) compared to the control (16±2) and 10% 0 (13±1) groups (figure 5). There were no significant differences in weekly 2 D urine potassium excretion (mmol/l2hrs, p  <  0.05, figure 6) between the three  groups whereas, the urine sodium (mmol/l2hrs) was significantly lower in the 10% D 0 group (0.24±0.01) compared to both the control (0.36±0.02) and 2 enalapril (0.42±0.06) groups (p <0.05, figure 7). The measurement of D 0 in 2 the urine by single-beam infrared spectrometry demonstrated a gradual increase in the D 0 level until it reached a plateau of 8% at 11 weeks of age (figure 8). 2 The glucose measurements (mmol/L) taken during the 7 week treatment period showed no significant difference between the control (7.8±0.2) group and the treatment groups (figure 9). However, the 10% D 0 group and the enalapril 2 group were significantly different from one another, 7.2±0.2 and 8.2±0.2, respectively. The triglyceride levels (mmol/L) were significantly decreased in the 10% D20 (0.43±0.04) and enalapril groups (0.44±0.03) as opposed to the control group (0.51±0.05) (figure 10).  There was no significant difference in  insulin levels (mU/L) between the control (62.6±6.4), 10% D20 (56.3±4.9), or enalapril (52.7±3.8) groups (figure 11).  The insulin:glucose (mU:mmol) ratio  also showed no significant difference between the groups (control, 7.2±0.6; 10% D20, 7.0±0.3; enalapril, 6.1±0.3, figure 12).  22 3.2.  Metformin Experiment  Preliminary experiments using 10 mg/kg/day metformin showed no significant difference in body weight, systolic blood pressure, water intake, urine volume, fasting plasma insulin, fasting plasma glucose, fasting plasma triglycerides, nor in the insulin glucose ratio between the control and treated groups. These results led to choosing a higher dose range (30, 100 and 300 mg/kg/day metformin) to determine the dose-response relationship of mefformin in lowering blood pressure and increasing insulin sensitivity in the SHR Body weight was not significantly different between the four groups (control, 30, 100, and 300 mg/kg/day metformin, p  <  0.05) during the 10 week  treatment period, although there was a significant increase in weight over the duration of the experiment (figure 13).  The systolic blood pressure was not  significantly different between the control (156±3) and treatment groups (154±3, 158±4, 160±4; 30 mg/kg/day, 100 mg/kg/day, and 300 mg/kg/day, respectively) throughout metformin treatment (figure 14).  The direct blood pressure  measurements taken at week 11 showed a significant decrease in systolic and diastolic blood pressure in the 300 mg/kg/day metformin treated group (166± 9/98±11) compared to the control (185±8/117±8) and other two treatment groups (192±7/122±4, 198±5/128±8; 30 and 100 mg/kglday, respectively) (figure 15, Table 1). The control group was not significantly different from the treated groups in 24 hour water intake (p  >  0.05, figure 16).  displayed no significant difference (p  >  The 24 hour urine volume also  0.05) between the control and treatment  groups (figure 17). Fasting plasma glucose (mmol/L) was monitored throughout the 10 week metformin treatment and indicated no significant difference between the control (5.7±0.2) and treatment groups (5.6±0.2, 5.7±0.2, 5.7±0.1, 30, 100 and 300  23 mg/kg/day metformin, respectively, figure 18). The final fasting glucose levels at week 11 showed that the treatment group receiving 300 mg/kg/day of metformin (9.0±1.5) had significantly higher fasting glucose levels than the control group (6.8±0.4) whereas the 30 and 100 mg/kg/day metformin treated groups (7.1±0.8 and 6.6±0.7, respectively) were not significantly different from control (Table 1). Measurement of the fasting plasma triglycerides (mmol/L) showed no significant difference between the four groups (0.56±0.02, 0.59±0.02, 0.51±0.02, 0.5±0.02, control, 30, 100, 300 mg/kg/day metformin, respectively, p  >  0.05, figure 19).  This was also verified by the final plasma analysis for triglycerides which also showed no significant difference between the control and treatment groups (Table 1). Fasting plasma insulin levels (mU/L) between the control (93±6) and treatment groups (133±29, 89±3, 138±27, 30, 100, 300 mg/kg/day metformin respectively, p  >  0.05) were also not significantly different from one another  (figure 20). However, the final plasma samples show that the group treated with 300 mg/kg/day metformin had significantly higher plasma insulin levels than the other three groups (Table 1). The insulin:glucose ratio (mU/mmol) between the four groups was not significantly different during the 10 week metformin treatment (figure 21). However, the final plasma samples show that the group treated with 300 mg/kg/day metformin had a significantly higher insulin:glucose ratio than the control and other two treatment groups (Table 1).  24 Figure 1.  Body weights (g) measured weekly.  There was no significant  difference between the control, 10% 2 D 0 , and 5Omg/L enalapril groups (p 0.05). Values are means ± S.E.M., n  =  8 for each group.  >  25  Weight (g) F3 C.n 0  -  01  o  0 0  0  0 0  (1,  0 1  -n C)  0  —I’ I  \ I  I-I I I  \  -‘  CD CD  -  o  c I  Cl,  .-  oJ 0  m  CD C) —  I  26 Figure 2.  Indirect systolic blood pressures (mmHg) measured weekly via tail  cuff. There is no significant difference between control and 10% 2 D 0 , while the enalapril group is significantly lower than both groups. S.E.M., n  =  8 for each group.  control and 10% 2 D 0 .  *  Values are means ±  indicates a significant difference (p  <  0.05) from  (N  a)  —  2 2  200  150  100  50 0  *  2  --—- —-  3  10% D20  Treatment (weeks)  4  Enalapnl  6  a  ————  7  *  •1  Figure 2. Systolic Blood Pressure  1  Control  Figure 3. Direct blood pressure (mmHg) recordings after six weeks of treatme,? There is no significant difference in systolic pressure (top of bar) between control and 10% 2 D 0 , while enalapril is significantly different from both groups (p  <  0.05). There is a significant difference in diastolic pressure (bottom of bar)  between control and enalapril. group. D 0 2 .  *  Values are means ± S.E.M., n  =  8 for each  indicates a significant difference (p <0.05) from the control and 10%  indicates a significant difference from control.  29  Blood pressure (mmHg) 0  r)  Ui  Ui 0  -  Ui  C) 0 z  0 0  Ui  •  Ui 0  -  Ui  HH CD  c) CD CD II  w 0  0 CD 0  0  CD  *  0  30 Figure 4. Twelve hour water intake (mL). No significant difference was found between the control and 10% D 0 groups, while the enalapril group was 2 significantly different from both. Values are means ± S.E.M., n group. D 0 2 .  *  indicates a significant difference (p  <  =  8 for each  0.05) from both control and 10%  1  CW)  z E  Cu  4—  1  *  /  /  ,  *  L a  a  j  *  I  ±  I  /1  I  5  T  I  4  -  I  3  10% 020  Treatment (weeks)  2  -  Enalapril  6  *  __..‘  7  j  *  Figure 4. Twelve Hour Water Intake 60 50 40 30 20 10  0  0  Control  32 Figure 5. Weekly urine volumes (mL) measured over 12 hours. There was no significant difference between the control and 10% D 0 groups, while the 2 enalapril group was significantly different from both. S.E.M., n  =  8 for each group.  both control and 10% 2 D 0 .  *  Values are means ±  indicates a significant difference (p  <  0.05) from  C’)  -J  0  >  .E D  I  *  1  a  — —  *  —  I  2  — • —  ——  -  *  I  3  — —  — ••  —•a  4 Treatment (weeks) 10% D20  *  .1—  I  5  —--—  *  6  Enalapril  ———  —  7  Figure 5. Twelve Hour Urine Volume 50  40  30  20  10  0  0  Control  34 Figure 6.  Urine potassium (mmol/12h) measured weekly multiplied by the 12  hour urine volume. There is no significant difference (p  >  0.05) between the  control, 10% 2 D 0 , and enalapril groups. Values are means ± S.E.M., n each group.  =  8 for  C)  c’J 0  E E E •0 U)  0  0  1.50  i.oo  0.50  0.00 0  -  5  ‘‘‘  Enalapril  6  I  —I  Figure 6. Urine Potassium  I  •  I  4  /  I  3  •  I  2  10% 020  Treatment (weeks)  1  Control  7  36 Figure 7. Urine sodium (mmoWl2h) measured weekly multiplied by the 12 hour urine volume. There is no significant difference (p  >  0.05) between the control  and enalapril treated group. The 10% D 0 group has significantly lower sodium 2 than both control and enalapril groups. Values are means ± S.E.M., n each group. (p <0.05).  *  =  8 for  indicates a significant difference from control and enalapril groups  (V)  C\I •1  I  0.75  0 25  0.00 0  -  I  2  I  /  :‘  /  T —r  F  I.  I  ——  3  —  -  ____  —  I  ——  I  6  *  I  5  —  Enalapril  4  10% D20  Treatment (weeks)  —  Figure 7. Urine Sodium  1  Control  — — —  —.  -  —————  7  38 Figure 8. Percentage of D 0 (v/v) in the urine measured weekly. There is an 2 obvious difference between 10% D 0 (open circles) with control and enalapril 2 (closed circles) where there was no detection of deuterium in the urine. The minimum level of detection with this method is 0.1%. S.E.M., n  =  8 for each group.  Values are means ±  0)  CV)  10—  :  T °  I  Figure 8. Percentage of D20 in Urine  T  Treatment (weeks)  40 Figure 9.  Fasting plasma glucose concentrations (mmol/L) measured weekly  The group treated with 10% D 0 has significantly lower plasma glucose 2 concentrations than the enalapril treated group.  There is no significant  difference between the control group and both treated groups. means ± S.E.M., n  =  8 for each group.  --  Values are  indicates a significant difference  between the 10% D 0 group and the enalapril group (p 2  <  0.05).  1  I  2  —  I  —  I  6  — — — — — — —  I  5  Enalapril  4  10% D20  Treatment (weeks)  3  7  Figure 9. Fasting Plasma Glucose 10-  : 5 0  Control  42 Figure 10. Fasting plasma triglyceride concentrations (mmolIL) measured weekly. Both treatment groups (10% D 0 and ena 1apr11) have significantly lower 2 triglyceride concentrations than the control group. n  =  8 for each group.  *  .  Values are means ± S.E.M.,  indicates a significant difference between the control and  treatment groups (p <0.05).  C.)  “1  .1  E U)  a)  -D  o F-  1  2  _.S__-.--*  1  4  10% D20  Treatment (weeks)  3  I  *  5  Enalapril  6  *  7  *  Figure 10. Fasting Plasma Triglycerides 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0  Control  44 Figure 11.  Fasting plasma insulin concentrations (mU/L) measured weekly.  There is no significant difference in insulin concentration between the control and treatment groups (p group.  >  0.05). Values are means ± S.E.M., n  =  8 for each  U) ‘1  100 90  : :: 40 30 20  1  I  6  I  7  Fasting Plasma Insulin  I  5  //  I  4  z::z; I  3  Treatment (weeks) 10% D20  Enalapril  2  z  Figure 11  0  Control  46 Figure 12.  lnsulin:glucose ratio (mU:mmol) is representative of insulin  resistance in the SHR. There is no significant difference in the insulin:glucose ratio between the control and treatment groups. Values are means ± S.E.M., n 8 for each group.  =  47  Insulin:Glucose (mU:mmol) 0  C.fl  0  0  ‘1  —a  cc  o o  II  —  I  I  CD  I  Ii I, ‘I’  II  I  I  \  I’  H  —  o  II  -‘  ft  I  -  CD  D  II  o  I’ I’ .1  CD CD  a.  It It  (0  C) —  “  ____  _  III  I’I •.11 ‘I  m  Ii  I, II  I  I  C) 0 0  CD  ‘I HI(  0)  H I  —  I  I  I  I  I  —  48 Figure 13. Body weights (g) measured weekly.  There was no significant  difference between the control, 30, 100, and 300 mg/kg/day metformin treated groups (p  >  0.05). Values are means ± S.E.M., n  =  6 for each group.  0)  400  300  200 C  1  —  3  i  4  5  6  _______  Treatment (weeks) Met 30  —  7  __•  Met 100  8  •  9  /  ‘I  10  —  Met 300  Figure 13. Weekly Body Weights  fr•  2  Control  —  11  50 Figure 14.  Indirect systolic blood pressure (mmHg) measured weekly via tail  cuff. There is no significant difference between the control, 30, 100, and 300 mg/kg/day metformin treated groups (p> 0.05). Values are means ± S.E.M., n 6 for each group.  =  i  ‘4)  200  100 C  :,  I  6  I  7  I  8  I  Met 300  9  j_.  Figure 14. Systolic Blood Pressures  I  5  ———  I  4  — —  I  3  ————  I  2  Treatment (weeks) Met 30  Met 100  1  Control  10  52 Figure 15. Direct blood pressure (mmHg) after ten weeks of treatment. There is no significant difference in systolic pressure (top of bar) between control, 30, and 100 mg/kg/day metformin treated groups, while the 300 mg/kg/day metformin treated group was significantly lower than the other groups (p <0.05). The diastolic pressure (bottom of bar) of the 300 mg/kg/day metformin was also significantly lower than the other three groups. Values are means ± S.E.M., n 6 for each group.  *  =  indicates significant difference in systolic pressure (p <0.05).  indicates significant difference in diastolic pressure.  () U)  0)  x E a) I.  U) U)  a) 0 0 0  0  225 200 175 150 125 100 75 50 25 -  L_ 0  J  V/A  Met 30  V)91  MetlOO  *  **  Met300  Figure 15. Direct Blood Pressures  Control  54 Figure 16. Twenty-four hour water intake (mL).  No significant difference was  found between the control and metformin treated groups. The 300 mg/kg/day metformin treated group had significantly lower water intake than the 30 mg/kg/day metformin treated group. Values are means ± S.E.M., n group.  •  indicates a signficant difference (p  mg/kg/day metformin treated groups.  =  6 for each  0.05) between the 30 and 300  U) U)  60  50  40  30 x 20  10  0 C  I  5  I  7  I  8  I  Met 100  6  —  9  Figure 16. 24 H Water Intake  I  4  *  I  3  *  I  2  Treatment (weeks) Met 30  1  Control  ————..  I  *  10  Met 300  —  *  11  56 Figure 17. Weekly urine volumes (mL) measured over 24 hours. There was no significant difference between the control and treatment groups (p  >  0.05). The  300 mglkglday metformin treated group had a significantly lower urine output than the 30 mg/kg/day metformin treated group over the treatment period. Values are means ± S.E.M., n difference (p groups.  ,  =  6 for each group.  *  indicates a signficant  0.05) between the 30 and 300 mg/kg/day metformin treated  ‘0  E  -J  G)  E  D  40  30  20  10  0 C  1%•  *  1  — —  0  *  3  — — —— —  I  5  I  — —  4  Met 30  —  I  —  I  9  *  I  8  -—  7  Met 100  — —  Treatment (weeks)  6  Figure 17. 24 H Urine Volume  *  I  2  Control  I.  %%%  10  Met 300  T  11  58 Figure 18. Fasting plasma glucose concentrations (mmol/L) measured weekly. There is no significant difference (p> 0.05) between the control group and the metformin treated groups, although there is a significant decrease in glucose concentration in all of the groups throughout the treatment period. Values are means ± S.E.M., n  =  6 for each group.  0)  U)  E  8  -  7  I  I  Met 300  9  — - —  8  Figure 18. Fasting Plasma Glucose  I  :  I  I  I  3  I  I  2  Met 100  1  -  S.  7%%..  6  4 C  Met 30  4 5 6 Treatment (weeks) Control  /  10  60 Figure 19.  Fasting plasma triglyceride concentrations (mmolIL) measured  weekly. No significant difference (p> 0.05) is observed between the control and treatment groups. Values are means ± S.E.M., n  =  6 for each group.  CD  -J  E  1.00 0.90 0.80 0.70 -  -.  -  Figure 19. Fasting Plasma Triglycerides  1  I  2  3  4  I  5  I  Met 100  6 Treatment (weeks) Met 30  7  8  —  Met 300  9  I  O6O  0.30 0.20 0.10 0.00 C  Control  10  62 Figure 20.  Fasting plasma insulin concentrations (mU/L) measured weekly.  There is no significant difference (p  >  0.05) in insulin concentrations between  the control and treatment groups. Values are means ± S.E.M., n group.  =  6 for each  (.3 (0  —I  D  c (0  200  160  120  80  4:  0 C  -  -  /  I  /  / / I.  /  3  -  -  —-1k “-——  \:  — a  —  I  —  —  7  I  — —a—  Met 100  4 5 6 Treatment (weeks) Met 30  8  S  I  I  I  10  /  9  Met 300  Figure 20. Fasting Plasma Insulin  I  2  IV.. I II_  1  Contro’  64 Figure 21.  lnsulin:Glucose ratio (mU/mmol) is representative of insulin  resistance in the SHR.  There is no significant difference (p  >  0.05) in the  insuliri:glucose ratio between the control and treatment groups.  Values are  means ± S.E.M., n  =  6 for each group.  LK) CD  40 35  15  20  25  30 E D  a)  U)  § -  ci)  10: 5 0 C  -  -  , /  I  1  /  ‘  2  /  /  /  -  -  ‘ ‘  —  I  -  I  4  I  3  .  ,‘ : :  5  —  - —  I  Met 100  6 Treatment (weeks) Met 30  7  •b__  8  — — - —  I  4.  9  I  ‘—  •  ——  Met 300  Figure 21. Insulin:Glucose Ratio  ——  Control  10  66 Table 1. Body weight, metabolic data, and blood pressure in the SHR at week 11 (final week) of metformin treatment.  67 Table 1. Body weight, metabolic data and blood pressures in the SHR at 11 weeks of metformin treatment Control Metformin Metformin Metformin 30mg/kg/day (n = 6) 100mg/kg/day 300mglkglday (n=6) (n=6) (n=6) Body weight (g) 359.7 ± 3.3 359 ± 7.8 359.7 ± 6.4 337.7 ± 6.5 * Fasting plasma 24.2 ± 3.2 23.4 ± 2.9 20.1 ± 2.3 39.0 ± 14.7t insulin (mUlL) Fasting plasma 6.8±0.4 7.1 ±0.8 6.6±0.7 9.0±1.5t glucose (mmollL) lnsulin:glucose 3.5 ± 0.3 3.3 ± 0.3 3.1 ± 0.3 5.0 ± 2.5t ratio (mU/mmol) Fasting plasma 0.45 ± 0.05 0.34 ± 0.06 0.39 ± 0.03 0.45 ± 0.09 triglyceride (mmoLfL) Systolic blood 185.3 ± 8.3 192.3 ± 6.9 198.4 ± 5.1 166.3 ± 9.9t pressure (mmHg) Diastolic blood 117.2 ± 7.8 122.0 ± 4.3 128.1 ± 7.6 97.7 ± 10.5t pressure (mmHg) 24h Water intake 40.5 ± 1.6 47.0 ± 3.5 38.5 ± 3.8t 36.7 ± 6.8t (mL) 24h Urine volume 23.3 ± 1.2 29.2 ± 2.5 21.8 ± 2.4t 20.0 ± 5.5t (mL) Mean values ± S.E.M. are shown t* significantly different from control (p < 0.05) significantly different from metformin 30 and metformin 100 (p < 0.05)  68 4.  DISCUSSION 4. 1.  Deuterium oxide and Enalapnl  Results from the deuterium oxide and enalapril study show that enalapril significantly lowers the systolic blood pressure (fig. 2) over the six week treatment period in the SHR. This was verified by the direct blood pressure measurement (fig. 3), which also showed a significant decrease in the systolic and diastolic blood pressure in the enalapril treated group compared to the control group. However, the insulin and glucose concentrations (fig. 11) were not significantly different from the control group, demonstrating that lowering the blood pressure in SHR does not have an appreciable effect on insulin resistance in this model. The measurement of insulin and glucose concentrations has previosusly been done by euglycemic clamping in rats (25, 43). The clamping technique measures insulin and glucose concentrations over a short period in the concious rat (approximately 180 mins.). In our experiment we used repeated measures of insulin and glucose concentrations, throughout the treatment period (weekly). The euglycemic clamp is a more sensitive method than the repeated measures but with repeated measures the ability to detect small changes in insulin requirements should be increased over time. The results above are in contrast earlier publications that suggest ACE inhibitors improve insulin resistance in hypertensive patients (71, 72, 93). Hypertensive patients were treated with captopril and measured for insulin promoted glucose uptake (71).  Insulin and glucose concentrations were  significantly reduced and insulin sensitivity was improved by 11 % with captopril treatment.  Similar results were seen in aged insulin-resistant hypertensive  patients who were treated with five different ACE-inhibitors: captopril, enalapril, quinapril, ramipril, and lisinopril (93).  69 It has been suggested that the potassium sparing (hyperkalemia) effect of ACE-inhibitors may play a role in improving insulin sensitivity.  Hyperkalemia  has been implicated in patients with renal failure who are being treated with ACE-inhibitors, due to decreased renal potassium clearance via reduced aldosterone levels (74, 75).  However, Scandling, et al. have shown that  treatment with enalapril does not acutely impair extrarenal  potassium  homeostasis in men with normal renal function (76). It has also been suggested that ACE-inhibitors induce better overall potassium conservation under everyday life conditions (67). Increases in potassium concentration have been shown to potentiate insulin release from the pancreas in rats (77).  Dietary-induced  potassium deficiency reduced insulin secretion in response to sustained hyperglycemia in healthy subjects (78). Thus, the potassium sparing effect of ACE-inhibitors may exert a positive influence on glucose tolerance, in combination with their known antihypertensive actions. Our results are supported by the view of Reaven and Chang, who suggest that hypertension per se does not cause insulin resistance in the SHR (94). Swislocki, et al. have also demonstrated that decreasing blood pressure in hypertensive patients does not necessarily affect abnormal insulin and glucose metabolism (95). Santoro, et al. showed that chronic ACE-inhibition does not interfere with insulin’s effect on glucose uptake (96), suggesting that ACE-inhibitors do not affect insulin resistance. Furthermore, in a comparison study between enalapril and captopril on insulin sensitivity in normotensive individuals, both ACE inhibitors caused an increase in fasting insulin concentrations (97); the opposite of what one would expect if insulin resistance was decreased. Non-obese, noninsulin-resistant patients with mi Id-to-moderate hypertension, treated with enalapril, had significantly lower blood pressure but there was no effect on  70 glucose tolerance (98). This study was performed with Japanese patients which may only be indicative of ethnic differences, however, the results suggest that ACE-inhibitors may improve insulin sensitivity only in insulin-resistant patients. Glucose intolerance or hyperglycemia is one of the abnormalities linked to Syndrome X. If hypertension directly causes insulin resistance we would expect to see a decrease in insulin and/or a decrease in glucose when the blood pressure is lowered. Insulin and glucose concentration, as a marker of insulin resistance, showed no significant difference between the three groups, suggesting that enalapril had no effect on the metabolic factors in the SHR (fig. 12). Therefore, we can conclude that 1) enalapril and D 0 do not have an effect 2 on insulin metabolism or 2) the SHR does not have insulin or glucose abnormalities associated with elevated blood pressure. Ten percent D 0 caused a decrease in fasting plasma glucose but no 2 change in fasting plasma insulin.  Despite this, the 10% D 0 group failed to 2  show a significant decrease in systolic blood pressure when compared to the enalapril treated and control groups (fig 2). This was confirmed by direct blood pressure measurements which were not significantly different in the 10% D 0 2 treated group when compared to the control (fig. 3). One possible explanation for these observations is a decrease in insulin resistance. These results do not support a causal link between insulin resistance and blood pressure. Our results are in contrast to the results published by Vasdev, et al. who demonstrated that 10% D 0 was effective in preventing the elevation of blood 2 pressure in SHR (64).  In both experiments the systolic blood pressure was  measured indirectly by a tail cuff method and each pressure value was an average of 3-4 recordings.  The only obvious difference was that in our  experiments the blood pressures were measured by an observer who was blinded as to the treatment.  71 We confirmed that 10% D 0 was being administered to the D 2 0 treated 2 group by measuring D 0 in the urine (fig. 8). The urine analysis, by infrared 2 spectrometer, for D 0 showed an 8% recovery of 2 2 D 0 , thus demonstrating that 0 was being ingested and equilibrating with total body water in the rats. The 2 D fact that it didn’t achieve 10% may be due to limitations in the method of detection and/or a contribution of water from food plus metabolism. As well as having a significant decrease in systolic blood pressure the enalapril treated group displayed a significantly higher water intake than the 0 groups (fig. 4). Result reported by McLennan, et al. (99) 2 control or 10% D demonstrated that SHR treated with 25 mg/kglday of enalapril drank significantly more than the untreated controls. This increase in water intake due to chronic converting enzyme inhibition has also been reported by Ferrone, et al. (100). The significant increase in urine volume is consistent with the higher water intake observed in the enalapril group (fig. 5) but it is not clear which is the primary event. The increased water intake may be secondary to increased urine volume. Enalapril prevents the conversion of angiotensin I to angiotensin II, by inhibiting the angiotensin converting enzyme (101). Angiotensin II is the primary regulator of aldosterone secretion and aldosterone secretion is known to stimulate sodium and water reabsorption (101).  Therefore, enalapril may  prevent sodium and water reabsorption in the SHR by inhibiting aldosterone action. This lack of reabsorption may lead to a primary increase in urine volume followed by an increase in water intake to maintain water homeostasis in the body. The mechanism of this effect has yet to be elucidated. The above experiment and other documented results (94) strongly suggest that lowering blood pressure in animals with hypertension and insulin resistance has no effect on elevated insulin concentrations and glucose intolerance. Similar results have been shown in human hypertensive patients  72 (95, 96). resistance.  It is therefore unlikely that increased blood pressure causes insulin In order to test whether insulin resistance causes hypertension,  SHR were treated with metformin.  4.2.  Mefformin Experiment  This second experiment examined if insulin resistance was the cause of hypertension by altering the blood glucose concentrations with metformin. Kaplan suggests that maneuvers that reduce hyperinsulinemia or improve insulin sensitivity may lower blood pressure (73). The dosing range used in this experiment was chosen to cover the normal dose of metformin given to patients (30 mg/kg/day, 82, 102) as well as supermaximal doses (100 and 300 mg/kg/day) given to SHR in previous studies (92). Over the ten week treatment period the systolic blood pressure was not significantly lowered by any dose of metformin (fig. 14). These results are in contrast to Morgan, et al. who showed a significant decrease in mean arterial pressure in SHR treated with 200-250 mg/kg/day metformin, intraperitoneal (92). In both methods, the measurement of blood pressure was done when the rats were conscious. The difference occurred in the length of metformin treatment; Morgan’s experiment was acute (7 days on metformin) whereas our experiment was chronic (10 weeks).  It is interesting to note that hypertensive patients  receiving 30 mg/kglday metformin have a significant decrease in blood pressure over a 6 week treatment period (91, 102) but in our experiments doses of 30, I 00, and 300 mg/kg/day metformin failed to reduce the blood pressure in the SHR. The reduction in direct blood pressure in the 300 mg/kg/day metformin treated group may be a manifestation of an interaction between the drug and the additional stress on the SHR due to the cannulation surgery prior to blood pressure measurement. It may also be due to an interaction between metformin and halothane because only 60-80% of absorbed halothane is eliminated  73 unchanged in the first twenty-four hours after inhalation (103). Therefore, the reduction in blood pressure seen in the higher doses of metformin is unlikely to represent a specific pharmacologic effect of the drug. It has been shown that mefformin enhances the basal rate of glucose transport (104) thus decreasing blood glucose concentrations in humans (91) at doses of approximately 30 mg/kg/day. Our results show no significant difference in fasting blood glucose (fig. 18), fasting blood insulin (fig. 20) concentrations, or insulin/glucose ratios (fig. 21) in the SHR over the dosing range. These results show that metformin has no effect on glucose homeostasis in this model. Explanations for this lack of effect include: 1) metformin does not have an effect on improving glucose concentration in the rat, 2) metformin does not have an effect in animals that do not express a diabetic state; i.e., SHR do not have insulin resistance, 3) our methods were not sensitive enough to detect an effect. The final blood samples taken at week 11 show significantly higher fasting glucose and insulin concentrations in the 300 mg/kg/day metformiri group compared to the control group. This effect is opposite to the expected effect of metformin, as discussed above. One of the reasons for this discrepancy may be due to the added effect of the high dose of the drug plus the invasive procedure performed on the rats prior to blood sampling. Rao suggests that stress from blood loss may be a source of error in the evaluation of glucose turnover and insulin sensitivity (105). Another explanation may be an interaction of the drug and anesthetic on glucose and insulin concentrations. It can be concluded that the elevated concentrations of glucose and insulin in the final plasma samples are most likely toxicological effects of the metformin treatment causing stress, interacting with the invasive procedure or anesthetic agent. 4.3.  Is the SHR an Effective Model of Insulin Resistance?  74 Although we failed to show an effect on insulin resistance as a result of blood pressure reduction and increased insulin sensitivity in the SHR, there is conflicting evidence as to whether this model accurately represents the insulin resistant state as seen in human patients (45, 46). As previously mentioned, the SHR has been described as an appropriate experimental model for both hypertension and insulin resistance (41, 42, 43). These studies suggest a link between high blood pressure and high insulin levels in this rat model. However, Horl, et al. showed no evidence of peripheral insulin resistance between SHR and WKY rats from the response of skeletal muscle to insulin.  After a 12h  fasting period, muscle glucagon and glucose levels were almost identical for the two groups of rats (46). These results suggest no difference between SHR and its control strain, which further suggests that the SHR is not insulin resistant because the WKY does not display insulin resistance. Furthermore, Buchanan, et al. used glucose clamp studies to show that insulin stimulated glucose uptake was not different between age-matched SHR and WKY rats (45). This evidence was seen in the SHR when their systolic blood pressure was significantly elevated, compared to the WKY. Therefore, the SHR may not be an effective model for studying the relationship between hypertension and insulin resistance as seen in human patients. It is of interest to note that there are also inconsistencies in the association between insulin resistance and hypertension in human subjects. O’Brien, et al. looked at patients with insulinoma to determine if hyperinsulinemia contributed to the pathogenesis of hypertension in the absence of insulin resistance (106). They showed no significant difference in systolic and diastolic blood pressure between the control patients and patients with insulinoma. The prevalence of hypertension was also similar between patients with insulinoma  75 and matched controls.  It was concluded that hyperinsulinemia could not be  implicated in the genesis of hypertension. 4.4.  Conclusions  1. Ten % 020 decreases fasting plasma glucose concentrations, thus possible causing a decrease in insulin resistance. 2. Despite this, chronic 10% D 0 has no effect on blood pressure or fasting 2 plasma insulin concentration in the SHR. 3. This suggests that insulin resistance does not cause increased blood pressure. 4. Enalapril decreases blood pressure but has no effects on glucose and insulin concentrations in the SHR, confirming that high blood pressure does not cause insulin resistance. 5. Enalapril causes a large increase in urine volume and water in the SHR. 6. 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