GLUCOCORTICOID REGULATION OF INSULIN-RECEPTOR AND SUBSTRATE IRS-1 TYROSINE PHOSPHORYLATION IN RAT SKELETAL-MUSCLE INVIVO

To test the hypothesis that glucocorticoid-induced insulin resistance might originate from abnormalities in insulin receptor signaling, we i nvestigated the effects of glucocorticoids on in vivo tyrosine phospho rylation of the insulin receptor and the insulin receptor substrate IR S-1 in rat skeletal muscle. Male Sprague-Dawley rats were treated with cortisone (100 mg/kg for 5 d) and compared to pair-fed controls. Cort isone treatment of rats resulted in both hyperglycemia and hyperinsuli nemia. Anesthetized animals were injected with 10 U/kg insulin via car diac puncture and, after 2 min, hindlimb muscles were removed, snap-fr ozen, and homogenized in SDS. Protein tyrosine phosphorylation was stu died by immunoblotting with phosphotyrosine antibody. Insulin receptor s and substrate IRS-1 were identified and quantified with specific ant ibodies. Cortisone treatment increased the amount of insulin receptor protein by 36%, but decreased the total level of receptor tyrosine pho sphorylation (69+/-4% of control, P < 0.05). The decreased level of re ceptor phosphorylation was explained by a reduced number of receptors containing phosphorylated tyrosine residues (64.6+/-5% of control, P< 0.05). Glucocorticoid excess decreased skeletal muscle IRS-1 content b y 50%, but did not significantly alter the total level of IRS-1 tyrosi ne phosphorylation. The apparent M(r) of IRS-1 was reduced by approxim ately 10 kD. Treatment with protein phosphatase-2A reduced IRS-1 M(r) in control but not in glucocorticoid-treated muscle indicating that th e lower M(r) likely results from lower phosphoserine and/or phosphothr eonine content. To investigate the role of hyperinsulinemia in the glu cocorticoid response, rats were made insulin-deficient with streptozot ocin (100 mg / kg, i.p.). Subsequent treatment with cortisone for 5 d had no effects on insulin levels, tyrosine phosphorylation of insulin receptors or IRS-1, or the M(r) of IRS-1. In conclusion, glucocorticoi d-treated skeletal muscle is characterized by: (a) decreased total tyr osine phosphorylation of insulin receptors as a result of a reduction in the pool of receptors undergoing tyrosine phosphorylation; (b) decr eased IRS-1 content and reduced serine and/or threonine phosphorylatio n of IRS-1. Glucocorticoid-induced hyperinsulinemia appears to be esse ntial for the development of these alterations.