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

Michael C. Allemand, Brian A. Irving, Yan Asmann, Katherine A. Klaus, Laura Tatpati, Charles C. Coddington, K Sreekumaran Nair

Research output: Contribution to journalArticle

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Abstract

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-1636/639 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 Ser636/639, 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 Ser636/639 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 Ser636/639 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 Ser636/639. These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance.

Original languageEnglish (US)
Article numbere4274
JournalPLoS One
Volume4
Issue number1
DOIs
StatePublished - Jan 26 2009

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polycystic ovary syndrome
Phosphorylation
Polycystic Ovary Syndrome
Skeletal Muscle Fibers
insulin resistance
testosterone
Insulin Resistance
Testosterone
Rats
phosphorylation
insulin
Insulin
rats
skeletal muscle
Skeletal Muscle
Muscle
serine
Serine
dosage
Intracellular Signaling Peptides and Proteins

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Effect of testosterone on insulin stimulated IRS1 ser phosphorylation in primary rat myotubes - A potential model for PCOS-related insulin resistance. / Allemand, Michael C.; Irving, Brian A.; Asmann, Yan; Klaus, Katherine A.; Tatpati, Laura; Coddington, Charles C.; Nair, K Sreekumaran.

In: PLoS One, Vol. 4, No. 1, e4274, 26.01.2009.

Research output: Contribution to journalArticle

Allemand, Michael C. ; Irving, Brian A. ; Asmann, Yan ; Klaus, Katherine A. ; Tatpati, Laura ; Coddington, Charles C. ; Nair, K Sreekumaran. / Effect of testosterone on insulin stimulated IRS1 ser phosphorylation in primary rat myotubes - A potential model for PCOS-related insulin resistance. In: PLoS One. 2009 ; Vol. 4, No. 1.
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AU - Coddington, Charles C.

AU - Nair, K Sreekumaran

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N2 - 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-1636/639 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 Ser636/639, 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 Ser636/639 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 Ser636/639 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 Ser636/639. These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance.

AB - 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-1636/639 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 Ser636/639, 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 Ser636/639 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 Ser636/639 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 Ser636/639. These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance.

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