| Partâ… Effects of testosterone on insulin sensitivity in C2C 12 myotubesOBJECTIVES To investigate the direct effect of testosterone on insulin sensitivity in C2C12 myotubes with different concentrations(10-11, 10-9, 10-8, 10-6, 10-5 M) treatment for short(30min) and for long(24hour) time.METHODS C2C 12 myoblasts was differentiated into matured myotubes and treated with different doses of testosterone(10-11, 10-9, 10-8, 10-6, 10-5 M) for 30 minutes and for 24 hours, then the affects of testosterone on insulin sensitivity were assessed as [3H]-2-deoxyglucose uptake.RESULTS (1) Exposure of C2C12 myotubes to testosterone did not affect the basal glucose uptake of C2C12 myotubes. Incubated cells with physiological concentrations(10-9, 10-8M) testosterone and lower than physiological concentrations (10-11M) for 30 minutes did not affect the insulin stimulated glucose uptake of the cell, P＞0.05.10-6M testosterone did not affect the insulin stimulated glucose uptake of the cell either, but when incubated the cell with 10-5M testosterone for 30 minutes, the insulin stimulated glucose uptake was significantly reduced, P=0.002. (2) Incubated cells with physiological concentrations(10-9, 10-8M) testosterone or lower than physiological concentrations (1011M) for 24 hours did not affect the insulin stimulated glucose uptake either, but 10-5M and 10-6M testosterone both could decreased the insulin stimulated glucose uptake of C2C12 myotubes.CONCLUSIONS Testosterone has direct effects on insulin sensitivity of C2C 12 myotubes, high concentration Testosterone can decrease insulin sensitivity of C2C12 myotubes when treatment for short(30min) or for long(24hour) time., testosterone can induce insulin resistance in skeletal cell. Partâ…¡ERK1/2 activation by testosterone inhibits insulin induced glucose uptake in C2C 12 myotubesOBJECTIVES Our previous studies indicated that treatment with high concentration of testosterone (10-6, 10-5 M) inhibited glucose uptake in C2C12 myotubes, while the underlined molecular mechanisms by which testosterone alters insulin signaling in C2C12 myotubes were unclear. The aims of this study were to investigate the effects of testosterone on expression and phosphorylation of ERK1/2 and IRS-1 as well as their mutual relations in C2C12 myotubes with different concentrations(10-11, 10-9, 10-8, 10-6, 10-5 M) treatment for short and for long time.METHODS (1) C2C12 myoblasts was differentiated into matured myotubes and cultureded with different doses of testosterone(1011, 10-9, 10-8, 10-6, 10-5 M) for 30 minutes and for 24 hours respectively; C2C12 myoblasts were also treated with 10-5M for different times(0min, 15min, 30min, 60min, 2h and 24h). The expression of ERK1/2 and p-ERK1/2 were detected by western blotting. (2)C2C 12 myoblasts were treated with 10-5M testosterone for 30min and 24h respectively in the absence or presence of insulin(at 100nM for 10min) and the expression of IRS-1,p-IRS-1 Ser307 and p-IRS-1 Tyr941 were detected by western blotting. (3) C2C 12 myoblasts pretreated with or without a specific MEK inhibitor PD98059, the effects of 10-5M testosterone on the expression of ERK1/2, p-ERK1/2, IRS-1,p-IRS-1 Tyr941 and p-IRS-1 Ser307 were measured. (4) C2C12 myoblasts pretreated with or without PD98059, the effects of 10-5M testosterone on [3H]-2-deoxyglucose uptake were measured.RESULTS (1)Testosterone enhanced serine phosphorylation of ERK1/2 in a doseand time-dependent manner. At 10-8M, testosterone promoted phosphorylation of ERK1/2.And At 10-5M, phosphorylation of ERK 1/2 were increased in 5min. and decreased from 2h. (2) The level ofp-IRS-1 Ser307 and p-IRS-1 Tyr941 in the untreated C2C 12 cells were hardly detected. Stimulated with 10-5M testosterone or 100nM insulin for 10min increased phosphorylation of IRS-1 at Ser307 or Tyr941 respectively(P＜0.05, respectively). However, C2C12 cells combined stimulated with 10-5M testosterone and 100nM insulin for 10min, phosphorylation of IRS-1 at Ser307 was still increased (P＜0.05), whereas phosphorylation of IRS-1 at Tyr941 was greatly decreased(P＜0.05), and there was no alteration of expression of total IRS-1 in 30min(P＜0.05) treatment. C2C 12 cells treated with 10-5M testosterone for 24h decreased the level of total IRS-1 but the phosphorylation of IRS-1 at Ser307 and at Tyr941(stimulated by 100nM insulin 10min) were unaffected. (3) Phosphorylation of ERK1/2 and Ser307IRS-1 increased by testosterone could be inhibited by PD98059 (P＜0.05), and the blockade effect of testosterone on insulin stimulated phosphorylation of Tyr941 IRS-1 could also be reversed by PD98059(P＜0.05). However, the effect of 10-5M testosterone for 24h on declination of protein level of IRS-1 could not be reversed by PD98059(P＞0.05). (4) the decreased glucose uptake of C2C 12 cells caused by testosterone could be only partially reversed by PD98059.CONCLUSIONS: Short-time testosterone treatment at high concentrations may phosphorylate ERK1/2 and IRS-1 at Ser307 sequentially, and then inhibit insulin stimulated phosphorylation of IRS-1 at Tyr941. The insulin signaling pathway is blocked and insulin stimulated glucose uptake is inhibited, and insulin resistance happens. Long-time testosterone treatment at high concentrations may decrease the expression of total IRS-1 and then block insulin signaling pathway and inhibit glucose uptake, and insulin resistance happens. Testosterone may cross talk with insulin signal pathway by phosphorylate ERK1/2 in C2C 12 myotubes.
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