Sp1-mediated transcription is involved in the induction of leptin by insulin-stimulated glucose metabolism

We have previouslydemonstrated that insulin-stimulated glucosemetabolism, andnot insulinper se, mediatesthe effects of insulintoincreasethetranscriptionalactivityoftheleptinpromoter in adipocytes.Here,wesoughtto identifythespecific cis-acting DNA elements required for the upregulation of leptin gene transcription in responseto insulin-mediated glucose metabolism. To accomplish this, 3T3-L1 cells and primary rat adipocytes were transfected with a series of luciferase reporter genes containing portions of the mouse leptin promoter. Using this method, we identified an element between K135 and K95 bp (relative to the transcriptional start site) that mediated transcription in response to insulin-stimulated glucosemetabolism in adipocytes. This effect was abolished by incubation with 2-deoxy-D-glucose, a competitive inhibitor of glucose metabolism. Gel shift electrophoretic mobility shift assays confirmed that the stimulatory effect of insulinmediated glucose metabolism on leptin transcription was mediated by a previously identified Sp1 site. Consistent with these findings, incubation of primary rat adipocytes with WP631, a specific inhibitor of specificity protein (Sp)1-dependent transcription, inhibited glucose- and insulin-stimulated, but not basal, leptin secretion. Together, these findings support a key role for Sp1 in the transcriptional activation of the leptin gene promoter by insulin-mediated glucose metabolism.