Effect of type-1 diabetes mellitus on the regulation of insulin and endothelin-1 receptors in rat hearts

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.     

Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.     

Regulation of insulin-like growth factor-1 by the renin-angiotensin system during regression of cardiac eccentric hypertrophy through angiotensin-converting enzyme inhibitor and AT1 antagonist

Angiotensin II (Ang II) mediates its effects through its non-tyrosine-kinase G protein coupled Ang-II type 1 receptor (AT1). Growing evidence indicates that a functional insulin-like growth factor-1 (IGF-1) tyrosine kinase receptor is required for Ang-II-induced mitogenesis. Along with Ang II, we have previously shown that changes in IGF-1 receptor binding at myofibers are causative agents for cardiac eccentric hypertrophy. This study investigated the interaction of the renin-angiotensin system with the IGF-1 receptor during the development and regression of cardiac hypertrophy. Alterations in IGF-1 binding were evaluated in the CHAPS-pretreated perfused heart. Four weeks of aortocaval shunt increased relative heart mass by 76% without a major change in body mass or systolic blood pressure. Binding studies showed that IGF-1 has a higher affinity for the cardiac myofibers of shunt than sham rats. Two weeks of treatment with the angiotensin-converting enzyme (ACE) inhibitor captopril (0.5 g/L in drinking water) or the AT1-antagonist losartan (10 mg/(kg x day)) reduced cardiac hypertrophy by 54 and 42%, respectively. However, while both ACE inhibition and AT1-antagonist treatments produced equivalent regression in ventricular hypertrophy, captopril was more efficacious than losartan in the regression of atrial hypertrophy. Regression of cardiac hypertrophy in the shunt by either captopril or losartan was accompanied with a reduction or normalization of the elevated IGF-1 affinity. Thus, the induction and regression of cardiac eccentric hypertrophy seems to be largely dependent on cross talk between the renin-angiotensin system and the IGF-1 axis at the receptor level.     

Effect of elevated blood pressure, renin and aldosterone on renal sodium handling in two-kidney Goldblatt hypertensive rats

The effect of elevated blood pressure, renin and aldosterone on renal Na+ retention in two-kidney Goldblatt hypertensive rats were investigated. The technique involved retrograde perfusion from the renal veins via the kidneys, and then through the renal arteries and dorsal aorta. Sodium retention in the stenosed kidney of 7 and 30-60 days post-stenosis hypertensive rats was 82 and 70% higher than in normotensive sham-operated rats respectively. Sodium rention in the clipped kidney, 1 day post-stenosis, was insignificant. However, the contralateral kidney of the 1 day post-stenosis rats retained 27% more Na+. The 1 and 7 days post-stenosis rats had higher plasma aldosterone concentrations than controls, while the 30-60 days post-stenosis rats showed lower levels. The plasma renin activity of the 1 day post-stenosis rats showed 65% higher activity than the sham controls with no significant change in the 30-60 days post-stenosis. Therefore Na+ retention may be mediated by aldosterone in the 7 days post-stenosis rats. Natriuresis in the non-stenosed kidneys of both the 7 and 30-60 days post-stenosis rats may be modulated by an increase in filtration rate due to hypertrophy.     

Role of ouabain and diuretics on sodium, potassium and chloride retention in perfused rat kidney

A novel in situ kidney perfusion technique is described in Sprague-Dawley rats. The procedure involves retrograde perfusion from the renal veins via the kidneys, and then through the renal arteries and dorsal aorta. Ouabain (15 mM) in perfusate increased Na retention by 92%, decreased K retention by 53% and produced no effect on Cl retention. Ethacrynic acid (1 mM) in perfusate decreased Na retention by 52%, increased K retention by 105% and decreased Cl retention by 27%. Furosemide (1.5 mM) in perfusate decreased Na retention by 52%, increased K retention by 47% and decreased Cl retention by 56%. The Na-K-ATPase pump localized at the peritubular side of the proximal tubule cell is ouabain sensitive and Mg dependent. An Na-K pump responsible for Na influx and K effux exists at the luminal side of the proximal tubule cell and is ethacrynic acid and furosemide sensitive.     

Binding and distribution of three prototype calcium channel blockers in perfused rat liver

This work represents a study of the binding and distribution of three different calcium channel blockers in the Sprague-Dawley rat liver, using an in situ perfusion technique. For this purpose, [3H] desmethoxyverapamil, [3H] PN200-110 (isradipine) and [3H] azidopine were used as binding probes interacting with calcium channels. The perfusion steps of the liver involved both portal vein and thoracic inferior vena cava cannulations as inlet and outlet respectively. The subhepatic inferior vena cava was ligated to prevent leakage of the perfusate. Buffer, containing the tracer drug, was administered via the portal vein at a rate of l mL/min and perfusate collected at the same rate within specified time intervals during 50 min. The concentration of the tracer solutes in the perfusate's outlet increased with time, and steady state was observed for all tracers at 40 min. The effect of adding cold isradipine to tracer desmethoxyverapamil, or cold verapamil to tracer PN200-110 were also assessed. First order rate constants for hepatocellular influx, efflux and calcium channel binding of the tracer substances were obtained using a simplified model from Goresky et al. [25]. These constants were mathematically manipulated and changed into permeability constants, second order binding constants, and residency times.Tracer solute influx across hepatocellular membranes is solubility-diffusion controlled, is inversely related to the molecular weights and is different in value from the efflux constants. Cold isradipine reduced the binding constant of desmethoxyverapamil by 36%, while cold verapamil reduced the binding constant of PN200-110 by 23%. Azidopine cellular distribution was low, however, binding to its receptor was analogous to desmethoxyverapamil and PN200-110. Moreover, PN200-110 had the highest residency time with no effect of cold verapamil on its receptor binding, while desmethoxyverapamil had the lowest residency time which significantly increased in the presence of cold isradipine.     

Effect of diabetes on natriuresis in the presence of ouabain and ethacrynic acid in perfused rat kidney

1. The effect of diabetes on renal sodium retention was investigated. 2. The technique involved retrograde perfusion from the renal veins via the kidneys, and then through the renal arteries and dorsal aorta. 3. Sodium retention by diabetic rat kidney was 58% lower than that in the normal rats. 4. Ouabain (15 mM) in perfusate increased sodium retention by 30% in normal rat kidney as compared to a 54% increase in diabetic rat kidney. 5. Ethacrynic acid (1 mM) in perfusate resulted in a 42% reduction in sodium retention in the normal rat kidney as compared to a 43% decrease in the diabetic rat kidney. 6. Control of hyperglycemia in diabetic rats with insulin therapy resulted in sodium retention that is not significantly different from that of normal rats. 7. The results suggest that diabetes has no effect on the peritubular ouabain-sensitive Na--K-ATPase pump, or the luminal ethacrynic acid-sensitive Na-K counter transport pump. Furthermore, the data suggest a reversible effect of diabetes on sodium retention during insulin therapy.     

Endothelium and myocyte cellular insulin receptor alterations in a rat model of myocardial infarction

Ischemic heart disease is considered to be one of the leading causes of death in adults. While extensive research on mechanisms contributing to the pathogenesis of myocardial infarction (MI) has been underway, it is not known whether insulin receptor characteristics and postreceptor signaling have been fully addressed as yet. Present work attempts to investigate whether the remodeling process effectively induces alteration(s) in insulin-binding characteristics at the coronary endothelium and cardiomyocytes using a rat heart model of MI. MI was induced by ligation of the left anterior descending coronary artery of adult male Sprague-Dawley rats. Two animal groups were used in the study: (i) sham-operated CHAPS-untreated and CHAPS-treated, and (ii) MI CHAPS-untreated and MI CHAPS-treated. A physical model describing 1:1 stoichiometry of reversible insulin binding to its receptors present on the endothelium and at cardiomyocytes after CHAPS treatment was considered for data analysis. Quantitation of the collected effluents after heart perfusion, the inlet at the aortic and outlet at the coronary sinus sites, were curve fitted using a first-order Bessel function, which determines the binding constants (k(n)), the reversible constant (k(-n)), the dissociation constant (k(d) = k(-n)/k(n)), and the residency time constant (tau = 1/k(-n)). In addition, hearts were excised, separated into right and left ventricles, and individually weighed, and areas of infarcted regions were measured. Results of the MI group showed significant increases in relative heart mass, left ventricle mass, and right ventricle mass normalized to total body mass. MI induced severe ischemia and irreversible myocardial injury as assessed by planimetry and histologic studies. The data showed differences in insulin receptor affinities at the endothelial and cardiac myocytes in the sham and in the MI-operated rats. The observed reduction in the binding affinity of insulin at the myocyte postinfarction may explain the pathogenic role of insulin in ischemic heart disease and, hence, resistance. Therefore, insulin administration during and post MI might be cardioprotective.     

Role of glucagon-like peptide-1 and its agonists on early prevention of cardiac remodeling in type 1 diabetic rat hearts

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted from intestinal L cells upon nutrients ingestion, and is currently used for treating diabetes mellitus. It plays an important role in receptor modulation and cross talk with insulin at the coronary endothelium (CE) and cardiomyocytes (CM) in diabetic type 1 rat heart model. We studied the effects of insulin, GLP-1 analogues (exendin-4), and dipeptidyl peptidase-IV (DPP-IV) inhibitor on GLP-1 cardiac receptor modulation. The binding affinity of GLP-1 to its receptor on CE and CM was calculated using a rat heart perfusion model with [(125)I]-GLP-1(7-36). Tissue samples from the heart were used for immunostaining and Western blot analyses. GLP-1 systemic blood levels were measured using ELISA. GLP-1 binding affinity (τ) increased on the CE (0.33 ± 0.01 vs. 0.25 ± 0.01 min; p < 0.001) and decreased on the CM (0.29 ± 0.02 vs. 0.43 ± 0.02 min; p < 0.001) in the diabetic non-treated rats when compared to normal. There was normalization of τ back to baseline on the CE and CM levels with insulin and DPP-IV inhibitor treatment, respectively. Histological sections and immunofluorescence showed receptor up-regulation in diabetic rats with significant decrease and even normalization with the different treatment strategies. Systemic GLP-1 levels increased after 14 days of diabetes induction (10 ± 3.7 vs. 103 ± 58 pM; p = 0.0005). In conclusion, there is a significant GLP-1 receptor affinity modulation on the CE and CM levels in rats with diabetes type 1, and a cross talk with GLP-1 analogues in early prevention of cardiac remodeling.     

Effects of Selenium and Exendin-4 on Glucagon-Like Peptide-1 Receptor, IRS-1, and Raf-1 in the Liver of Diabetic Rats

Selenium and exendin-4 exert antidiabetic effects by unknown mechanisms. Herein, we investigated their effects on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and Raf-1 in the livers of rats with streptozotocin-induced diabetes. Diabetic rats were injected intraperitoneally with exendin-4 (0.03?μg/kg body weight) twice daily or treated with 5?ppm selenium as sodium selenite in drinking water for 4?weeks. Both selenium and exendin-4 reduced the hyperglycemia in diabetic rats. Induction of diabetes mellitus resulted in decreased level of GLP-1R and increased levels of IRS-1 and Raf-1 in the liver. Treatment of diabetic rats with selenium or exendin-4 resulted in increased level of GLP-1R and decreased levels of IRS-1 and Raf-1 in the liver, compared with the levels in diabetic rats. Therefore, the antidiabetic actions of selenium and exendin-4 involve their effects on GLP-1R, IRS-1, and Raf-1 levels in the liver.