Leptin Interferes with Adrenocorticotropin/3_,5_-Cyclic Adenosine Monophosphate (Camp) Signaling, Possibly through a Janus Kinase 2- Phosphatidylinositol 3-Kinase/Akt-Phosphodiesterase 3-Camp Pathway, to down-Regulate Cholesterol Side-Chain Cleavage Cytoc

Context: Obesity has adverse effects on adrenocortical functions. Adipocyte-derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism. Objective: We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI-H295 tumor cell line. Methods: Levels of progesterone, cortisol, and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450 side-chain cleavage (P 450scc), Janus kinase 2(JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450 scc and leptin receptors were measured by RT-PCR and realtime PCR. P450scc promoter activity was analyzed with a luciferase reporter system. Results: Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin- induced steroid secretion. The concomitant inhibitions by leptin on cholera toxininduced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/ cholera toxininduced CYP11A1 promoter activity via a known cAMP-responsive region located between Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/ cholera toxin-induced cAMP levels, CYP11A1 promoter activity, and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or phosphatidylinositol 3- kinase/Akt ( wortmannin) as well as inhibitors of cAMP- degrading phosphodiesterases ( PDEs), including nonspecific 3 -isobutyl-1-methylxanthine and PDE3- specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE- nonhydrolyzable N6- monobutyryl-cAMP. Conclusions: Leptin interferes with ACTH/ cAMP signaling , possibly through a cAMP-degrading mechanism involving activation of JAK2 , phosphatidylinositol 3-kinase, and PDE3, to down-regulate P 450scc expression and consequent adrenal steroidogenesis.