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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 Effectsof Deutenum oxide, Enalapril, and MetforminBySharon MaxwellB.Sc. (Hons.), Queen’s University, 1991A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMasters of ScienceinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF PHARMACOLOGY & THERAPEUTICSFACULTY OF MEDICINEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASeptember, 1994© Sharon Maxwell, 1994In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Tht-\ OcCDOcjThe University of British ColumbiaVancouver, CanadaDate IDE-6 (2/88)IIABSTRACTElevated blood pressure has been recognized as a marker for disease since theearly I 800s. It is commonly divided into two categories: primary hypertension andsecondary hypertension. Primary hypertension is defined as hypertension whereinheritable and/or environmental factors are unknown whereas, secondaryhypertension is defined as hypertension caused by a known congenital or acquireddisease. Primary hypertension is discussed in this paper.In an attempt to better understand the pathophysiology of hypertension, acommon syndrome in patients was described called Syndrome X. Patients with thissyndrome have resistance to insulin-stimulated glucose uptake, glucose intolerance,hyperinsul inemia, increased very-low-density lipoprotein triglyceride, decreased high-density lipoprotein cholesterol, and hypertension. In an attempt to better understandthe relationship between elevated insulin concentrations (hyperinsulinemia) andelevated blood pressure, experiments were designed using spontaneouslyhypertensive rats (SHR) as a genetic model of hypertension. Agents which lower bloodpressure (deuterium oxide and enalapril) and an agent which lowers plasma glucoseconcentrations were used to try to elucidate the relationship between insulin resistanceand hypertension. If insulin resistance and hypertension are causally related onewould expect that by pharmacologically altering one of the abnormalities a similardirection and magnitude of effect would occur in the other.Two experiments were performed. The first experiment examined the effects of10% 020 and 50 mg/L enalapril on hemodynamic and metabolic factors in the SHR.IIIThe second experiment examined a dose range (10, 30, 100, and 300 mg/kg/day) ofmetformin in SHR and its effects on hemodynamic and metabolic factors. In bothexperiments body weight, systolic blood pressure, insulin, glucose and triglycerideconcentrations in plasma, water intake, and urine volume were recorded weekly. At theend of each experiment direct blood pressures were recorded from the iliac artery.In the D20 and enalapril experiment, enalapril significantly lowered the systolicpressure compared to the control and 10% D20 groups. There was no significantdifference in the insulin (mU/L) or glucose (mmol/L) concentrations between the threegroups and the insulin:glucose ratio (mU/mmoL) was not significantly different betweenthe groups. These results suggest that there is no effect on insulin or glucoseconcentrations when the blood pressure is lowered in the SHR.In the metformin experiment, metformin did not significantly lower the systolicblood pressure during the treatment period. There was also no significant difference infasting plasma insulin and glucose concentrations. The insulin:glucose ratio alsoshowed no significant difference between the groups.Conclusions:1. Ten % D20 decreases fasting plasma glucose concentrations, thus possiblecausing a decrease in insulin resistance.2. Despite this, chronic 10% D20 has no effect on blood pressure or fasting plasmainsulin concentration in the SHR.3. This suggests that insulin resistance does not cause increased blood pressure.iv4. Enalapril decreases blood pressure but has no effects on glucose and insulinconcentrations in the SHR, confirming that high blood pressure does not causeinsulin 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 glucoseconcentrations or blood pressure in SHR.7. The SHR may not be an appropriate model for studying the link betweenhypertension and insulin resistance.VTABLE OF CONTENTSAbstract iiList of Contents vList of TablesList of Figures viiiAcknowledgements ixCHAPTER 11. INTRODUCTION1.1 Hypertension- A general background I1.2 Hyperinsulinemia and Hypertension 21.3 Genetic Model of Hypertension - The spontaneously hypertensiverat (SHR) 61.3.1. The Control Strain - Wistar-Kyoto Rats (WKY) 101.4 Pharmacological agents - Deuterium oxide, Enalapril, Metformin 101.4.1. Deuterium oxide 101.4.2. Enalapril 131.4.3. Metformin 141.5 Objectives 16CHAPTER-22. METHODS2.1 Deuterium oxide and Enalapril 16vi2.2Metformin 182.3 Statistics 20CHAPTER -33. RESULTS3.1 Deuterium oxide and Enalapril 203.2 Metformin experiment 22CHAPTER-44. DISCUSSION4.1 Deuterium oxide and Enalapril 684.2 Mefformin experiment 724.3 Is the SHR an Effective Model of Insulin Resistance? 734.4 Conclusion 755. REFERENCES 76LIST OF TABLESTable Page1. Hemodynamic and metabolic data, Metformin experiment 67viiVIIILIST OF FIGURESFigure Page1. Body weights, D20 and enalapril 252. Indirect systolic blood pressure, D20 and enalapril 273. Dircet blood pressure, D20 and enalapril 294. Twelve hour water intake, D20 and enalapril 315. Twelve hour urine volume, 020 and enalapril 336. Urine potassium, D20 and enalapril 357. Urine sodium, D20 and enalapril 378. Percentage of D20 in urine, D20 and enalapril 399. Fasting plasma glucose concentrations, D20 and enalapril 4110. Fasting plasma triglyceride concentrations, D20 and enalapril 4311. Fasting plasma insulin concentrations, D20 and enalapril 4512. lnsulin:Glucose ratio, 020 and enalapril 4713. Body weight, Metformin 4914. Indirect systolic blood pressure, Metformin 5115. Direct blood pressure, Metformin 5316. Twenty-four hour water intake, Metformin 5517. Twenty-four hour urine volume, Metformin 5718. Fasting plasma glucose concentration, Metformin 5919. Fasting plasma triglyceride, Metformin 6120. Fasting plasma insulin, Metformin 6321. Insulin:Glucose Ratio, Metformin 65ixACKNOWLEDGMENTSThe author would like to thank Dr. J.M. Wright for all of his guidance andencouragement; Drs. M.C. Sutter, R.A. Wall, and C.C.Y. Pang for their advice and asmembers of the Supervisory Committee.The author would also like to thank Christina and Kenneth Poon for their helpwith the surgeries performed in this experiment.A special thanks to the departmental secretaries for their assistance.11. INTRODUCTION1.1. Hypertension - A general backgroundElevated blood pressure was recognized as a disease entity in 1827 (1).In 1895, Allbutt called a rise in blood pressure without proteinuria “senileplethora”, which was later revised to “hyperpiesis” (2). The term “hyperpiesis”was modified to “essentille hypertonie” by Frank, in 1911, and was translated toessential hypertension (3). Today, primary hypertension is most commonly usedto describe elevated blood pressure.In 1955, Pickering characterized primary hypertension as high bloodpressure with hypertensive cardiovascular hypertrophy and proposed that it wasdependent on inheritance and environment (4). Thus, primary hypertension wasthought to be initiated by a polygenic and multifactorial cause. Secondaryhypertension was defined as hypertension caused by a known congenital oracquired disease such as renovascular hypertension or primaryhyperaldosteronism. This is in comparison to primary hypertension where theinheritable and/or environmental factors are unknown (5).More recently, primary hypertension has been postulated to be caused bygenetic factors (6). This differs from secondary hypertension caused byenvironmental factors or disease (6-8). There is probably an interactionbetween environmental and genetic factors such as salt, alcohol, obesity, lowexercise, etc. Genetic hypertension is thought to be caused by abnormalities inarterial smooth muscle causing increased peripherial vascular resistance; bloodpressure increases steeply at 30 to 50 years of age, without any knownenvironmental factors.In humans, hypertension can be further divided into four groups on thebasis of the blood pressure measurements. Borderline hypertension is defined2by systolic pressure between 140-159 mmHg and diastolic pressures of 90-94mmHg. Mild and moderate hypertension are defined by systolic pressures of160-219 mmHg and diastolic pressures of 95-114 mmHg. Lastly, systolicpressures greater than 220 mmHg and diastolic pressures greater than 115mmHg are indicative of severe hypertension (9).Hypertension is a particular problem because it is usually asymptomaticand it is the most common cardiovascular disorder in North America, affectingmore than I in 10 persons (10). It is important to control elevated bloodpressure because it can lead to a greater risk of stroke, heart failure, renaldisease, 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 antihypertensivedrugs (9).The pathophysiology of hypertension is complex and not well understood.Recently, Reaven in an attempt to better understand the pathophysiology ofhypertension, described a common syndrome in patients called Syndrome X.Patients with this syndrome have resistance to insulin-stimulated glucoseuptake, glucose intolerance, hyperinsul inemia, increased very-low-densitylipoprotein triglyceride, decreased high—density lipoprotein cholesterol, andhypertension (12). The mechanistic reason for this association betweenhypertension and hyperinsulinemia has not been elucidated. A review of therelationship between insulin resistance and hypertension follows.1.2. Hyperinsulinemia and HypertensionIn healthy individuals actions of insulin are numerous but in generalinsulin is secreted from the pancreas after meals and promotes the storage ofcarbohydrates, protein, and fat (13). Specific actions of insulin include3increased glucose entry in adipose tissue and muscle as well as an increase intriglyceride deposition in adipose tissue. Insulin is synthesized in the 13-cells ofthe Islet of Langerhans in the pancreas and once it is secreted it has a half-lifeof approximately 5 minutes. The degradation of insulin mostly occurs in the liverand kidneys but almost all tissues have the ability to metabolize insulin (13).Thus, in normal individuals insulin secretion is triggered by a rise in bloodglucose associated with food intake. The release of insulin then promotesglucose uptake into specific tissues where the glucose is metabolized.Insulin resistance has been described in a variety of ways, but in generalit is a reduction in the response to insulin (14). Glucose uptake requires aspecific concentration of insulin to promote the transfer of glucose into a cell.Binding of insulin to specific surface receptors triggers unknown intracellularmessages which in turn activate glucose transporters that transport glucoseacross the cell membrane (15). The molecular biology of insulin resistance hasrecently been described and it has been shown that the impairment of insulinaction can be attributed to decreased insulin receptor number and post bindingdefects of insulin action (16). A malfunction at any stage of this process couldresult in resistance to insulin-stimulated glucose uptake.Insulin resistance is present in all patients with non-insulin dependentdiabetes mellitus (NIDDM) (12) and is frequently combined with a defect ininsulin secretion (17). Obesity is a common precursor of NIDDM and iscommonly associated with insulin resistance (17-19). A relationship also existsbetween NIDDM and hypertension (Syndrome X) (20-22).A common pathogenic link between diabetes and hypertension may beinsulin resistance (20). It has been demonstrated that hypertensive patients, onaverage, are more insulin resistant than a control population in the absence ofobesity or NIDDM (23-25). Bonora, et al. conducted studies involving 247 non-4obese and 120 obese non-diabetic subjects (24). All subjects underwent astandard oral glucose tolerance test which included the measurement of plasmainsulin concentrations. One single blood pressure measurement was obtained Ito 2 hours following the glucose load. Results showed a significant relationshipbetween either systolic or diastolic blood pressure and both fasting and post-glucose plasma insulin. It was suggested that the post-glucose plasma insulinresponse was independently associated with blood pressure in the non-obesesubjects, while the association between plasma insulin and blood pressure inobesity was mainly mediated by factors such as age and body weight. It wasconcluded that insulin may play a role in the regulation of blood pressure in theabsence of obesity (24).Pollare, et al. studied the relationship between abnormalities incarbohydrate metabolism and hypertension in 143 newly detectedhypertensives, which were divided into obese and non-obese groups, and 51normotensive controls (25). The euglycemic clamp technique was used (initiallydescribed by Defronzo, et al. 26) to calculate steady state plasma insulin andglucose concentrations. The non-obese hypertensive group had significantlyincreased fasting plasma insulin values compared with the control group. Theobese hypertensive group had significantly higher plasma insulin valuescompared to both control and non-obese groups. These results suggest that theabnormalities of carbohydrate, insulin, and lipid metabolism in primaryhypertensive patients may occur independently from obesity (25).In 1985, Mancini, et a). compared obese normotensive and obesehypertensive patients (26). The patients were subjected to an oral glucose.tolerance test (OGTT) by giving 75g of glucose as a 33% solution and takingblood samples for glucose and insulin measurements at 0 up to 240 minutesafter the glucose load. The results of this study showed a significant increase in5serum insulin in the hypertensive group. The authors also concluded thatimpaired glucose tolerance was more common in the obese hypertensive groupalthough this finding was based on one data point at 120 minutes after the oralload. It was concluded that in obese patients, high blood pressure wasindependently associated with impaired glucose tolerance and higher fastingserum insulin levels. The results from this experiment suggest that hypertensionand insulin resistance occur independently of obesity, however because of thesmall number of patients and the other criticisms mentioned above it is notconclusive.As previously mentioned, Reaven defined the relationship between insulinresistance and hypertension as Syndrome X. Various studies have been carriedout since this publication that confirm these results. Glucose clamp studies haveshown the presence of insulin resistance in elderly patients with hypertension ascompared to normotensive controls (27). Oral glucose loads have been given tomiddle-aged hypertensive patients who showed exaggerated glucose and insulinresponses (28), which could be indicative of insulin resistance. The bulk of dataconfirms a relationship between these two abnormalities. However, it is notknown whether this is a causal relationship or if these two abnormalities developindividually, possibly from an early genetic defect.The primary site of insulin resistance in hypertensive patients is theskeletal muscle (29,30). Direct measurements in the forearm of hypertensivepatients has displayed an impairment in insulin action at the muscle tissue level(29). Julius, et al. postulated that the pressure-induced restriction of themicrocirculation, associated with hypertension, would limit nutritional flow andthereby impair glucose uptake in the skeletal muscle. Thus decreased skeletalmuscle blood supply may be a possible link between insulin resistance andhypertension (30).6Hwang, et al. conducted an experiment to try to determine if hypertensioncould 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 stateplasma insulin and glucose, and biochemical measurements were taken.Systolic blood pressure was significantly higher in the fructose fed group incomparison to controls. Hyperinsulinemia and hypertriglyceridemia were alsoassociated with the increase in blood pressure. However, when clonidine(antihypertensive agent) was added to the drinking water the fructose inducedhypertension was inhibited, but the increases in plasma insulin and triglyceridewere not effected. This evidence suggests that elevated blood pressure is notthe 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 asignificant difference in the steady state plasma insulin (SSPI) levels whencompared to normotensive and non-treated hypertensive men. This indicatesthat lowering blood pressure with an antihypertensive agent does notnecessarily effect plasma insulin levels (32). Thus, results from both animalexperiments and human patients suggest that the metabolic abnormalitiesassociated with insulin resistance are not altered when elevated blood pressureis lowered by antihypertensive agents.1.3. Genetic Model of Hypertension- The spontaneously hypertensiverat (SHR)The spontaneously hypertensive rat (SHR) is the most commonly usedexperimental model of inherited hypertension since its development 27 yearsago by Okamoto and Aoki (33). The development of this strain of rat began frominbred Wistar rats, who were sent from the United States to Kyoto University.7These rats (Wistar-Kyoto, WKY) were then outbred within a closed colony andwere subsequently screened for elevated blood pressure (34). A single male ratwas found with a systolic blood pressure in the range of 150-175 mmHg. Therewas a subsequent mating of this male rat with a female rat whose systolic bloodpressure ranged between 130-140 mmHg, which was above the average of thecolony. There was further inbreeding from three generations of offspring untilthe rats developed blood pressures of > 150 mmHg (34). Okamoto and Aokidefined these rats as arbitrarily hypertensive (33). Blood pressures rose witheach generation of hypertension and the development of high blood pressurebegan to occur at a younger age (34). In 1969, after 20 generations ofinbreeding a line of SHRs had become fixed. However, some of these SHRswere released to other laboratories before the 20th generation. Therefore,“genetic variation exists between colonies of SHRs because they either arosefrom different inbred SHR strains released prior to the strain reaching genetichomogeneity, or because of the genetic drift that is known to occur within andbetween colonies arising from the same inbred strain” (34). Differences betweenthe colonies are more likely trait difference, which are not related to thedevelopment of hypertension (35). Therefore, even though there may be slightdifferences between colonies, the SHR is an appropriate model for hypertensionand may provide clues as to the basis of primary hypertension in humans asexplained below.The similarities of genetic hypertension between man and rat has beenexplored by Trippodo and Frohlich (36). Certain important differences arerecognized; 1) in the human population of primary hypertension, hypertensivepatients tend to be heavier than those without hypertension whereas SHRnormally weigh less than their normotensive controls, 2) the SHR may havealtered thyroid function which does not occur in most primary hypertensive8patients, and 3) the rat has relative resistance to developing significantatherosclerosis, in contrast to primary hypertensive patients whose elevatedblood pressure facilitates the onset of atherogenesis (36). The SHR developsincreased arterial pressure as early as 3 weeks of age and it continues toincrease until approximately 20-28 weeks of age. The onset and rate ofdevelopment of arterial pressure in humans is not clearly defined.Despite these differences, the SHR has a number of similarities to humanprimary hypertension. Increased total peripheral resistance and normal cardiacoutput are two hemodynamic factors that are found in established hypertensionin both humans and SHRs. Moreover, both SHR and human patients displayeither normal or slightly reduced blood volume, an elevated heart rate, and theprogressive development of hypertrophy in the left ventricle. The persistence ofvascular resistance in both cases may lead to impaired myocardial functionwhich can result in congestive heart failure. Thus, the hemodynamic alterationsin both forms of hypertension appear to follow a very similar course (36).The participation of renal factors and their effect on arterial bloodpressure continues to be studied. Studies have shown that renal blood flow isusually normal or decreased with a normal or slightly reduced glomerularfiltration rate and increased filtration fraction in both humans with uncomplicatedhypertension (37) and SHR (38). Renal vascular resistance is also elevated inboth forms of hypertension (36).Other similarities such as increased venoconstriction and increasedsympathetic nerve activity have been implicated in both humans with primaryhypertension and in the SHR, but the exact mechanism of how these factorseffect blood pressure in man and rat is still being investigated (36).Because the similarities outweigh the differences, the SHR is consideredto be an appropriate model for studying the mechanism of hypertension in man.9However, is it also an appropriate model for studying the relationship betweenprimary hypertension and insulin resistance? A number of studies have beenpublished which support the view that SHRs develop an insulin resistant statesimilar to that seen in human hypertensive patients (39-42). In a preliminaryreport Mondon and Reaven showed that abnormalities in insulin secretion,action, and catabolism existed in rats with spontaneous hypertension (39). Thisevidence was supported in a later publication which demonstrated cellularresistance to glucose uptake in adipocytes from SHR (40). In 1989, Mondon, etal. suggested that high plasma insulin concentrations (hyperinsulinemia)associated with insulin resistance may be due to a decreased removal of insulinby skeletal muscle and the kidneys rather than impaired hepatic removal ofinsulin (41). The presence of peripheral insulin resistance in the SHR wasobserved, specifically in the skeletal muscle (42, 43). Further study of thisrelationship suggests that SHR release insulin normally, but they exhibit reducedtissue sensitivity to insulin (46) and that this reduced sensitivity is a primaryrather than a secondary event in hypertension (47). Experiments by Swislockiand Tsuzuki contribute to the previous findings that SHR are suitable models forinsulin resistance and primary hypertension through the expression of insulinresistance in terms of glucose and fatty acid metabolism in SHR (48). Thus it issuggested that the SHR, as well as being hypertensive, may also havemetablolic abnormalities.However, some evidence exists that contradicts the presence of insulinresistance in SHR (44, 45). Gaboury, et al. demonstrated that the action ofinsulin on glucose metabolism is not impaired in the SHR at a time when theirblood pressure is clearly elevated (44). Buchanan, et al. showed no significantdifference between SHR and their control strain WKY in the response of skeletal10muscle to insulin (45). Therefore, it is not clear whether insulin resistance is aconsistent finding in the SHR.Does this potential insulin resistant state in the SHR parallel the one seenin human patients? A review by Gerald Reaven concludes that theabnormalities of glucose, insulin, and lipoprotein metabolism that occur inhypertensive patients also occur in the SHR (49). Therefore, it appears that theSHR is the best animal model for studying the relationship between elevatedblood 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 controlstrain for the SHR. The WKY differs from the SHR because the increase inarterial blood pressure occurs at a slower rate in the WKY and reaches itsmaximum at approximately 6-10 weeks of age (36). The mean arterial pressureof the WKY reaches 115-130 mmHg, while the average mean arterial pressureof the SHR is between 190-200 mmHg depending on the colony. However,there is some speculation as to the validity of this normotensive control becauseit was not developed simultaneously with the SHR (36, 50, 51). Genetic“fingerprint” patterns were examined from both SHR and WKY (50). Resultsshowed 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 forinvestigating the pathogenesis of hypertension. Therefore, in the followingexperiments WKY rats were not studied.1.4. Pharmacological agents - Deuterium oxide, Enalapril, Mefformin1.4.1. Deuterium Oxide11Deuterium oxide (D20) is a stable nonradioactive isotope of water, whichhas been studied in mammals since the late 1950’s. In 1958, a study wasconducted in rats to determine the effect of D20 on glomerular filtration andrenal plasma flow (52). The rats were given 50 mole % D20 as drinking waterfor 38 days. Results showed a decrease in both filtration rate and renal plasmaflow to about 40% of rats on normal water. It was also noted that when the ratswere returned to normal tap water the filtration rate and renal plasma flowreturned to its normal state. They concluded that the effect of D20 may havebeen due to a disturbance of adrenal function. A couple of years later, theeffects of D20 were studied in heart, and voluntary muscle at concentrationsvarying between 99.8 to 25 % in drinking water (53). D20 decreased the forceand velocity of contraction in both the heart and voluntary muscle. Furtherinvestigations in frog muscle, suggested that the contractile proteins could beaffected by deuterium (54).Muscles of the barnacle were used by Kaminer and Kimura to test theirhypothesis that calcium release was prevented by D20, in the coupling ofexcitation and contraction (55). Aequorin, a protein which luminesces in thepresence of calcium, was used to determine the amount of calcium present inthe muscle tissue after exposure to 99.9 % D20. The results showed that in thepresence of 020 no calcium was released and therefore no contractile responseobserved. It has been suggested that D20 depresses the mobilization ofcalcium ions by lowering the rate of release of calcium ions, decreasing amountof calcium release, and reducing diffusion of calcium ions (56, 57).Recent studies with D20 in Sprague-Dawley rats have demonstrated thatD20 affects vascular muscle relaxation (58). It was suggested that these resultsoccurred through action on the sarcoplasmic reticulum calcium mobilization or12contractile proteins. Therefore, D20 may have multiple sites of action onvascular smooth muscle.Deuterium oxide has been shown to affect both insulin and glucose inexperimental rats. Experiments with D20 in Sprague-Dawley rats have shownthat 50% D20 in the drinking water decreases blood glucose over a period of 35days. It appears that the D20 treatment slows down gluconeogensis, thus bloodsugar cannot be maintained at a normal range (59). It has also been shown thatD20 inhibits insulin release, probably through its stabilizing action on themicrotubular system of the 13-cell (60). D20 may also mimic the action of insulinby increasing glucose metabolism in adipose tissue (61). This specific action ofD20 is of interest because D20 may also promote glucose uptake in the skeletalmuscle by acting like insulin at this site.Vasdev, et at. hypothesized that D20 may help prevent the developmentof hypertension, by preventing the abnormal contractile activity of the vascularsmooth muscle associated with this abnormality (62). Twenty-five percent D20was given to male Dahl salt-sensitive rats for four weeks. The D20 treatmentcaused a significant decrease in the systolic blood pressure compared to thenon treated rats. It was suggested that the antihypertensive effect of D20 wasthe result of increased blockage of calcium channels by bound deuterium ions.A further study showed that D20 (25%) prevented hypertension inspontaneously hypertensive rats (SHR) compared to their control strain WistarKyoto (WKY) (63). It was also demonstrated that 25% D20 normalized elevatedcalcium uptake in the aorta. It was again postulated that the blood pressurelowering effect of D20 was the result of bound deuterium ions in the vascularcalcium channels.An investigation of the dose-dependent effect of D20 in drinking water onsystolic blood pressure and aortic calcium uptake was conducted in SHR to13determine the minimum effective dose of D20 (64). SHR were treated for 7weeks with 5%, 10%, and 20% D20. 10% and 20% D20 prevented the increasein systolic blood pressure. These two groups also displayed normal values ofaortic calcium uptake. It was concluded that 10% D20 was the minimum doserequired to completely prevent the development of hypertension and elevatedaortic calcium uptake in SHR. Because of its potential effect on both glucosemetabolism and blood pressure we decided to use 10% D20 as anantihypertensive agent to try to determine the relationship between elevatedblood pressure and insulin resistance.1.4.2. EnalaprilEnalapril is an angiotensin-converting enzyme (ACE) inhibitor and is oneof 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, thuspreventing the pressor action of Ang II on the arteriolar smooth muscle (66).There is a decrease in arteriolar resistance and arteriolar pressure. There isalso a decreased production of aldosterone because of the lack of Ang II actionto increase aldosterone secretion. The lack of aldosterone prevents sodiumretention 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 theangiotensin-converting enzyme than its predecessor, captopril (67). It is rapidlyabsorbed from the gastrointestinal tract and reaches peak serum concentrationsin about one hour. Its half-life is approximately 11 hours, which is more thantwice as long as the half-life of captopril. Because of the time needed forhydrolysis by the liver to convert it to its active form, the onset of action of14enalapril is slow (two to four hours). The excretion of enalapril and enalaprilat isunchanged in the urine.The efficacy of ACE-inhibitors in hypertensive patients has been welldocumented (65-67). The antihypertensive effects of this class of drugs are alsoseen in the spontaneously hypertensive rat (68-70). ACE-inhibitor treatment inyoung SHR for 4 weeks was sufficient to prevent the full expression of genetichypertension (68). As in humans, ACE-inhibitors exert their antihypertensiveeffect in SHR by blocking the renin-angiotensin system (69). Enalapril, at a doseof approximately 25 mg/kg/day in the drinking water, significantly reduced meanarterial pressure in the SHR compared to the control group receiving normal tapwater (70). Therefore, enalapril was used in our experiments to compare it’santihypertensive effects to the antihypertensive effects of deuterium oxide.1.4.3. MetforminMetformin is an oral hypoglycemic agent, widely used in Europe andCanada for the treatment of NIDDM. It is composed of two guanidine moleculesthat are linked together with the elimination of an amino group, thus it is in thedrug class of the biguanides with other agents such as buformin and phenformin(79). Metformin is not metabolized. Its absorption is slow (approx. 6 hours) andit 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 3hours and plasma concentration at a steady state ranges from about I to 2 ig/mL (80).Metformin differs from sulfonylureas in a number of various respects. Itdoes not undergo biotransformation and is not bound to plasma proteins. It iseliminated solely by the kidney and rarely cauSes hypoglycemia. Generally, itdoes not cause weight gain. The doses of metformin given range from 500 to1000 mg, up to three times a day and it is usually given with meals (80).15The mechanism of action of metformin is not completely understood.However, studies show that it does not stimulate the release of insulin (81-85) asdo the sulfonylureas. Metformin has been shown to reduce basal hepaticglucose production and improve oral glucose tolerance without increasingglucose uptake in patients with NIDDM (80). It also improves insulin-inducedwhole-body glucose uptake in these patients. Metformin causes a decrease infasting blood glucose, insulin and C-peptide concentrations in plasma (86). Italso increases insulin action at the cellular level (83) without raising the plasmainsulin concentrations (82).A number of studies have attempted to elucidate the mechanism of actionof metformin. One suggestion is that metformin’s action is due to a post receptorevent that causes glucose lowering and that the effects of metformin on insulinbinding are indirect (87). It is also postulated that the basis for the hypoglycemiceffect of metformin is at the level of the skeletal muscle, where it increasesglucose transport across the cell membrane (88). In muscle cells, mefformin hasbeen shown to stimulate specific glucose transporters; GLUTI and GLUT4 (88-90). Metformin has also been shown to increase insulin stimulated glucosetransport by potentiating GLUTI and GLUT4 transporters in the plasmamembrane in rat adipocytes (89). It has been suggested that the increasedglucose uptake caused by metformin results from an increase in glucosetransporter number without the need to invoke a modification of intrinsictransporter activity (90). These results suggest that metformin stimulatesglucose transport in muscle cells independently of insulin. Therefore, insulinand metformin may be exerting their effects through different subcellularpathways (90). Although the effects of metformin on glucose transportersenhances our knowledge of its mechanism of action, it is still not fullyunderstood how metformin lowers plasma glucose.16Due to its ability to lower plasma glucose levels without increasing insulinlevels, metformin has been used to study the relationship between insulinresistance and hypertension. The effect of metformin on blood pressure andmetabolism was studied by Landin, et at. in nine non-obese men withhypertension to try to determine the role of insulin resistance (91). They showedthat metformin treatment (30 mg/kg/day) significantly lowered blood pressureafter six weeks. Two months after the removal of the drug the blood pressureincreased, suggesting insulin resistance plays a role in the etiology ofhypertension. 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, wedecided to examine these effects of metformin in the SHR for an extendedtreatment period (10 weeks). Given the usual dose of metformin in patients is 30mg/kg/day, four doses at 10, 30, 100, and 300 mg/kg/day, were used to studythe effects of chronic metformin treatment in SHR.1.5. ObjectivesTo try to elucidate the relationship between insulin resistance andhypertension using the spontaneously hypertensive rat by studying agents whichprimarily lower blood pressure (deuterium oxide, enalaprit) and possibly have aneffect on glucose metabolism and an agent which primarily lowers plasmaglucose levels (metformin) and possibly has an effect on blood pressure. Ifinsulin resistance and hypertension are causally related one would expect thatby pharmacologically altering one of the abnormalities a similar direction andmagnitude of effect would occur in the other.2. METHODS2.1. Deuterium oxide and Enalapril17Animals:Twenty-four male spontaneously hypertensive rats (SHR) were obtainedfrom Charles River Canada (200-220g). These rats were maintained on a12/1 2h light/dark cycle and food and water were available ad libitum. For oneweek prior to experimental onset the rats were acclimatized to restraining tubesfor subsequent blood pressure measurement via tail cuff (approximately 15mm ./day).Experimental Setup:At eight weeks of age ( 200g), the 24 male SHR were randomly dividedinto three groups: control, 10% D20, and 50 mg/L enalapril (n=8). Drugtreatment was given through the drinking water a by single-blind experimentalprotocol. Water bottles were filled and coded by an individual who was notinvolved in any of the measurements. In addition all plasma analyses were doneon coded samples. During the six week treatment period body weight, urinevolume, and water intake were recorded weekly. Blood samples were taken viathe tail and systolic blood pressure was measured by tail cuff weekly. Systolicblood pressure was taken in the morning (09:00 - 12:00) and all blood sampleswere also taken in the morning following a 12-14 hour fast. After the six weektreatment period direct blood pressure was measured by iliac artery cannulationunder pentobarbital anesthesia (0.1 mg/I OOg). An intracardiac blood sample wasobtained for plasma biochemistry prior to sacrifice.Measurements/Analysis:Weekly blood samples (O.5mL) were collected by loosely wrapping eachrat in a towel, to restrict movement, with their tail exposed. Approximately 1mmof the tail tip was cut-off to allow for bleeding. Blood was collected in I.OmLeppindorf tubes which were coated with heparin. The tail tip was subsequentlysubmerged into a 3% hydrogen peroxide solution for antiseptic purposes. Blood18samples were then centrifuged for 10-15 mm. and the plasma removed andtransferred 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. Theratio of insulin to glucose (mU:mmol) was used from each rat as an indicator ofinsulin resistance.The SHR were housed overnight (12-14h) in metabolic cages formeasurement of water intake and urine output. Urine samples were analyzed forD20 content with a single-beam infrared spectrometer (MIRAN I FF, TheFoxboro Co.) and for sodium and potassium levels with a flame photometer.Systolic blood pressure was recorded indirectly from the tail artery using apneumatic pulse transducer (Narco Bio Systems Inc.). Blood pressure wasrecorded as an average of three measurements.Cannulae were inserted into the iliac artery while rats were underpentobarbital anesthesia. Systolic and diastolic pressure were recorded after a10 minute wait to allow for pressure stabilization.2.2. MetforminAnimals:Twenty-four male spontaneously hypertensive rats (SHR) were obtainedfrom Charles River Canada (200-220g). These rats were maintained on a12112h light/dark cycle and food and water were available ad libitum. For oneweek prior to experimental onset the rats were acclimatized to restraining tubesfor subsequent blood pressure measurement via tail cuff (approximately 15min./day).Experimental Setup:Preliminary experiments with 10 mg/kg/day metformin followed the sameexperimental protocol as below.19At eight weeks of age ( 200g), the 24 male SHR were randomly dividedinto four groups: control, 30 mglkg/day, 100 mglkg/day and 300 mg/kg/daymetformin (n=6). Drug treatment was given through the drinking water. Duringthe 10 week treatment period body weight, urine volume, and water intake wererecorded weekly. Blood samples were taken via the tail and systolic bloodpressure was measured by tail cuff weekly. Systolic blood pressure was taken inthe morning (09:00 - 12:00) and all blood samples were taken in the morningfollowing a 12-14 hour fast. After the 10 week treatment period direct bloodpressure was measured in concious rats by an iliac artery cannula (see below).This technique was used to eliminate the effect of the anesthetic on bloodpressure as was observed in the first experiment.Measurement/Analysis:Weekly blood samples (0.5mL) were collected by loosely wrapping eachrat in a towel, to restrict movement, with their tail exposed. Approximately 1 mmof the tail tip was cut-off to allow for bleeding. Blood was collected in 1.OmLeppindorf tubes which were coated with heparin. The tail tip was subsequentlysubmerged into a 3% hydrogen peroxide solution for antiseptic purposes. Bloodsamples were then centrifuged for 10-15 mm. and the plasma removed andtransferred 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 themeasurement of water intake and urine output.Systolic blood pressure was recorded indirectly from the tail artery using apneumatic pulse transducer (Narco Bio Systems Inc.). Blood pressure wasrecorded as an average of three measurements.For direct blood pressure measurements, cannulae were inserted into theiliac artery while rats were under halothane anesthesia. Two small incisions20were made, one in the back of the neck between the ears and the other in thethigh region above the iliac artery. A cannula was then run subcutaneously fromthe neck to the thigh region. The iliac artery was exposed and cannulated. Bothincisions were sutured and the rats were placed back in their cages. After a 24hour recovery period, direct systolic and diastolic pressures were recorded. Theprotruding cannula at the neck was attached to a Grass transducer and directblood pressure was recorded after a 10 minutes wait to allow for pressurestabilization. After the direct blood pressure was recorded the rats werereanesthetized and the chest wall retracted to expose the heart for anintracardiac puncture ( 2mL of plasma was collected).2.3. StatisticsA one-way ANOVA and an unpaired two tailed student’s t-test were usedto compare direct blood pressure between the groups. A repeated measuresANOVA with Duncan’s multiple range test was used to compare the weeklydifferences in body weight, urine volume, water intake, insulin, glucose,triglycerides, and indirect systolic blood pressures. P <0.05 was accepted as asignificant difference and all results are recorded as mean ± S.E.M. Allexperimental methods were pre approved by the Animal Care Committee ofU.B.C.3. RESULTS3.1. Deuterium oxide and EnalaprilBody weight was not significantly different between the three groups,although there was a significant increase in weight during the six treatmentweeks (p <0.05, figure 1). Systolic blood pressure (mmHg) rose in the controlgroup from I 39±2 to I 54±10 and this increase in blood pressure was notsignificantly prevented by 10% D20 (132±4 to 161±5) but was prevented by21enalapril (127±6 to 113±6, p < 0.05, figure 2). The direct blood pressure(mmHg) measurements by iliac artery cannulation also confirm that the systolicand diastolic blood pressure of the enalapril group (133±2/92±4) wassignificantly lower than both the control (156±11/107±11) and 10% D20 groups(144±8/97±6) (figure 3).The enalapril group had significantly higher 12 hour water intake (44±3mL) compared to the control (20±3 mL) and the 10% D20 (17±4 mL) groups(figure 4). Urine output (mL) was also significantly higher over the six weektreatment period in the enalapril (34±1) compared to the control (16±2) and 10%D20 (13±1) groups (figure 5). There were no significant differences in weeklyurine potassium excretion (mmol/l2hrs, p < 0.05, figure 6) between the threegroups whereas, the urine sodium (mmol/l2hrs) was significantly lower in the10% D20 group (0.24±0.01) compared to both the control (0.36±0.02) andenalapril (0.42±0.06) groups (p <0.05, figure 7). The measurement of D20 inthe urine by single-beam infrared spectrometry demonstrated a gradual increasein the D20 level until it reached a plateau of 8% at 11 weeks of age (figure 8).The glucose measurements (mmol/L) taken during the 7 week treatmentperiod showed no significant difference between the control (7.8±0.2) group andthe treatment groups (figure 9). However, the 10% D20 group and the enalaprilgroup 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 the10% D20 (0.43±0.04) and enalapril groups (0.44±0.03) as opposed to thecontrol group (0.51±0.05) (figure 10). There was no significant difference ininsulin levels (mU/L) between the control (62.6±6.4), 10% D20 (56.3±4.9), orenalapril (52.7±3.8) groups (figure 11). The insulin:glucose (mU:mmol) ratioalso 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).223.2. Metformin ExperimentPreliminary experiments using 10 mg/kg/day metformin showed nosignificant difference in body weight, systolic blood pressure, water intake, urinevolume, fasting plasma insulin, fasting plasma glucose, fasting plasmatriglycerides, nor in the insulin glucose ratio between the control and treatedgroups. These results led to choosing a higher dose range (30, 100 and 300mg/kg/day metformin) to determine the dose-response relationship of mefforminin lowering blood pressure and increasing insulin sensitivity in the SHRBody weight was not significantly different between the four groups(control, 30, 100, and 300 mg/kg/day metformin, p < 0.05) during the 10 weektreatment period, although there was a significant increase in weight over theduration of the experiment (figure 13). The systolic blood pressure was notsignificantly 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 pressuremeasurements taken at week 11 showed a significant decrease in systolic anddiastolic 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 in24 hour water intake (p > 0.05, figure 16). The 24 hour urine volume alsodisplayed no significant difference (p > 0.05) between the control and treatmentgroups (figure 17).Fasting plasma glucose (mmol/L) was monitored throughout the 10 weekmetformin 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 30023mg/kg/day metformin, respectively, figure 18). The final fasting glucose levels atweek 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±0.8and 6.6±0.7, respectively) were not significantly different from control (Table 1).Measurement of the fasting plasma triglycerides (mmol/L) showed no significantdifference 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 alsoshowed no significant difference between the control and treatment groups(Table 1). Fasting plasma insulin levels (mU/L) between the control (93±6) andtreatment groups (133±29, 89±3, 138±27, 30, 100, 300 mg/kg/day metforminrespectively, p > 0.05) were also not significantly different from one another(figure 20). However, the final plasma samples show that the group treated with300 mg/kg/day metformin had significantly higher plasma insulin levels than theother three groups (Table 1). The insulin:glucose ratio (mU/mmol) between thefour groups was not significantly different during the 10 week metformintreatment (figure 21). However, the final plasma samples show that the grouptreated with 300 mg/kg/day metformin had a significantly higher insulin:glucoseratio than the control and other two treatment groups (Table 1).24Figure 1. Body weights (g) measured weekly. There was no significantdifference between the control, 10% D20, and 5Omg/L enalapril groups (p >0.05). Values are means ± S.E.M., n = 8 for each group.25Weight (g)- F301 0 C.n 0 (1,o 0 0 0 00 1-nC)0—I’I\II-I \I -‘I CDCD-o cI.- oJCl, 0mCDC)—I26Figure 2. Indirect systolic blood pressures (mmHg) measured weekly via tailcuff. There is no significant difference between control and 10% D20, while theenalapril group is significantly lower than both groups. Values are means ±S.E.M., n = 8 for each group. * indicates a significant difference (p < 0.05) fromcontrol and 10% D20.Figure2.SystolicBloodPressure(N200—————150a)22•1--—--—-a*100*500123467Treatment(weeks)Control10%D20EnalapnlFigure 3. Direct blood pressure (mmHg) recordings after six weeks of treatme,?There is no significant difference in systolic pressure (top of bar) betweencontrol and 10% D20, 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. Values are means ± S.E.M., n = 8 for eachgroup. * indicates a significant difference (p <0.05) from the control and 10%D20. indicates a significant difference from control.Blood pressure (mmHg) 29CDc)CDCDIIw00CD00CD0- • -0 r) Ui 0 UiUi 0 Ui 0 Ui 0 UiHHC)0z*30Figure 4. Twelve hour water intake (mL). No significant difference was foundbetween the control and 10% D20 groups, while the enalapril group wassignificantly different from both. Values are means ± S.E.M., n = 8 for eachgroup. * indicates a significant difference (p < 0.05) from both control and 10%D20.Figure4.TwelveHourWaterIntake1CW)**60Laa50/1**/±j,I--T*__..‘j/z40E304—Cu20100IIII01234567Treatment(weeks)Control10%020Enalapril32Figure 5. Weekly urine volumes (mL) measured over 12 hours. There was nosignificant difference between the control and 10% D20 groups, while theenalapril group was significantly different from both. Values are means ±S.E.M., n = 8 for each group. * indicates a significant difference (p < 0.05) fromboth control and 10% D20.Figure5.TwelveHourUrineVolumeC’)50***40I•———-—*a•—•a.1—-J——————•*————300>.E20D100III01234567Treatment(weeks)Control10%D20—--—Enalapril34Figure 6. Urine potassium (mmol/12h) measured weekly multiplied by the 12hour urine volume. There is no significant difference (p > 0.05) between thecontrol, 10% D20, and enalapril groups. Values are means ± S.E.M., n = 8 foreach group.Figure6.UrinePotassiumC)1.50-•/—I‘‘‘c’J•i.oo0EEE•0U)0.50000.00IIIII01234567Treatment(weeks)Control10%020Enalapril36Figure 7. Urine sodium (mmoWl2h) measured weekly multiplied by the 12 hoururine volume. There is no significant difference (p > 0.05) between the controland enalapril treated group. The 10% D20 group has significantly lower sodiumthan both control and enalapril groups. Values are means ± S.E.M., n = 8 foreach group. * indicates a significant difference from control and enalapril groups(p <0.05).Figure7.UrineSodium(V)0.75/C\I-I.•1I-:‘F____————————.-I025/T———r——*—————0.00IIIII01234567Treatment(weeks)Control10%D20Enalapril38Figure 8. Percentage of D20 (v/v) in the urine measured weekly. There is anobvious difference between 10% D20 (open circles) with control and enalapril(closed circles) where there was no detection of deuterium in the urine. Theminimum level of detection with this method is 0.1%. Values are means ±S.E.M., n = 8 for each group.Figure8.PercentageofD20inUrine0)CV)10—:TT°ITreatment(weeks)40Figure 9. Fasting plasma glucose concentrations (mmol/L) measured weeklyThe group treated with 10% D20 has significantly lower plasma glucoseconcentrations than the enalapril treated group. There is no significantdifference between the control group and both treated groups. Values aremeans ± S.E.M., n = 8 for each group. -- indicates a significant differencebetween the 10% D20 group and the enalapril group (p < 0.05).Figure9.FastingPlasmaGlucose10-:—————————5IIII01234567Treatment (weeks)Control10%D20Enalapril42Figure 10. Fasting plasma triglyceride concentrations (mmolIL) measuredweekly. Both treatment groups (10% D20 and ena 1apr11) have significantly lowertriglyceride concentrations than the control group.. Values are means ± S.E.M.,n = 8 for each group. * indicates a significant difference between the control andtreatment groups (p <0.05).Figure10.FastingPlasmaTriglyceridesC.)“10.900.80.10.70E0.60U)a)-D0.50o*0.40*_.S__-.---F-0.301**0.20I01234567Treatment(weeks)Control10%D20Enalapril44Figure 11. Fasting plasma insulin concentrations (mU/L) measured weekly.There is no significant difference in insulin concentration between the controland treatment groups (p > 0.05). Values are means ± S.E.M., n = 8 for eachgroup.Figure11FastingPlasmaInsulinU)‘110090::::z//z::z;403020IIIII01234567Treatment(weeks)Control10%D20Enalapril46Figure 12. lnsulin:glucose ratio (mU:mmol) is representative of insulinresistance in the SHR. There is no significant difference in the insulin:glucoseratio between the control and treatment groups. Values are means ± S.E.M., n =8 for each group.47Insulin:Glucose (mU:mmol)0 C.fl 00‘1—acco_____II-‘o III—I CDIIiI,II ‘I’ I II’\— H-I fto CDDIII’ a.o CD ItCD It C)I’(0.1—“ C)_____ I’.11 I I 0III • I‘I 0CDm IiI,II‘I0) HI(H—I I—III I48Figure 13. Body weights (g) measured weekly. There was no significantdifference between the control, 30, 100, and 300 mg/kg/day metformin treatedgroups (p > 0.05). Values are means ± S.E.M., n = 6 for each group.Figure13. WeeklyBodyWeights0)400‘I——_______•/—__•300ifr•—200C1234567891011Treatment (weeks)ControlMet 30Met 100Met 30050Figure 14. Indirect systolic blood pressure (mmHg) measured weekly via tailcuff. There is no significant difference between the control, 30, 100, and 300mg/kg/day metformin treated groups (p> 0.05). Values are means ± S.E.M., n =6 for each group.Figure14.SystolicBloodPressures‘4)200—————————j_.i:,100IIIIIIIIC12345678910Treatment(weeks)ControlMet30Met100Met30052Figure 15. Direct blood pressure (mmHg) after ten weeks of treatment. There isno significant difference in systolic pressure (top of bar) between control, 30,and 100 mg/kg/day metformin treated groups, while the 300 mg/kg/daymetformin 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 alsosignificantly 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)225Figure15.DirectBloodPressuresControlV/AMet30V)91MetlOOMet3000)xEa)I.U)U)a)0000*J200175150125100755025-0 L_**54Figure 16. Twenty-four hour water intake (mL). No significant difference wasfound between the control and metformin treated groups. The 300 mg/kg/daymetformin treated group had significantly lower water intake than the 30mg/kg/day metformin treated group. Values are means ± S.E.M., n = 6 for eachgroup. • indicates a signficant difference (p 0.05) between the 30 and 300mg/kg/day metformin treated groups.Figure16.24HWaterIntakeU)U)6050————..40—*30**x20*10IIIIIIII0C1234567891011Treatment(weeks)ControlMet30Met100—Met30056Figure 17. Weekly urine volumes (mL) measured over 24 hours. There was nosignificant difference between the control and treatment groups (p > 0.05). The300 mglkglday metformin treated group had a significantly lower urine outputthan the 30 mg/kg/day metformin treated group over the treatment period.Values are means ± S.E.M., n = 6 for each group. * indicates a signficantdifference (p , 0.05) between the 30 and 300 mg/kg/day metformin treatedgroups.Figure17.24HUrineVolume‘040%%%T30I.-JEG)ED200———————————1%•——10****0IIIIIIC1234567891011Treatment(weeks)ControlMet30Met100-—Met30058Figure 18. Fasting plasma glucose concentrations (mmol/L) measured weekly.There is no significant difference (p> 0.05) between the control group and themetformin treated groups, although there is a significant decrease in glucoseconcentration in all of the groups throughout the treatment period. Values aremeans ± S.E.M., n = 6 for each group.Figure18.FastingPlasmaGlucose0)U)87%%..ES.6-:/-4IIIIIIIIC12345678910Treatment(weeks)ControlMet30Met100—-—Met30060Figure 19. Fasting plasma triglyceride concentrations (mmolIL) measuredweekly. No significant difference (p> 0.05) is observed between the control andtreatment groups. Values are means ± S.E.M., n = 6 for each group.Figure19.FastingPlasmaTriglyceridesCD1.000.900.80-J0.70--E-.O6O0.300.200.100.00IIIIC12345678910Treatment(weeks)ControlMet30Met100—Met30062Figure 20. Fasting plasma insulin concentrations (mU/L) measured weekly.There is no significant difference (p > 0.05) in insulin concentrations betweenthe control and treatment groups. Values are means ± S.E.M., n = 6 for eachgroup.(.3(0200-Figure20.FastingPlasmaInsulinIV..III_//I.//Treatment(weeks)S/I160-—IDc(0/120804:0-—-1k-“-——\:——a———a——IIIIC12345678910IIContro’Met30Met100Met30064Figure 21. lnsulin:Glucose ratio (mU/mmol) is representative of insulinresistance in the SHR. There is no significant difference (p > 0.05) in theinsuliri:glucose ratio between the control and treatment groups. Values aremeans ± S.E.M., n = 6 for each group.Figure21.Insulin:GlucoseRatioLK)CD403530ED25/‘—a)/‘U)/§20.,‘——‘-15-,--::•b__ci)//-II•10:I4.‘———5-IIII0C12345678910Treatment(weeks)ControlMet30—-—Met100——-—Met30066Table 1. Body weight, metabolic data, and blood pressure in the SHR at week 11(final week) of metformin treatment.67Table 1. Body weight, metabolic data and blood pressures in the SHR at 11 weeks of metformintreatmentControl Metformin Metformin Metformin(n = 6) 30mg/kg/day 100mg/kg/day 300mglkglday(n=6) (n=6) (n=6)Body weight (g) 359 ± 7.8 359.7 ± 3.3 359.7 ± 6.4 337.7 ± 6.5 *Fasting plasma 24.2 ± 3.2 23.4 ± 2.9 20.1 ± 2.3 39.0 ± 14.7tinsulin (mUlL)Fasting plasma 6.8±0.4 7.1 ±0.8 6.6±0.7 9.0±1.5tglucose (mmollL)lnsulin:glucose 3.5 ± 0.3 3.3 ± 0.3 3.1 ± 0.3 5.0 ± 2.5tratio (mU/mmol)Fasting plasma 0.45 ± 0.05 0.34 ± 0.06 0.39 ± 0.03 0.45 ± 0.09triglyceride(mmoLfL)Systolic blood 185.3 ± 8.3 192.3 ± 6.9 198.4 ± 5.1 166.3 ± 9.9tpressure (mmHg)Diastolic blood 117.2 ± 7.8 122.0 ± 4.3 128.1 ± 7.6 97.7 ± 10.5tpressure (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 shownt significantly different from control (p < 0.05)* significantly different from metformin 30 and metformin 100 (p < 0.05)684. DISCUSSION4. 1. Deuterium oxide and EnalapnlResults from the deuterium oxide and enalapril study show that enalaprilsignificantly lowers the systolic blood pressure (fig. 2) over the six weektreatment period in the SHR. This was verified by the direct blood pressuremeasurement (fig. 3), which also showed a significant decrease in the systolicand diastolic blood pressure in the enalapril treated group compared to thecontrol group. However, the insulin and glucose concentrations (fig. 11) werenot significantly different from the control group, demonstrating that lowering theblood pressure in SHR does not have an appreciable effect on insulin resistancein this model.The measurement of insulin and glucose concentrations has previosuslybeen done by euglycemic clamping in rats (25, 43). The clamping techniquemeasures insulin and glucose concentrations over a short period in the conciousrat (approximately 180 mins.). In our experiment we used repeated measures ofinsulin and glucose concentrations, throughout the treatment period (weekly).The euglycemic clamp is a more sensitive method than the repeated measuresbut with repeated measures the ability to detect small changes in insulinrequirements should be increased over time.The results above are in contrast earlier publications that suggest ACEinhibitors improve insulin resistance in hypertensive patients (71, 72, 93).Hypertensive patients were treated with captopril and measured for insulinpromoted glucose uptake (71). Insulin and glucose concentrations weresignificantly reduced and insulin sensitivity was improved by 11 % with captopriltreatment. Similar results were seen in aged insulin-resistant hypertensivepatients who were treated with five different ACE-inhibitors: captopril, enalapril,quinapril, ramipril, and lisinopril (93).69It has been suggested that the potassium sparing (hyperkalemia) effect ofACE-inhibitors may play a role in improving insulin sensitivity. Hyperkalemiahas been implicated in patients with renal failure who are being treated withACE-inhibitors, due to decreased renal potassium clearance via reducedaldosterone levels (74, 75). However, Scandling, et al. have shown thattreatment with enalapril does not acutely impair extrarenal potassiumhomeostasis in men with normal renal function (76). It has also been suggestedthat ACE-inhibitors induce better overall potassium conservation under everydaylife conditions (67). Increases in potassium concentration have been shown topotentiate insulin release from the pancreas in rats (77). Dietary-inducedpotassium deficiency reduced insulin secretion in response to sustainedhyperglycemia in healthy subjects (78). Thus, the potassium sparing effect ofACE-inhibitors may exert a positive influence on glucose tolerance, incombination with their known antihypertensive actions.Our results are supported by the view of Reaven and Chang, who suggestthat hypertension per se does not cause insulin resistance in the SHR (94).Swislocki, et al. have also demonstrated that decreasing blood pressure inhypertensive patients does not necessarily affect abnormal insulin and glucosemetabolism (95).Santoro, et al. showed that chronic ACE-inhibition does not interfere withinsulin’s effect on glucose uptake (96), suggesting that ACE-inhibitors do notaffect insulin resistance. Furthermore, in a comparison study between enalapriland captopril on insulin sensitivity in normotensive individuals, both ACEinhibitors caused an increase in fasting insulin concentrations (97); the oppositeof what one would expect if insulin resistance was decreased. Non-obese, non-insulin-resistant patients with mi Id-to-moderate hypertension, treated withenalapril, had significantly lower blood pressure but there was no effect on70glucose tolerance (98). This study was performed with Japanese patients whichmay only be indicative of ethnic differences, however, the results suggest thatACE-inhibitors may improve insulin sensitivity only in insulin-resistant patients.Glucose intolerance or hyperglycemia is one of the abnormalities linked toSyndrome X. If hypertension directly causes insulin resistance we would expectto see a decrease in insulin and/or a decrease in glucose when the bloodpressure is lowered. Insulin and glucose concentration, as a marker of insulinresistance, 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 D20 do not have an effecton insulin metabolism or 2) the SHR does not have insulin or glucoseabnormalities associated with elevated blood pressure.Ten percent D20 caused a decrease in fasting plasma glucose but nochange in fasting plasma insulin. Despite this, the 10% D20 group failed toshow a significant decrease in systolic blood pressure when compared to theenalapril treated and control groups (fig 2). This was confirmed by direct bloodpressure measurements which were not significantly different in the 10% D20treated group when compared to the control (fig. 3). One possible explanationfor these observations is a decrease in insulin resistance. These results do notsupport a causal link between insulin resistance and blood pressure.Our results are in contrast to the results published by Vasdev, et al. whodemonstrated that 10% D20 was effective in preventing the elevation of bloodpressure in SHR (64). In both experiments the systolic blood pressure wasmeasured indirectly by a tail cuff method and each pressure value was anaverage of 3-4 recordings. The only obvious difference was that in ourexperiments the blood pressures were measured by an observer who wasblinded as to the treatment.71We confirmed that 10% D20 was being administered to the D20 treatedgroup by measuring D20 in the urine (fig. 8). The urine analysis, by infraredspectrometer, for D20 showed an 8% recovery of D20, thus demonstrating thatD20 was being ingested and equilibrating with total body water in the rats. Thefact that it didn’t achieve 10% may be due to limitations in the method ofdetection and/or a contribution of water from food plus metabolism.As well as having a significant decrease in systolic blood pressure theenalapril treated group displayed a significantly higher water intake than thecontrol or 10% D20 groups (fig. 4). Result reported by McLennan, et al. (99)demonstrated that SHR treated with 25 mg/kglday of enalapril drank significantlymore than the untreated controls. This increase in water intake due to chronicconverting enzyme inhibition has also been reported by Ferrone, et al. (100).The significant increase in urine volume is consistent with the higher waterintake observed in the enalapril group (fig. 5) but it is not clear which is theprimary event. The increased water intake may be secondary to increased urinevolume. Enalapril prevents the conversion of angiotensin I to angiotensin II, byinhibiting the angiotensin converting enzyme (101). Angiotensin II is the primaryregulator of aldosterone secretion and aldosterone secretion is known tostimulate sodium and water reabsorption (101). Therefore, enalapril mayprevent sodium and water reabsorption in the SHR by inhibiting aldosteroneaction. This lack of reabsorption may lead to a primary increase in urine volumefollowed by an increase in water intake to maintain water homeostasis in thebody. The mechanism of this effect has yet to be elucidated.The above experiment and other documented results (94) stronglysuggest that lowering blood pressure in animals with hypertension and insulinresistance has no effect on elevated insulin concentrations and glucoseintolerance. Similar results have been shown in human hypertensive patients72(95, 96). It is therefore unlikely that increased blood pressure causes insulinresistance. In order to test whether insulin resistance causes hypertension,SHR were treated with metformin.4.2. Mefformin ExperimentThis second experiment examined if insulin resistance was the cause ofhypertension by altering the blood glucose concentrations with metformin.Kaplan suggests that maneuvers that reduce hyperinsulinemia or improve insulinsensitivity may lower blood pressure (73).The dosing range used in this experiment was chosen to cover the normaldose of metformin given to patients (30 mg/kg/day, 82, 102) as well assupermaximal 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 notsignificantly lowered by any dose of metformin (fig. 14). These results are incontrast to Morgan, et al. who showed a significant decrease in mean arterialpressure in SHR treated with 200-250 mg/kg/day metformin, intraperitoneal (92).In both methods, the measurement of blood pressure was done when the ratswere conscious. The difference occurred in the length of metformin treatment;Morgan’s experiment was acute (7 days on metformin) whereas our experimentwas chronic (10 weeks). It is interesting to note that hypertensive patientsreceiving 30 mg/kglday metformin have a significant decrease in blood pressureover 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 theSHR. The reduction in direct blood pressure in the 300 mg/kg/day metformintreated group may be a manifestation of an interaction between the drug and theadditional stress on the SHR due to the cannulation surgery prior to bloodpressure measurement. It may also be due to an interaction between metforminand halothane because only 60-80% of absorbed halothane is eliminated73unchanged in the first twenty-four hours after inhalation (103). Therefore, thereduction in blood pressure seen in the higher doses of metformin is unlikely torepresent a specific pharmacologic effect of the drug.It has been shown that mefformin enhances the basal rate of glucosetransport (104) thus decreasing blood glucose concentrations in humans (91) atdoses of approximately 30 mg/kg/day. Our results show no significant differencein fasting blood glucose (fig. 18), fasting blood insulin (fig. 20) concentrations, orinsulin/glucose ratios (fig. 21) in the SHR over the dosing range. These resultsshow that metformin has no effect on glucose homeostasis in this model.Explanations for this lack of effect include: 1) metformin does not have an effecton improving glucose concentration in the rat, 2) metformin does not have aneffect in animals that do not express a diabetic state; i.e., SHR do not haveinsulin resistance, 3) our methods were not sensitive enough to detect an effect.The final blood samples taken at week 11 show significantly higherfasting glucose and insulin concentrations in the 300 mg/kg/day metformiri groupcompared to the control group. This effect is opposite to the expected effect ofmetformin, as discussed above. One of the reasons for this discrepancy may bedue to the added effect of the high dose of the drug plus the invasive procedureperformed on the rats prior to blood sampling. Rao suggests that stress fromblood loss may be a source of error in the evaluation of glucose turnover andinsulin sensitivity (105). Another explanation may be an interaction of the drugand anesthetic on glucose and insulin concentrations. It can be concluded thatthe elevated concentrations of glucose and insulin in the final plasma samplesare 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?74Although we failed to show an effect on insulin resistance as a result ofblood pressure reduction and increased insulin sensitivity in the SHR, there isconflicting evidence as to whether this model accurately represents the insulinresistant state as seen in human patients (45, 46). As previously mentioned, theSHR has been described as an appropriate experimental model for bothhypertension and insulin resistance (41, 42, 43). These studies suggest a linkbetween high blood pressure and high insulin levels in this rat model. However,Horl, et al. showed no evidence of peripheral insulin resistance between SHRand WKY rats from the response of skeletal muscle to insulin. After a 12hfasting period, muscle glucagon and glucose levels were almost identical for thetwo groups of rats (46). These results suggest no difference between SHR andits control strain, which further suggests that the SHR is not insulin resistantbecause the WKY does not display insulin resistance. Furthermore, Buchanan,et al. used glucose clamp studies to show that insulin stimulated glucose uptakewas not different between age-matched SHR and WKY rats (45). This evidencewas seen in the SHR when their systolic blood pressure was significantlyelevated, compared to the WKY. Therefore, the SHR may not be an effectivemodel for studying the relationship between hypertension and insulin resistanceas seen in human patients.It is of interest to note that there are also inconsistencies in theassociation between insulin resistance and hypertension in human subjects.O’Brien, et al. looked at patients with insulinoma to determine if hyperinsulinemiacontributed to the pathogenesis of hypertension in the absence of insulinresistance (106). They showed no significant difference in systolic and diastolicblood pressure between the control patients and patients with insulinoma. Theprevalence of hypertension was also similar between patients with insulinoma75and matched controls. It was concluded that hyperinsulinemia could not beimplicated in the genesis of hypertension.4.4. Conclusions1. Ten % 020 decreases fasting plasma glucose concentrations, thus possiblecausing a decrease in insulin resistance.2. 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