Abstract-Clinical reports indicate that patients with primary aldosteronism commonly have impaired glucose tolerance; however, the relationship between aldosterone and insulin signaling pathway has not been clarified. In this study, we examined the effects of aldosterone treatment on insulin receptor substrate-1 expression and insulin signaling pathway including Akt phosphorylation and glucose uptake in rat vascular smooth muscle cells. Insulin receptor substrate-1 protein expression and Akt phosphorylation were determined by Western blot analysis with anti-insulin receptor substrate-1 and phosphorylated-Akt antibodies, respectively. Glucose metabolism was evaluated using 3 H-labeled 2-deoxy-D-glucose uptake. Aldosterone (1-100 nmol/L) dose-dependently decreased insulin receptor substrate-1 protein expression with a peak at 18 hours (nϭ4). Aldosterone-induced degradation of insulin receptor substrate-1 was markedly attenuated by treatment with the selective mineralocorticoid receptor antagonist eplerenone (10 mol/L; nϭ4). Furthermore, degradation was blocked by the Src inhibitor PP1 (20 mol/L; nϭ4). Treatment with antioxidants, N-acetylcysteine (10 mmol/L), or ebselen (40 mol/L) also attenuated aldosterone-induced insulin receptor substrate-1 degradation (nϭ4). In addition, proteasome inhibitor MG132 (1 mol/L) prevented insulin receptor substrate-1 degradation (nϭ4). Aldosterone treatment abolished insulin-induced Akt phosphorylation (100 nmol/L; 5 minutes; nϭ4). Furthermore, aldosterone pretreatment decreased insulin-stimulated (100 nmol/L; 60 minutes; nϭ4) glucose uptake by 50%, which was reversed by eplerenone (10 mol/L; nϭ4). These data indicate that aldosterone decreases insulin receptor substrate-1 expression via Src and reactive oxygen species stimulation by proteasome-dependent degradation in vascular smooth muscle cells; thus, aldosterone may be involved in the pathogenesis of vascular insulin resistance via oxidative stress. Key Words: aldosterone Ⅲ oxidative stress Ⅲ insulin receptor substrate-1 Ⅲ insulin resistance Ⅲ type 2 diabetes mellitus Ⅲ metabolic syndrome Ⅲ eplerenone I nsulin resistance is a key attribute of type 2 diabetes and the metabolic syndrome. 1,2 Systemic glucose metabolism is maintained in the liver and skeletal muscle, and in the insulin resistant state, insulin-stimulated glucose uptake is attenuated and accompanied with subsequent increases in blood glucose concentration. High blood glucose concentration induces secretion of insulin from the pancreas and results in hyperinsulinemia. Both hyperglycemia and hyperinsulinemia affect the vasculature and are associated with microangiopathy, including retinopathy and nephropathy, and macroangiopathy, including cardiovascular disease and atherosclerosis. 3 Insulin resistance in the vasculature might also affect systemic glucose metabolism. However, the involvement of normal insulin signaling contributes to arteriosclerosis. 4,5 On the other hand, serine phosphorylation and degradation of insulin receptor substrate-1 (IRS-1) is a possible...

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, plays a role in endothelial dysfunction, an initial step of atherosclerosis. Advanced glycation end products (AGEs) also contribute to accelerated atherosclerosis. However, a pathophysiological crosstalk between ADMA and AGEs remains unclear. In this study, we investigated the relationship between ADMA and AGE level in patients with end-stage renal disease (ESRD) due to diabetic nephropathy. We also examined whether and how AGEs increased ADMA generation by cultured endothelial cells (ECs). Plasma ADMA levels were positively associated with serum AGE level and were inversely correlated with endothelial function determined by flow-mediated vasodilatation. AGEs dose dependently increased reactive oxygen species (ROS) generation in ECs, which was blocked by antisense DNA raised against receptor for AGEs (RAGE). Furthermore, AGEs decreased messenger RNA (mRNA) level of dimethylarginine dimethylaminohydrolase (DDAH)-II, an enzyme for ADMA degradation, reduced its total enzymatic activity and resultantly increased ADMA, all of which were completely blocked by an antioxidant, N-acetylcysteine. These results suggest that the AGE-RAGE-mediated ROS generation could be involved in endothelial dysfunction in diabetic ESRD patients partly by increasing the ADMA generation via suppression of DDAH activity in ECs.

BackgroundStudies were performed to determine if early treatment with an angiotensin II (Ang II) receptor blocker (ARB), olmesartan, prevents the onset of microalbuminuria by attenuating glomerular podocyte injury in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with type 2 diabetes mellitus.MethodsOLETF rats were treated with either a vehicle, olmesartan (10 mg/kg/day) or a combination of nonspecific vasodilators (hydralazine 15 mg/kg/day, hydrochlorothiazide 6 mg/kg/day, and reserpine 0.3 mg/kg/day; HHR) from the age of 7–25 weeks.ResultsOLETF rats were hypertensive and had microalbuminuria from 9 weeks of age. At 15 weeks, OLETF rats had higher Ang II levels in the kidney, larger glomerular desmin-staining areas (an index of podocyte injury), and lower gene expression of nephrin in juxtamedullary glomeruli, than nondiabetic Long-Evans Tokushima Otsuka (LETO) rats. At 25 weeks, OLETF rats showed overt albuminuria, and higher levels of Ang II in the kidney and larger glomerular desmin-staining areas in superficial and juxtamedullary glomeruli compared to LETO rats. Reductions in mRNA levels of nephrin were also observed in superficial and juxtamedullary glomeruli. Although olmesartan did not affect glucose metabolism, it decreased blood pressure and prevented the renal changes in OLETF rats. HHR treatment also reduced blood pressure, but did not affect the renal parameters.ConclusionsThis study demonstrated that podocyte injury occurs in juxtamedullary glomeruli prior to superficial glomeruli in type 2 diabetic rats with microalbuminuria. Early treatment with an ARB may prevent the onset of albuminuria through its protective effects on juxtamedullary glomerular podocytes.

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Ischemia/reperfusion injury is the leading cause of acute tubular necrosis. Nitric oxide has a protective role against ischemia/reperfusion injury; however, the role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, in ischemia/reperfusion injury remains unclear. ADMA is produced by protein arginine methyltransferase (PRMT) and is mainly degraded by dimethylarginine dimethylaminohydrolase (DDAH). Here we examined the kinetics of ADMA and PRMT and DDAH expression in the kidneys of ischemia/reperfusion-injured mice. After the injury, DDAH-1 levels were decreased and renal and plasma ADMA values were increased in association with renal dysfunction. Renal ADMA was correlated with 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress. An antioxidant, N-acetylcysteine, or a proteasomal inhibitor, MG-132, restored these alterations. Infusion of subpressor dose of ADMA exacerbated renal dysfunction, capillary loss, and tubular necrosis in the kidneys of ischemia/reperfusion-injured wild mice, while damage was attenuated in DDAH transgenic mice. Thus, ischemia/reperfusion injury–induced oxidative stress may reduce DDAH expression and cause ADMA accumulation, which may contribute to capillary loss and tubular necrosis in the kidney.