Changes of insulin-mediated protein kinases phosphorylation and the expression of IGFBP-3 in skeletal muscle of streptozotocin-diabetic mice.

UNLABELLED: The purpose of the present study was to investigate potential changes in expression and activation of Ser/Thr protein kinases as well as in the level of insulin-like growth factor-binding proteins (IGFBPs) in skeletal muscle of streptozotocin (STZ)-diabetic mice. We have examined the basal and insulin-mediated phosphorylation of protein kinase B (PKB), protein kinase Czeta (PKCzeta), p70(S6k), mitogen-activated protein kinase (MAPK)/p90(rsk) pathway and the expression of IGFBP-3, -4, and -5 in mice selected for body weight gain (line C) and reduction (line L). Apart from IGFBP-3 level, which was higher in C line, the diabetes-associated changes in signaling components examined in present work were similar in both lines of mice. The expression of PKB in skeletal muscle was similar in control and diabetic mice. Insulin increased the Ser473 phosphorylation of PKB in both experimental groups however, in diabetic mice the insulin-dependent PKB phosphorylation was more evident in comparison to control group. Neither protein level nor insulin-stimulated p70(S6k) activation were modified by STZ-diabetes. Basal PKC phosphorylation was augmented in muscle of diabetic mice and it was not increased following insulin injection. No apparent differences in levels of p42(MAPK), p44(MAPK) and p90(rsk) protein in gastrocnemius muscles between control and STZ-treated mice were observed. Basal phosphorylation of p90(rsk) in diabetic mice was markedly elevated in comparison to the control. In muscle of C-line mice, insulin stimulated the p90(rsk) activity to the same extent in both experimental groups (+22% over appropriate basal value). Insulin-mediated stimulation of p90(rsk) in muscle of L-line mice amounted to +26% and +14%, for control and diabetic mice, respectively. Protein level of IGFBP-3 in muscle of diabetic C-line mice was augmented by approx. 28% when compared to the control, whereas the expression of IGFBP-4 and -5 was not modified by STZ-diabetes. IN CONCLUSION: diabetes-associated changes in the insulin signaling in skeletal muscle involve: 1) enhanced insulin-dependent phosphorylation of PKB; 2) increased basal phosphorylation of PKC and its resistance to stimulatory action of insulin; 3) increased basal phopshorylation of p90(rsk), and 4) augmented IGFBP-3 protein level, which can potentially contribute to disruption of anabolic signals in this tissue.

The impairment of IGF-I-stimulated protein synthesis and activation of protein kinase B, p70(S6k), MAP kinase, and p90(rsk) in mouse C2C12 myogenic cells exposed to high glucose and high insulin.

The aim of the present study was to examine the effect of high glucose alone and in combination with high insulin on IGF-I-stimulated protein synthesis and the activation of IGF-I signaling pathways in mouse C2C12 myogenic cells. Experiments were performed on mouse C2C12 myoblasts subjected to differentiation under normal glucose (5 mmol/I), 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 IGF-I resistance, which was manifested by the abolition of the stimulatory effect on protein synthesis. IGF-I 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 IGF-I was markedly decreased as compared with control (differentiation under normal glucose). Neither the p70(S6k) protein content nor the pattern of IGF-I-mediated kinase activation was affected by pretreatment with high glucose, however high glucose and high insulin in combination caused an impairment of the p70(S6k) phosphorylation, in relation to the control. An increase in p42(MAPK) phosphorylation occurred under normal glucose conditions after the stimulation with IGF-I. The MAP kinase was not phosphorylated in response to IGF-I in cells preincubated with high glucose alone or in combination with high insulin. The pattern of p90(rsk) activation by IGF-I was not modified by pretreatment with high glucose, however no activation of p90(rsk) was found in cells pretreated with high glucose and high insulin in combination. In conclusions: 1) high glucose abolishes the stimulatory action of IGF-I on protein synthesis and it does not affect the activation of PKB, p70(S6k), and p90(rsk) in mouse C2C12 myogenic cells, 2) high glucose with high insulin in combination also abolish the stimulatory effect of IGF-I, but this phenomenon is accompanied by attenuated PKB and p70(S6k) activation and the lack of activation of p90(rsk), 3) apart from PKB, p70(S6k) and p90(rsk), other kinases are probably involved in the regulation of IGF-I-mediated protein synthesis in myogenic cells.