Abstract
It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin (Standaert, M. L., Farese, R. V., Cooper, R. D., and Pollet, R. J. (1988) J. Biol. Chem. 263, 8696-8705). In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle. Exposure to PLC-Cp activated glycogen phosphorylase and potentiated twitch tension in response to electrical stimulation, providing evidence that PLC-Cp increases cytoplasmic Ca2+ concentration. Dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, completely blocked both the activation of phosphorylase and the stimulation of glucose transport by PLC-Cp. These findings provide evidence that an increase in cytoplasmic Ca2+ concentration is involved in the activation of glucose transport in skeletal muscle by PLC-Cp.
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