A key metabolic action of insulin is the stimulation of non-oxidative glucose utilization in skeletal muscle, by increasing both glucose uptake and glycogen synthesis. The molecular mechanism underlying this process has been investigated using a variety of experimental systems. We report here the use of cultured human myoblasts to study insulin control of glycogen synthesis in humans. In these cells insulin stimulates glycogen synthesis approx. 2.2-fold, associated with a similar activation of glycogen synthase (GS) which occurs within 5-10 min of the addition of insulin. Insulin also causes inactivation of glycogen synthase kinase-3 (GSK-3) and activation of protein kinase B, both processes being sufficiently rapid to account for the effects of insulin on GS. Activation by insulin of the protein kinases p70s6K, p90s6K and extracellular signal-regulated kinase 2 (ERK2) is observed, but is significantly slower than the activation of GS. Selective inhibitors of the p70s6K pathway (rapamycin), the ERK2/p90s6K pathway (PD98059) and phosphatidylinositol 3-kinase (wortmannin) have been used to probe the contribution of these components to insulin signalling in human muscle. Wortmannin blocks activation of both glycogen synthesis and GS and inactivation of GSK-3. PD98059 is without effect on these events, while rapamycin is without effect on inactivation of GSK-3 but partially blocks activation of glycogen synthesis and GS. Taken together, these findings suggest that protein kinase B is responsible for the inactivation of GSK-3, but that an additional rapamycin-sensitive mechanism may contribute to the activation of GS and stimulation of glycogen synthesis.
In hepatocytes glycogen storage is stimulated by insulin and this effect of insulin is counteracted by epidermal growth factor (EGF). The mechanism by which insulin stimulates glycogen synthesis in liver is unknown. We investigated the involvement of candidate protein kinases in insulin signalling in hepatocytes. Both insulin and EGF activated extracellular regulated kinase 2 (ERK-2), p70rsk and protein kinase B (PKB) and inactivated glycogen synthase kinase-3 (GSK-3). Whereas EGF caused a greater activation of ERK-2 than insulin, the converse was true for PKB. The stimulation by insulin of ERK-2 was blocked by a mitogen-activated protein (MEK) inhibitor (PD 98059) and of p70rsk by rapamycin. However, these inhibitors, separately or in combination, did not block the stimulation of glycogen synthesis by insulin, indicating that activation of these kinases is not essential for the stimulation of glycogen synthesis by insulin. Mono Q fractionation of hepatocyte extracts resolved a single myelin basic protein (MBP) kinase peak from extracts of EGF-treated cells (peak 1, eluting at 200 mmol/l NaCl) and two peaks from insulin-treated cells (peak 1 eluting at 200 mmol/l NaCl and peak 2 eluting at 400 mmol/l NaCl). In the combined presence of insulin and EGF, activation of peak 2 was abolished. In situ MBP kinase assays and immunoblotting established that peak 1 coincides with ERK-2 and peak 2 is not an activated form of ERK-1 or ERK-2. It is concluded that PKB, which is activated to a greater extent by insulin than EGF, and peak 2, which is activated by insulin and counteracted by EGF, are possible candidates in mediating the stimulation of glycogen synthesis by insulin.
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