u-0126 and Hyperinsulinism

Glomerular hyperfiltration and mesangial expansion have been described in mouse models of a hyperinsulinemic early stage of type 2 diabetes mellitus (DM). Large-conductance Ca(2+)-activated K(+) channels (BK) have been linked to relaxation of human mesangial cells (MC) and may contribute to MC expansion and hyperfiltration. We hypothesized that high insulin levels increase BK activity in MC by increasing the number and/or open probability (P(o)) of BK in the plasma membrane. With the use of the patch-clamp technique, BK activity was analyzed in cultured MC exposed to normal insulin (1 nM) and high insulin (100 nM) for a 48-h period. The mean P(o) and the percentage of patches (cell attached) with detected BK increased by 100% in the insulin-treated cells. Real-time PCR revealed that insulin increased mRNA of BK-alpha. Western blot revealed an insulin-stimulated increase in BK-alpha from both total cellular and plasma membrane protein fractions. The mitogen-activated protein kinase (MAPK) inhibitors PD-098059 and U-0126 attenuated the insulin-induced increase in BK-alpha expression. PD-098059 inhibited insulin-stimulated phosphorylation of extracellular signal-regulated kinase 1/2 in MC. An insulin-stimulated increase also was found for total cellular BK-beta(1), the accessory subunit of BK in MC. A similar increase in BK-alpha mRNA and protein was evoked by an insulin-like growth factor I analog. Glomeruli, isolated from hyperinsulinemic early stage type 2 DM mice, exhibited increased BK-alpha mRNA by real-time PCR and protein by immunohistochemical staining and Western blot. These results indicate that insulin activates BK in the plasma membrane of MC and stimulates, via MAPK, an increase in cellular and plasma membrane BK-alpha.

Topics: Animals; Butadienes; Cells, Cultured; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Dietary Fats; Enzyme Inhibitors; Flavonoids; Humans; Hyperinsulinism; Hypoglycemic Agents; Insulin; Insulin-Like Growth Factor I; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channel beta Subunits; Male; MAP Kinase Signaling System; Mesangial Cells; Mice; Mice, Inbred C57BL; Nitriles; Patch-Clamp Techniques; RNA, Messenger

Pancreatic islet fibrosis observed in Type 2 diabetes is one of the major factors leading to progressive beta-cell loss and dysfunction. Despite its importance, the mechanism of islet-restricted fibrogenesis associated with pancreatic stellate cell (PSC) activation and proliferation remains to be defined. Therefore, we studied whether the islet-specific environment represented by hyperglycemia and hyperinsulinemia had additive effects on the activation and proliferation of cultured rat PSCs. Cells were stimulated to activate and proliferate with glucose and insulin, either individually or concomitantly. Both stimuli promoted PSC proliferation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation independently, but an additive effect was also demonstrated. Blockade of ERK signaling by the mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, suppressed both glucose- and insulin-induced ERK 1/2 phosphorylation and PSC proliferation. Glucose and insulin-induced ERK 1/2 phosphorylation also stimulated connective tissue growth factor gene expression. Thus, hyperglycemia and hyperinsulinemia are two crucial mitogenic factors that activate and proliferate PSCs, and the presence of both states will amplify this response.

Topics: Animals; Blood Glucose; Blotting, Western; Butadienes; Cell Proliferation; Cells, Cultured; Connective Tissue Growth Factor; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibrosis; Gene Expression; Glucose; Hyperglycemia; Hyperinsulinism; Immediate-Early Proteins; Insulin; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Male; MAP Kinase Signaling System; Nitriles; Pancreas; Phosphorylation; Rats; Rats, Sprague-Dawley