We have previously reported that glucose metabolism mediates the effects of insulin to increase leptin gene expression and leptin secretion by isolated adipocytes. The aim of the present study was to investigate the role of transcription and translation in the regulation of basal and insulin-stimulated leptin production. The short-term (4 h) and long-term (24-48 h) effects of actinomycin D, a transcriptional inhibitor, and cycloheximide, an inhibitor of protein synthesis, on leptin gene expression and leptin secretion by isolated adipocytes were determined. Actinomycin D (5 microg/ml) increased both basal and insulin-stimulated (1.6 nM) leptin secretion at 4 and 24h (193+/-14.9% and 153.8+/-10.4% of respective controls at 24h, both p<0.001). Similar effects of actinomycin D were observed on basal and insulin-stimulated leptin mRNA levels. 5,6-dichlororibofuranosyl benzimidazole (DRB), another inhibitor of transcription, also increased basal (175.4+/-18.2% of control; p<0.01) and insulin-stimulated leptin secretion (141.0+/-11.1% of insulin-treated cells; p<0.05) at 24 h. The effect of actinomycin D and DRB to increase basal leptin secretion observed at 4 and 24 h was not present at 48 h when actinomycin D and DRB both markedly inhibited insulin-stimulated leptin secretion (to 36+/-16%, p<0.05 and 21.9+/-5.6% of control, for actinomycin D and DRB, respectively, both p<0.001). Neither actinomycin D nor DRB had any effect on adipocyte glucose utilization between 24 and 48 h. The observed effects of inhibitors of transcription on leptin gene expression and leptin secretion are consistent with a long-term transcriptional mechanism for insulin-stimulated glucose metabolism to increase leptin production. Cycloheximide treatment (10 microg/ml) abolished the effects of insulin to stimulate leptin secretion (29+/-11% of control, p<0.01) during the first 4 h of treatment and at all later time points, which indicate that de novo protein synthesis is required for insulin-mediated glucose metabolism to increase leptin secretion.