Leptin is a regulator on placenta and conceptus during pregnancy. Hyperinsulinism and hypoxia induce partially overlapping pathophysiological disturbances during pregnancy. As insulin and hypoxia are known inducers of leptin secretion, we asked whether these two stimuli have synergistic effects. By analyzing mRNA levels of leptin after stimulation of BeWo cells with insulin in the presence or absence of oxygen, we found a supraadditive effect when incubating hypoxic cells with insulin. As shown. by Western-blot of hypoxia-inducible-factor-1alpha (HIF-1alpha), the additive effects of these stimuli were not mediated by an increased stabilization of the HIF-complex. We therefore asked what elements of the leptin promoter are responsible for these effects. When deleting a 0.6 kb fragment of the cloned leptin promoter, a so far unknown loss of insulin-dependent activation of transcription, as well as a loss of the supraadditive effect of insulin and hypoxia could be observed. These results provide strong evidence that insulin and hypoxia act as agonists on the human leptin transcription but on two different regulatory elements. (C) 2003 Elsevier Science (USA). All rights reserved.