It was recently shown that leptin impairs insulin signalling, i.e. insulin receptor autophosphorylation and insulin-receptor substrate (IRS)-1 phosphorylation in rat-1 fibroblasts, NIH3T3 cells and HepG2 cells. To evaluate whether leptin might impair the effects of insulin in muscle tissue we studied the interaction of insulin and leptin in a muscle cell system, i.e. C2C12 myotubes. Preincubation of C2C12 cells with leptin (1-500 ng/ml) did not significantly affect insulin stimulated glucose transport and glycogen synthesis (1.8 to 2 fold stimulation); however, leptin by itself (1 ng/ml) was able to mimic approximately 80-90% of the insulin effect on glucose transport and glycogen synthesis. Both glucose transport as well as glycogen synthesis were inhibited by the phosphatidylinositol-3 (PI3)-kinase inhibitor wortmannin and the protein kinase C inhibitor H7 while no effect was observed with the S6-kinase inhibitor rapamycin. We determined whether the effect of leptin occurs through activation of IRS-1 and PI3-kinase. Leptin did not stimulate PI3-kinase activity in IRS-1 immunoprecipitates; however, PI3-kinase activation could be demonstrated in p85 alpha immunoprecipitates (3.04 +/- 1.5 fold of basal). In summary the data provide the first evidence for a positive crosstalk between the signalling chain of the insulin receptor and the leptin receptor. Leptin mimics in C2C12 myotubes insulin effects on glucose transport and glycogen synthesis most likely through activation of PI3-kinase. This effect of leptin occurs independently of IRS-1 activation in C2C12 cells.