Decreased in situ insulin receptor dephosphorylation in hyperglycemia-induced insulin resistance in rat adipocytes

The regulation of insulin receptor (IR) tyrosine (tyr) phosphorylation is a key step in the control of insulin signaling. Augmented IR tyr dephosphory lation by protein tyrosine phosphatases (PTPs) may contribute to insulin re sistance. To investigate this possibility in hyperglycemia-induced insulin resistance, primary cultured rat adipocytes were rendered insulin-resistant by chronic exposure (18 h) to 15 mmol/l glucose combined with 10(-7) mol/l insulin. Insulin-resistant adipocytes showed a decrease in insulin sensiti vity and a maximum response of 2-deoxyglucose uptake, which was associated with a decrease in maximum insulin-stimulated IR tyr phosphorylation in sit u. To assess tyr dephosphorylation, IRs of insulin-stimulated permeabilized adipocytes were labeled with [gamma-P-32]ATP and chased for 2 min with unl abeled ATP in the presence of EDTA. In a nonradioactive protocol, insulin-s timulated adipocytes were permeabilized and exposed to EDTA and erbstatin f or 2 min, and IRs mere immunoblotted with anti-phosphotyrosine (pY) antibod ies. Both methods showed a similar diminished extent of IR tyr dephosphoryl ation in resistant cells. Immunoblotting of four candidate IR-PTPs demonstr ated no change in PTP1B or the SH2 domain containing phosphatase-2 (SHP-2) whereas a significant decrease in leukocyte antigen-related phosphatase (LA R) (51 +/- 3% of control) and an increase in PTP-alpha (165 +/- 16%) were f ound. Activity of immunoprecipitated PTPs toward a triple tyr phosphorylate d IR peptide revealed a correlation with protein content for PTP1B, SHP-2, and LAR but a decrease in apparent specific activity of PTP-alpha. The data indicate that decreased IR tyr phosphorylation in hyperglycemia-induced in sulin resistance is not due to enhanced dephosphorylation. The diminished I R tyr dephosphorylation observed in this model is associated with decreased LAR protein content and activity.