Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance

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.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.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.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

Single-dose intramuscular methotrexate treatment of cervical ectopic pregnancy. A case report

BACKGROUNDCervical ectopic pregnancies (CEPs) are increasingly being managed conservatively. However, the efficacy of specific conservative approaches such as the single-dose intramuscular (IM) methotrexate (MTX) therapy has not been evaluated. We reportthe successful management of 2 consecutive CEPs in the same patient with single-dose IM MTX therapy, and review published cases of CEPs treated with a similar regimen, to determine the efficacy of this approach.CASEIn our case, both CEPs resolved with single-dose IM MTX, and the patient subsequently had a successful term delivery. A Medline/OVID English language search, covering the period 1982-2012, identified 38 additional cases of CEPs treated with single-dose IM MTX. Of the total 40 cases analyzed, including our 2 cases, 30 (75%) were successfully treated, with complete remission. Of the 27 cases with no embryonic cardiac activity, 25 (93%) were successfully treated with single-dose IM MTX.CONCLUSIONAppropriately selected cases of CEPs can be successfully managed with single-dose IM MTX with very low complication rates and preservation of future fertility potential

There was no contamination of non-myoblast cells in our primary culture of rat skeletal muscle cells.

<p>Cells were positively stained with MHCI-antibody (Green Florescence).</p

The effect of insulin in S6K Thr³⁸⁹ phosphorylation with or without pre-exposure of low and high dose testosterone.

<p>(A). The effect of insulin alone on S6K Thr³⁸⁹ phosphorylation after insulin treatment for 10, 20, 30, 60, and 120 minutes; Insulin significantly increased S6K Thr³⁸⁹ phosphorylation after 60 minutes; (B). Cells were pre-exposed to low-dose (20ng/ml) or high-dose (200ng/ml) of testosterone for 16 hours before insulin treatment. Compared to non-testosterone treated cells, S6K Thr³⁸⁹ phosphorylation was significantly increased with low-dose testosterone treatment. Representative western blots are shown above the bar graphs. The four experimental groups are: control cells without insulin (C−I), control cells plus insulin (C+I), testosterone exposed cells without insulin (T−I), and testosterone exposed cells plus insulin (T+I). The phosphorylated signal of S6K Thr³⁸⁹ is normalized to total S6K in the sample.</p

The effect of insulin in Akt Ser⁴⁷³ phosphorylation with or without pre-exposure of low and high dose testosterone.

<p>(A). The effect of insulin alone on Akt phosphorylation after insulin treatment for 10, 20, 30, 60, and 120 minutes; Insulin significantly increased Akt Ser⁴⁷³ phosphorylation at all time points except 20 minutes; (B). Cells were pre-exposed to low-dose (20ng/ml) or high-dose (200ng/ml) of testosterone for 16 hours before insulin treatment. No change in Akt Ser⁴⁷³ phosphorylation was observed between T treated and non-T treated cells. Representative western blots are shown above the bar graphs. The four experimental groups are: control cells without insulin (C−I), control cells plus insulin (C+I), testosterone exposed cells without insulin (T−I), and testosterone exposed cells plus insulin (T+I). The phosphorylated signal of Akt Ser⁴⁷³ is normalized to total Akt in the sample.</p

The effect of insulin in mTOR Ser²⁴⁴⁸ phosphorylation with or without pre-exposure of low and high dose testosterone.

<p>(A). The effect of insulin alone on mTOR Ser²⁴⁴⁸ phosphorylation after insulin treatment for 10, 20, 30, 60, and 120 minutes; Insulin had no effect on mTOR Ser²⁴⁴⁸ phosphorylation at all time points; (B). Cells were pre-exposed to low-dose (20ng/ml) or high-dose (200ng/ml) of testosterone for 16 hours before insulin treatment. Compared to non-testosterone treated cells, mTOR Ser²⁴⁴⁸ phosphorylation was significantly increased with low-dose testosterone treatment. Representative western blots are shown above the bar graphs. The four experimental groups are: control cells without insulin (C−I), control cells plus insulin (C+I), testosterone exposed cells without insulin (T−I), and testosterone exposed cells plus insulin (T+I). The phosphorylated signal of mTOR Ser²⁴⁴⁸ is normalized to total mTOR in the sample.</p