The long-term goal of this project is to understand the neurobiological mechanisms that are responsible for food intake and body weight regulation. Leptin, a product of the obese gene, is one of the most important peripheral satiety factors that inhibits food intake and body weight by acting primarily in the hypothalamic region of the brain. However, the molecular mechanisms by which this hormone acts in the hypothalamus are not well defined. In this regard, since the leptin receptor is a member of the family of cytokine receptors, leptin activates the JAK -STAT signaling pathway in the hypothalamus. Our recent study demonstrates that leptin may act via an alternative-signaling pathway, involving reduction of cAMP levels through activation of a phosphodiesterase, PDE3B. Specifically, leptin induces PDE3B activity and reduces cAMP levels in the hypothalamus, and the PDE3 inhibitor, cilostamide, reverses the effect of leptin on food intake and body weight in rats. While several leptin-sensitive orexigenic and anorectic neurons in the hypothalamus comprise the neural circuitry that governs food intake and body weight regulation, but the extent to which PDE3B activation is altered in these neurons in leptin signaling is unclear. It is still unknown which cells in particular are cilostamide responsive or which cells show altered cAMP levels in response to leptin. Thus, to further demonstrate that PDE3B-cAMP pathway is an integral part of leptin signaling in the hypothalamus, following Specific Aims will be addressed:
Aim 1 : To identify primary hypothalamic sites of PDE3B dependent leptin signaling: examine the effects of PDE3 inhibition in specific hypothalamic sites on leptin action.
Aim 2 : To identify the neuronal network that mediates PDE3B activation-dependent leptin signaling.
Aim 3 : To identify upstream signaling components that leads to leptin-induced activation of PDE3B and reduction of cAMP. The action of PDE3B and PI3K will be blocked by intracerebroventricular administration of specific inhibitors. PDE3B, PDE3A, PDE4, PKB, PI3K activity will be measured by enzyme assays. Cyclic AMP levels will be measured by RIA. RNAse protection assays and in situ hybridization will be used to examine changes in PDE3B, NPY, MCH, GAL, Orexin, POMC and NT gene expression. JAK2, STAT3 and PKB protein levels will be measured by Western blotting. DNA-binding activity of STAT3 will be examined by electrophoretic mobility shift assay. These studies will further our understanding of leptin signaling in the hypothalamus in relation to feeding, and therefore will be relevant to the development of therapeutic approaches to eating disorders. ? ?
