High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes:: possible implications for insulin resistance in type 2 diabetes

Objective: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. Design and methods: Rat adipocytes were cultured for 24h in different glucose concentrations with 10(4) muU/ml of insulin or without insulin. After washing, I-125-insulin binding, basal and acutely insulin-stimulated D-[C-14] glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. Results: High glucose (15 and 2 5 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by similar to40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by similar to 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by similar to20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination. shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by similar to 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. Conclusions: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose. it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.