Hypertension and diabetes mellitus, which often coexist beyond the chance occurrence, are considered to be important risk factors for cardiovascular morbidity and mortality (1-3). Thus efficient antihypertensive therapy in diabetic patients would be positively beneficial as far as the progression of cardiovascular complications of diabetes are concerned. The objective of any antihypertensive agent to be used in these conditions should not only be reduction in increased blood pressure but also the reversion of diabetes-induced cardiovascular changes, such as macrovascular and microvascular complications and cardiomyopathy, along with satisfactory control over metabolic disequilibrium (4).
Vasodilator like hydralazine (5), prazosin (6), and calcium channel blockers like nifedipine (7) have been shown to prevent STZ-induced cardiomyopathy, cardiac dysfunction, and hyperlipidemias. Cardioselective β-adrenoceptor blockers like atenolol, however, did not improve diabetes-induced cardiac dysfunctions, cardiomyopathy, and hyperlipidemia (6).
Enalapril is widely used as monotherapy for the treatment of essential hypertension (8). It is orally active and reduces blood pressure without any effect on heart rate. It has been reported to cause an improvement in cardiac performance, and no serious adverse effects have been reported (9). Several results from short-term studies have suggested that angiotensin-converting enzyme (ACE) inhibitors may be advantageous over other conventional antihypertensive agents in improving cardiac functions (10). Increasing attention is also being directed toward the effect of antihypertensive drugs on insulin sensitivity in diabetic patients. ACE inhibitors have been shown to improve this sensitivity (11). They may beneficially influence the lipid profile (12).
Hebden et al. (13) reported that streptozotocin (STZ)-diabetic deoxycorticosterone acetate (DOCA)-hypertensive rats may be a useful model for the clinical condition in humans in which diabetes mellitus, hypertension, and atherosclerosis can occur simultaneously. There are, however, controversial reports on the effectiveness of ACE inhibitors in DOCA-hypertensive rats (14-16). In light of these reports, we studied the effect of long-term treatment with enalapril on cardiovascular complications in STZ-induced diabetes and DOCA-induced hypertension in rats.
MATERIALS AND METHODS
Induction of diabetes mellitus and hypertension
Healthy female albino rats of the Wistar strain, weighing 170-200 g, were used in the experiments. Diabetes was induced by a single tail-vein injection of STZ (45 mg/kg) dissolved in citrate buffer (pH 4.5). Hypertension in rats was induced by subcutaneous administration of DOCA in the dose of 5 mg/kg/day, and the DOCA administration continued throughout the 6-week study period. DOCA-Hypertensive animals were fed with 2% salt solution in drinking water. Animals that exhibited blood pressure >150 mm Hg after 10 days of DOCA treatment were considered hypertensive. Animals showing glycosuria (>2%) 48 h after the injection of STZ were considered diabetics for further experiments. Other animals (nondiabetics) administered citrate buffer were considered control.
The animals from the first group were divided into four subgroups: Diabetic, Diabetic enalapril treated, Diabetic hypertensive and Diabetic hypertensive enalapril treated. Similarly the animals from the second group (Nondiabetic) were divided into other subgroups: Control, Enalapril treated, Hypertensive, and Hypertensive enalapril treated. Enalapril was given by oral gavage daily in the dose of 5 mg/kg for 6 weeks. They were monitored throughout the 6-week study period for water intake, food intake, and changes in body weight, blood pressure, heart rate, and mortality. Measurement of blood pressure was done by the tail-cuff method by using a Harvard blood pressure monitor.
Blood sample collection and serum analysis
At the end of 6 weeks, blood samples were carefully collected from the retroorbital plexus of the eye. About 4- to 5-ml blood samples were collected in centrifuge tubes and allowed to clot for 30 min at room temperature. The tubes then were centrifuged at 3,000 rev/min for 30 min. Supernatant clear serum was separated and transferred to small test tubes sealed with aluminum foil and frozen until analysis. Serum samples were analyzed for insulin by radioimmunoassay by using the kit from Bhabha Atomic Research Center, Bombay, India. Serum cholesterol and triglycerides were analyzed by using their respective standard assay kits from Miles India Ltd. Glucose estimation was done in whole blood by using glucose oxidase peroxidase kit from Miles India Ltd., Baroda, India.
Recording of cardiac function
One day after collection of blood samples, animals were killed, hearts were quickly dissected out from the rats and placed in Chenoweth Koelle solution maintained at 37 ± 1°C. They were mounted as per the modified Neely's Working Heart model (5). The aortic outflow was connected to a compliance chamber containing 2-3 ml of air. Hearts were subjected to an afterload of 75 cm water. Hearts were allowed to stabilize for 10 min at the perfusion pressure of 10 cm water. The left ventricular developed pressures (LVDPs) were then recorded at different atrial filling pressures (5-25 cm water). The atrial filling pressure was changed stepwise (5 cm water each time) by changing the height of constant-level reservoirs.
Measurement of myocardial angiotensin-converting enzyme levels
A part of the frozen left ventricular portion of the myocardium was used for the estimation of myocardial ACE levels. The frozen left ventricular portion was brought to room temperature and blotted to remove excess water and ∼25 mg of the tissue with 1 ml of buffer (0.05 M TRIS buffer, pH 7.0, 0.3% bovine serum albumin, 75 mM NaCl, 50 mM ZnSO4) was homogenized. The homogenate was centrifuged at 12,000 rev/min for 20 min, and the supernatant was taken to estimate the ACE levels. ACE levels were measured by the ACE diagnostic kit (Sigma, St. Louis, MO, U.S.A.), and ACE levels were expressed as units/100 g of myocardial tissue.
The results were analyzed statistically by analysis of variance (ANOVA) followed by Tukey's multiple-range test. A value of p < 0.05 was considered statistically significant.
Effect of enalapril treatment on body weight and cardiovascular parameters
Diabetic animals showed a significant loss of the body weight. This loss in body weight was prevented in diabetic rats by enalapril treatment. This loss in body weight was partially prevented in diabetic hypertensive rats (Table 1). A 33% mortality was observed in the diabetic normotensive group. No mortality was observed in any of the other groups.
Mean blood pressure of diabetic animals, diabetic hypertensive animals, and that of nondiabetic hypertensive animals was found to be increased significantly as compared with control animals after 6 weeks of the treatment period. The increase in blood pressure in diabetic hypertensive as well as in hypertensive animals was found to be significantly greater as compared with diabetic animals at the end of 6 weeks. Enalapril treatment prevented the increase in blood pressure in all these groups of animals (Table 1).
Bradycardia was observed in the control diabetic and diabetic hypertensive groups of animals (Table 1). Enalapril treatment significantly prevented this bradycardia in diabetic and diabetic hypertensive animals. Other groups (hypertensive and hypertensive enalapril-treated animals) did not show significant changes in heart rate as compared with the control animals. Control animals treated with enalapril also did not show significant changes in heart rate (Table 1).
LVDP was found to be reduced at higher filling pressure in hearts from diabetic animals as compared with those from controls. Enalapril treatment prevented the depressed LVDP in diabetic animals (Fig. 1a). Hearts from diabetic hypertensive animals showed significantly higher LVDP as compared with diabetic animals but lower than control animals. Depression of cardiac function in these animals also was prevented by enalapril treatment (Fig. 1b). Nondiabetic hypertensive hearts demonstrated a slight increase in LVDP as compared with control hearts, but it was not significant (p > 0.05). Treatment with enalapril in the nondiabetic hypertensive group did not show any alteration in LVDP. Diabetic and diabetic hypertensive animals showed an increase in the index of hypertrophy, calculated as wet heart weight/body weight ratio, and enalapril treatment was found to prevent this hypertrophy of heart in both the groups of animals (Table 1).
Glucose and insulin levels
Injection of STZ produced a significant increase in blood glucose levels in all diabetic animals. The control diabetic hypertensive animals showed a significant reduction in blood glucose level as compared with the diabetic control. However, in nondiabetic hypertensive animals, the glucose level did not alter significantly. Enalapril treatment failed to alter the blood glucose level significantly in any of the groups (Table 2).
Injection of STZ also produced a state of hypoinsulinemia in diabetic and diabetic hypertensive animals as compared with their respective control animals. However, significantly higher insulin levels were observed in diabetic hypertensive animals as compared with diabetic animals. DOCA administration in nondiabetic rats produced a significant increase in insulin levels. Enalapril treatment, however, did not cause any alteration in insulin levels in any of the treated groups (Table 2).
Lipid profile and cardiac ACE levels
Cholesterol, triglyceride, and total lipid levels in serum were found to be increased in diabetic as well as diabetic hypertensive animals as compared with controls (Table 2). DOCA administration also produced an increase in cholesterol and triglyceride levels, but it was not as high as observed in diabetic animals. Enalapril treatment produced a significant decrease in increased cholesterol and triglycerides in diabetic and diabetic hypertensive animals. Total serum lipid levels were also found to be decreased by enalapril; however, the difference was not found to be significant.
Cardiac ACE levels were found to be significantly increased in the hearts obtained from diabetic rats as compared with controls. This increased in ACE level was found to be prevented by pretreatment with enalapril (Table 2). Administration of DOCA in control and diabetic rats did not produce any alteration in cardiac ACE levels.
In this investigation, 45 mg/kg STZ was found to produce not only hyperglycemia, hypoinsulinemia, and hyperlipidemia but also a significant loss of body weight (Tables 1 and 2). These results are consistent with those reported earlier (7). Administration of DOCA in nondiabetic animals did not produce any change in blood glucose levels. However, administration of DOCA in diabetic animals caused reduction in increased blood glucose levels in these animals without any alteration in insulin level. These finding are consistent with those reported by Dai and McNeill (17). It is possible that DOCA increases insulin sensitivity in diabetics. Enalapril failed to reduce the blood glucose levels in any of the groups of animals. Enalapril does not seem to alter the glucose tolerance or insulin release or sensitivity. It has been reported by many workers that ACE inhibitors like captopril and lisinopril improve insulin sensitivity, which leads to enhanced insulin-mediated glucose disposal, causing reduction in blood glucose level (15).
Enalapril treatment prevented an increase in blood pressure in all the diabetic or hypertensive animals or both. A number of factors are involved in the pathogenesis of hypertension in diabetes mellitus such as sodium retention, extracellular fluid volume expansion, altered activity of sympathetic nervous and renin-angiotensin systems, and increased vascular reactivity toward noradrenaline and angiotensin II (18). Administration of DOCA to animals with increased sodium supplementation caused the development of hypertension after 10 days. The mechanism by which DOCA causes an increase in blood pressure may be due to sodium retention and the expansion of plasma volume. The other mechanisms responsible for DOCA-salt-induced hypertension may be activation of brain renin-angiotensin system (19), vasopressin (20), or an increase in basal plasma noradrenaline levels (21). The inhibition of ACE seems to be the main mechanism involved. Along with reduction in vasoconstrictor agency, the probable increase of bradykinin and in turn that of vasodilatory eicosanoids may be involved in reducing the increased blood pressure.
Bradycardia has been frequently observed in STZ-diabetic rats (22). In our investigation. diabetic animals were found to have bradycardia as compared with control. DOCA administration did not produce any changes in heart rate in any groups.
Cardiac functions were found to be lower in diabetic animals. This was observed at higher filling pressure. Treatment with enalapril was able to prevent cardiac depression in both diabetic and diabetic hypertensive animals. Treatment with DOCA in nondiabetic animals by itself did not produce an alteration in LVDP in response to an increase in left atrial filling pressure. However, the cardiac depression observed in diabetic animals was found to be prevented when these animals were administered DOCA. Dai and McNeill (17) also reported that DOCA-induced hypertension did not significantly aggravate the severity of myocardial dysfunction associated with STZ-induced diabetes. The overall cardiovascular function involves the integrated influence of intrinsic myocardial performance, peripheral vascular properties, and modulations by nervous reflexes (23). DOCA is known to cause an increase in sympathetic activity, a shift in baroreflex responses, and an increase in cardiac afterload (11). It is possible that the attenuated hypertensive effect of DOCA in STZ-diabetic rats may be responsible for this protective effect (in severity of cardiac dysfunction) in diabetic rats.
Among various other factors responsible for a decrease in cardiac function, hyperlipidemia and atherosclerosis also appear to be of prime importance. Patients with diabetes are found to have increased plasma levels of triglycerides, cholesterol, free fatty acids, and phospholipids. Insulin has an inhibitory action on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key rate-limiting enzyme in the metabolism of cholesterol rich low-density lipoprotein (LDL) particles. In our study, diabetic animals showed hypoinsulinemia, which may be responsible for the increase in triglyceride levels. It was observed that triglycerides and cholesterol were increased in diabetic and diabetic hypertensive animals. Enalapril treatment reduced triglycerides, cholesterol, and LDL in diabetic and diabetic hypertensive animals.
The synthesis of endogenous angiotensinogen, renin, and ACE has been reported within blood vessels and the heart (24-26). In experiments with pressure-overload left ventricular hypertrophy, ACE activity in the ventricle has been reported to be increased under the condition of increased pressure loading (27). The expression of ACE messenger RNA (mRNA) was reported to be increased threefold in hearts with ventricular hypertrophy, which subsequently increases ACE and angiotensin II. Enalapril in our investigation was found to produce a decrease in the index of hypertrophy, as calculated by the ratio of wet heart weight to body weight, which was higher in diabetic and diabetic hypertensive animals. The increase in hypertrophy was found to be associated with the increase in ACE levels in hearts from diabetic and diabetic hypertensive animals. The regression of cardiac hypertrophy and decrease in ACE levels caused by enalapril treatment in diabetic and diabetic hypertensive rats suggest a direct role of ACE activity in cardiac hypertrophy, which may be different from its hemodynamic effects. This is further supported by the earlier reports that indicated that the renin-angiotensin system is one of the most important systems for the pathogenesis of cardiac interstitial fibrosis. Furthermore, in experimental studies, angiotensin II showed mitogenic properties and was reported to promote growth of cardiac myocytes (28). Studies with ACE inhibitors like captopril and enalapril confirmed that these agents cause regression of cardiac hypertrophy in animal models of hypertension.
In conclusion, our data suggest that treatment with enalapril prevented an increase in blood pressure and heart weight. Decrease in the heart rate, reduction in LVDP, and increase in intracardiac activity were observed in diabetic rats; these were also prevented by enalapril treatment. Enalapril had no effect on plasma glucose and did not modify plasma insulin levels in diabetic animals. The effect of STZ and DOCA together were not additive on the investigated parameters, and enalapril was similarly efficient in diabetic and diabetic hypertensive animals.
Acknowledgment: This work was supported by a research grant from the Council of Scientific and Industrial Research, New Delhi. The enalapril was generously supplied by ICI Pharmaceuticals, London, U.K., as a gift. We sincerely acknowledge both of them.
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