PROJECT SUMMARY/ABSTRACT The incretin-mediated potentiation of glucose-stimulated insulin secretion (GSIS) accounts for 50% of postprandial insulin secretion and is essential for physiologic glycemic regulation. Upon nutrient stimulus, the two principal incretin hormones glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted, respectively, from intestinal endocrine K- and L-cells and reach β-cells via the circulation, where they bind their receptors, GIP receptor (GIPR) and GLP-1 receptor (GLP-1R) to amplify GSIS. While circulating incretin hormone levels are not impacted by T2DM, incretin effects on β-cell GSIS are significantly diminished in T2DM patients. However, the precise mechanisms underlying diabetes-associated blunting of incretin signaling in β-cells remain unclear. In T2DM, pharmacologic means to increase circulating GLP-1 levels improves GSIS and glycemic control. In contrast, the GIP-mediated incretin effect is notably absent in T2DM; and pharmacologic means to increase circulating GIP concentrations is not met with any GSIS improvement. The molecular mechanisms underlying diminished response to GLP-1 and absent response to GIP in T2DM remain poorly understood. T2DM is in part linked to a chronic inflammatory state with increased levels of local and circulating inflammatory cytokines that negatively impact insulin action in peripheral tissues and negatively impact the endocrine pancreas. We have therefore examined the role of pro-inflammatory cytokines that are elevated in T2DM on incretin signaling in islet -cells. Conversely, we have also interrogated the anti-inflammatory cholinergic signaling system through nicotinic acetylcholine receptors (nAchR) in modulating incretin signaling. Based on our preliminary studies, we hypothesize that TNFα - signaling through its receptor TNFR1 on β- cells - activates the Ser/Thr G-protein receptor kinase 2 (GKR2) in a non-canonical manner to suppress Gαs- activation and cAMP synthesis by ligand activated GIPR and by GLP-1R. Thus, our findings establish a mechanistic link between inflammatory cytokines in T2DM with diminished incretin signaling in β-cells. Our preliminary studies also indicate that in a murine model of T2DM, stimulating the α7 nicotinic acetylcholine receptor (α7-nAchR) reactivates GIPR signaling in β-cells to potentiate GSIS and improve glycemia. Our mechanistic studies indicate that α7-nAchR signaling phosphorylates (TNFα-activated) GRK2 at serine 670 to counteract its inhibitory effects on GIPR and GLP-1R-mediated cAMP synthesis. We now seek to expand our novel and exciting findings specifically a) to understand the role of TNFR1- GRK2-mediated signaling on in vivo β-cell function, on GIP and GLP-1R action as well as how TNFR1 signaling modulates α-cell gene expression in the context of obesity and T2DM; b) to specifically understand the role of GRK2 in mediating resistance to β-cell incretin action in T2DM; and c) to understan...