Orthovanadate decreases the leptin content in isolated mouse fat pads via proteasome activation

When isolated mouse fat pads were incubated with insulin or sodium orthovanadate (vanadate) for up to 4 h, the intracellular leptin content was increased by insulin, while it was decreased by vanadate. Bupranolol, a β3-adrenergic receptor antagonist, prevented both effects of vanadate, i.e., the decrease in intracellular leptin and increase in cellular cAMP content, while BRL 37344, a β3-adrenergic receptor antagonist mimicked the action of vanadate. H-89 prevented the vanadate-induced decrease in intracellular leptin, suggesting the involvement of a cAMP-dependent protein kinase (PKA). No detectable difference in the incorporation of [3H]leucine into leptin was observed between incubations of the fat pads with and without vanadate, suggesting that the action of vanadate is independent of decreasing synthesis. Similar concentrations of MG-132, a membrane-permeable proteasome inhibitor, prevented the vanadate-induced decrease in both intracellular leptin content and leptin secretion, suggesting the involvement of the proteasome in the vanadate action. The proteasome fraction separated from the vanadate-treated fat pads increased the degradation of exogenous [125I]leptin in the presence of an ATP-regenerating system together with an ubiqutination system. The endopeptidase activity against Cbz–Leu–Leu–Glu–β-naphthylamine also was increased by the proteasome fraction. MG-132 prevented both increased effects. The 8-Br–cAMP-treated proteasome fraction increased the degradation of the exogenous leptin. H-89 prevented the effect of 8-Br–cAMP. These results indicate that vanadate decreases the intracellular leptin content by increased degradation via a cAMP/PKA-dependent process involving proteasome activation.