Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress - Evidence for cross-talk between the insulin and SAPK2/p38 mitogen-activated protein kinase signaling pathways

We have investigated the cellular mechanisms that participate in reducing i nsulin sensitivity in response to increased oxidant stress in skeletal musc le. Measurement of glucose transport and glycogen synthesis in L6 myotubes showed that insulin stimulated both processes, by 2- and 5-fold, respective ly. Acute (30 min) exposure of muscle cells to hydrogen peroxide (H2O2) blo cked the hormonal activation of both these processes. Immunoblot analyses o f cell lysates prepared after an acute oxidant challenge using phospho-spec ific antibodies against c-Jun N-terminal kinase (JNK), p38, protein kinase B (PKB), and p42 and p44 mitogen-activated protein (MAP) kinases establishe d that H2O2 induced a dose dependent activation of all five protein kinases , In vitro kinase analyses revealed that 1 mM H2O2 stimulated the activity of JNK by similar to 8-fold, MAP-KAP-K2 (the downstream target of p38 MAP k inase) by similar to 12-fold and that of PKB by up to 34-fold. PKB activati on was associated with a concomitant inactivation of glycogen synthase kina se-3, Stimulation of the p38 pathway, but not that of JNK, was blocked by S E 202190 or 8B203580, while that of p42/p44 MAP kinases and PKB was inhibit ed by PD 98059 and wortmannin respectively. However, of the kinases assayed , only p38 MAP kinase was activated at H2O2 concentrations (50 mu M) that c aused an inhibition of insulin-stimulated glucose transport and glycogen sy nthesis. Strikingly, inhibiting the activation of p38 MAP kinase using eith er SE 202190 or SE 203580 prevented the loss in insulin-stimulated glucose transport, but not that of glycogen synthesis, by oxidative stress, Our dat a indicate that activation of the p38 MAP kinase pathway plays a central ro le in the oxidant-induced inhibition of insulin-regulated glucose transport , and unveils an important biochemical link between the classical stress-ac tivated and insulin signaling pathways in skeletal muscle.