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 Ser(473), mTOR-Ser(2448), and S6K-Thr(389) 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 Ser(473) phosphorylation. Cells pre-exposed to low-dose testosterone had significantly increased insulin-induced mTOR-Ser(2448) and S6K-Thr(389) 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-Ser(2448) and S6K-Thr(389) 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.
Allemand
Michael C
MC
Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Mayo Clinic Graduate School of Medicine, Rochester, Minnesota, United States of America.
Irving
Brian A
BA
Asmann
Yan W
YW
Klaus
Katherine A
KA
Tatpati
Laura
L
Coddington
Charles C
CC
Nair
K Sreekumaran
KS
D000818:Animals; D002454:Cell Differentiation; D005260:Female; D007328:Insulin / Q000378:metabolism*; D007333:Insulin Resistance; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D010766:Phosphorylation; D011085:Polycystic Ovary Syndrome / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D012694:Serine / Q000737:chemistry*; D058570:TOR Serine-Threonine Kinases; D013739:Testosterone / Q000378:metabolism*
D007328:Insulin; D013739:Testosterone; D012694:Serine; D011494:Protein Kinases; C546845:mTOR protein, rat; C494920:Akt1 protein, rat; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; C526901:Rps6kb1 protein, rat; D058570:TOR Serine-Threonine Kinases