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 downregulate cholesterol side-chain cleavage cytochrome P450 enzyme in human adrenocortical NCI-H295 cell line

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 (P450scc), Janus kinase 2 (JAK2), Akt, and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time 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 toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP-responsive region located between -1.7 and -1.5 kb. 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 P450scc expression and consequent adrenal steroidogenesis.