Insulin-stimulated glucose metabolism plays a key role in the regulation of leptin mRNA expression and protein secretion. However, it is not known whether stimulation of leptin production by glucose metabolism is regulated at the level of promoter activation or at a step distal to the promoter. Therefore, in order to investigate the transcriptional regulation of the leptin promoter by insulin-stimulated glucose metabolism, 3T3-L1 cells were transfected with a plasmid containing the leptin promoter driving a luciferase reporter gene. Leptin promoter activity was increased after 48 hours of treatment by 219 +/- 64 (p = 0.028) and 225 +/- 69% (p = 0.046) at insulin concentrations of 16 and 160 nM, respectively. The activation of the leptin promoter induced by insulin (16 nM) was markedly inhibited by 2-deoxy-D-glucose (2-DG, 50 mg/dl), a competitive inhibitor of glucose metabolism. The increment of insulin-stimulated leptin promoter activation was reduced by 52 +/- 11% (p = 0.028 vs insulin alone). The activity of a control plasmid (pGL2-Control) was unaffected by insulin or 2 DG. These results provide strong evidence that insulin-stimulated glucose metabolism, and not insulin per se, mediates the effects of insulin to increase the transcriptional activity of the leptin promoter.