GPCR Regulation of insulin secretion by modulation of the release machinery

Vanderbilt University, Nashville TN

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Abstract

? DESCRIPTION (provided by applicant): One of the earliest and most important pathophysiological signs of type 2 diabetes (T2D) is impaired insulin release. A number of therapies used in the clinic, such as GLP1- agonists and DPP-4 inhibitors, act to sensitize the release machinery and enhance insulin release from the beta cell. Gi/o-coupled G-protein coupled receptors (GPCRs) inhibit insulin release through liberating G protein ?? subunits, which can inhibit calcium entry through voltage-gated calcium channels. We have demonstrated that G?? can also directly inhibit exocytosis at a point distal to Ca2+ entry by binding to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Beta cell ?2A adrenergic receptors (2AAR) profoundly inhibit insulin release by this mechanism. This proposal aims to investigate the role of the ?2AAR in the inhibition of insulin release in animal models of T2D. We hypothesize that overactive inhibition of insulin secretion by Gi/o- coupled GPCRs is a part of the impaired insulin secretion in the pathogenesis of T2D. No studies have previously investigated whether blocking this inhibitory mechanism is an alternative approach for enhancing insulin secretion in an animal model of T2D, the diet-induced obese (DIO) aged mouse. In Aim 1, we will test whether 2AAR-mediated inhibition is enhanced in islets isolated from DIO mice. In Aim 2, we will determine whether negating the impact of G?? on the exocytotic fusion machinery can improve GSIS in DIO mice. We have both small molecule inhibitors of the G??-SNARE interaction, as well as a mutant mouse that is resistant to G??'s inhibitory effect. The truncated SNAP25 mutant mouse (SNAP253) has an impaired ability to interact with G while exocytosis itself remains intact. We will assess GSIS in isolated islets in vitro as well as in vivo using hyperglycemic clamps from both lean and DIO WT and SNAP253 mice. In Aim 3, we will determine whether dual addition of low levels of secretogogues and G??-SNARE inhibitors synergize with each other, potentially providing us with a therapeutic window of selectivity for enhanced insulin secretion over secretion events in other cell types. Regulation of the exocytotic fusion machinery by G?? is a new mechanism that targets the very final step of insulin secretion. Importantly, our studies have the potential to open up a new therapeutic approach to impaired insulin release. Reversing the inhibitory signals at the exocytotic machinery could amplify insulin release while still maintaining the critical physiological control f insulin release by glucose and other modulators.