Supplementary Material for: Hypothalamic Phosphodiesterase 3B Pathway Mediates Anorectic and Body Weight-Reducing Effects of Insulin in Male Mice

2016-10-14T08:50:52Z (GMT) by Sahu M. Anamthathmakula P. Sahu A.
<br><strong><em>Background:</em></strong> Insulin action in the hypothalamus plays a critical role in the regulation of energy homeostasis, yet the intracellular signaling mechanisms mediating insulin action are incompletely understood. Although phosphodiesterase 3B (PDE3B) mediates insulin action in the adipose tissue and it is highly expressed in the hypothalamic areas implicated in energy homeostasis, its role, if any, in mediating insulin action in the hypothalamus is unknown. We tested the hypothesis that insulin action in the hypothalamus is mediated by PDE3B. <b><i>Methods:</i></b> Using enzymatic assay, we examined the effects of peripheral or central administration of insulin on hypothalamic PDE3B activity in adult mice. Western blotting and immunohistochemistry also examined p-Akt and p-STAT3 levels in the hypothalamus. Effects of leptin on these parameters were also compared. We injected cilostamide, a PDE3 inhibitor, prior to central injection of insulin and examined the 12- to 24-hour food intake and 24-hour body weight. Finally, we examined the effect of cilostamide on insulin-induced proopiomelanocortin <i>(Pomc)</i>, neurotensin <i>(Nt)</i>, neuropeptide Y <i>(Npy)</i> and agouti-related peptide <i>(Agrp)</i> gene expression in the hypothalamus by qPCR. <b><i>Results:</i></b> Peripheral or central injection of insulin significantly increased PDE3B activity in the hypothalamus in association with increased p-Akt levels but without any change in p-STAT3 levels. However, leptin-induced increase in PDE3B activity was associated with an increase in both p-Akt and p-STAT3 levels in the hypothalamus. Prior administration of cilostamide reversed the anorectic and body weight-reducing effects as well as stimulatory effect of insulin on hypothalamic <i>Pomc</i> mRNA levels. Insulin did not alter <i>Nt</i>,<i> Npy</i> and <i>Agrp</i> mRNA levels. <b><i>Conclusion:</i></b> Insulin induction of hypothalamic PDE3B activity and the reversal of the anorectic and body weight-reducing effects and stimulatory effect of insulin on hypothalamic <i>Pomc</i> gene expression by cilostamide suggest that activation of PDE3B is a novel mechanism of insulin signaling in the hypothalamus.