Effect of testosterone on insulin stimulated IRS1 ser phosphorylation in primary rat myotubes - A potential model for PCOS-related insulin resistance

Background: Polycystic ovary syndrome (PCOS) is characterized by a hyperandrogenic state and frequently develops skeletal muscle insulin resistance. We determined whether testosterone adversely affects insulin action by increasing serine phosphorylation of IRS-1^636/639 in differentiated rat skeletal muscle myotubes. The phosphorylation of Akt, mTOR, and S6K, downstream targets of the PI3-kinase-IRS-1 complex were also studied. Methods: Primary differentiated rat skeletal muscle myotubes were subjected to insulin for 30 min after 16-hour pre-exposure to either low (20 ng/ml) or high (200 ng/ml) doses of testosterone. Protein phosphorylation of IRS-1 Ser^636/639, Akt Ser⁴⁷³, mTOR-Ser²⁴⁴⁸, and S6K-Thr³⁸⁹ were measured by Western blot with signal intensity measured by immunofluorescence. Results: Cells exposed to 100 nM of insulin had increased IRS-1 Ser^636/639 and Akt Ser⁴⁷³ phosphorylation. Cells pre-exposed to low-dose testosterone had significantly increased insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation (p,0.05), and further increased insulin-induced IRS-1 Ser^636/639 phosphorylation (p = 0.042) compared to control cells. High-dose testosterone pre-exposure attenuated the insulin-induced mTOR-Ser²⁴⁴⁸ and S6K-Thr³⁸⁹ phosphorylation. Conclusions: The data demonstrated an interaction between testosterone and insulin on phosphorylation of intracellular signaling proteins, and suggests a link between a hyperandrogenic, hyperinsulinemic environment and the development of insulin resistance involving serine phosphorylation of IRS-1 Ser^636/639. These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance.