SUN-482 Nitric Oxide Induces Spexin (Spx), Galanin Receptor 2 (GalR2), and Galanin Receptor 3 (GalR3) mRNA Independently of the cGMP/PKG Pathway and Enhances C/EBP-β Binding to the Spx 5’ Regulatory Region

Abstract

Spexin (SPX) is a recently discovered peptide that binds to galanin receptors 2 (GalR2) and 3 (GalR3). Emerging evidence implicates SPX as a modulator of satiety and energy balance as circulating SPX levels are dysregulated in obesity. However, it is currently unknown how SPX and its receptors are regulated in the hypothalamus, a brain region critical for energy homeostasis. As protein kinase signalling is involved in the regulation of many hypothalamic neuropeptides and receptors, we hypothesized that SPX and its receptors would be regulated by at least one of the protein kinases: PKA, PKC or PKG. We therefore examined hypothalamic Spx, GalR2 and GalR3 mRNA expression in response to several protein kinase activators in a clonal adult-derived hypothalamic cell model, mHypoA-59, using quantitative reverse transcription PCR (qRT-PCR). Treatments over 24 hours revealed that Spx, GalR2 and GalR3 mRNA were all induced with 100 µM sodium nitroprusside (SNP), a nitric oxide donor that can activate the PKG pathway. However, direct stimulation of PKG with increasing concentrations of 8-Bromo-cGMP failed to induce Spx, GalR2 or GalR3 suggesting that the regulation of these genes by nitric oxide was independent of the canonical cGMP/PKG pathway. Characterization of the 5’ regulatory region of Spx led to identification of putative response elements for C/EBP-β and OCT-1. Interestingly, treatment with 100 µM SNP resulted in the induction of C/ebp-β, but not Oct-1 mRNA. Subsequent chromatin immunoprecipitation studies revealed that 100 µM SNP enhanced binding of C/EBP-β, but not OCT-1 to the 5’ regulatory region of Spx, suggesting that increased binding of C/EBP-β is involved in the induction of Spx mRNA in the response to SNP. The results thus far suggest that nitric oxide induces Spx, GalR2 and GalR3 mRNA independently of PKG and that C/EBP-β may be involved in the induction of Spx. Elucidating the pathways that regulate gene expression of SPX and its receptors in the hypothalamus will contribute to current models of energy balance and provide insight into how components of this regulatory circuit may be dysregulated in obesity.