Regulation of Glucose Transport and Glycogen Synthesis in L6 Muscle Cells during Oxidative Stress

Abstract

We have investigated the cellular mechanisms that participate in reducing insulin sensitivity in response to increased oxidant stress in skeletal muscle. Measure- ment of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both proc- esses, by 2- and 5-fold, respectively. Acute (30 min) ex- posure of muscle cells to hydrogen peroxide (H2O2) blocked the hormonal activation of both these proc- esses. Immunoblot analyses of cell lysates prepared af- ter an acute oxidant challenge using phospho-specific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-acti- vated protein (MAP) kinases established that H2O2 in- duced a dose-dependent activation of all five protein kinases. In vitro kinase analyses revealed that 1 mM H2O2 stimulated the activity of JNK by ?8-fold, MAP- KAP-K2 (the downstream target of p38 MAP kinase) by ?12-fold and that of PKB by up to 34-fold. PKB activa- tion was associated with a concomitant inactivation of glycogen synthase kinase-3. Stimulation of the p38 path- way, but not that of JNK, was blocked by SB 202190 or SB203580, while that of p42/p44 MAP kinases and PKB was inhibited by PD 98059 and wortmannin respec- tively. However, of the kinases assayed, only p38 MAP kinase was activated atH2O2 concentrations (50 ? M) that caused an inhibition of insulin-stimulated glucose trans- port and glycogen synthesis. Strikingly, inhibiting the activation of p38 MAP kinase using either SB 202190 or SB 203580 prevented the loss in insulin-stimulated glu- cose transport, but not that of glycogen synthesis, by oxidative stress. Our data indicate that activation of the p38 MAP kinase pathway plays a central role in the oxidant-induced inhibition of insulin-regulated glucose transport, and unveils an important biochemical link between the classical stress-activated and insulin sig- naling pathways in skeletal muscle. A