The aim of the present study was to examine the effect of high glucose alone and in combination with high insulin on insulin-stimulated protein synthesis and the activation of insulin signaling pathways in mouse C2C12 myogenic cells. The experiments were performed on mouse C2C12 myoblasts subjected to differentiation under normal glucose (5 mmol/l), high glucose alone (15 mmol/l), or in combination with high insulin (50 nmol/l). Six-day differentiation under high glucose alone or in combination with high insulin resulted in insulin resistance, manifested by the abolition of the stimulatory effect on protein synthesis. High glucose concentration in the culture medium did not affect the protein kinase B (PKB) cellular content in C2C12 myogenic cells, whereas in cells preincubated with a combination of high glucose and high insulin a slight but significant increase (of 19%) in PKB protein content was determined. Insulin caused the activation of protein kinase B (PKB) in control C2C12 myogenic cells. Pretreatment with high glucose did not affect PKB phosphorylation whereas in cells differentiated under high glucose and high insulin PKB activation by insulin was markedly attenuated as compared with the control (differentiation under normal glucose). Neither the p70S6k protein content nor the pattern of insulin-mediated kinase activation was affected by pretreatment with high glucose, however high glucose and high insulin in combination caused an impairment in the p70S6k phosphorylation when compared to the control. High glucose exerted no significant changes in MAP kinase protein content, however concomitant treatment with high glucose and high insulin resulted in the decrease in p42MAPK and p44MAPK proteins (by 20%). During the whole period of observation, p42MAPK exhibited basal phosphorylation that was not modified in the presence of insulin. However, the phosphorylation of p42MAPK was profoundly impaired in cells preincubated with high glucose alone or in combination with high insulin. In conclusion: 1) high glucose abolishes the stimulatory action of insulin on protein synthesis without changes in PKB and p70S6k activation; 2) high glucose and high insulin in combination abolish the stimulatory effect of insulin dependent on PKB- and p70S6k; 3) the stimulatory action of insulin on protein synthesis in C2C12 myogenic cells depends on basal phosphorylation of p42MAPK.
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