Type 2 Diabetes ensues when pancreatic beta cells dysfunction, die, or both. Beta cells function to secrete insulin, triggered by glucose to generate glycolytic and other oxidative events to stimulate an increase the ratio of ATP relative to ADP within the cell. Insulin is then released upon engagement of the exocytosis SNARE proteins. However, this increased metabolic function, in turn, leads to increased levels of reactive oxygen and nitrogen species (ROS and RNS, respectively), and while this is normal, under pathophysiological conditions such as hyperglycemia, nitrositive stress occurs and insulin exocytosis fails, but the mechanistic reason for failure is unknown. One clue may be in the generation of post-translational protein modifications induced by nitrosative stress. This concept is supported by new preliminary data which shows glucose-induced nitrosylation and nitration of two known islet beta cell exocytosis proteins, Syntaxin 4 and Munc18c, respectively. The objective of this application is to delineate the roles of RNS-induced post-translational modifications modulate and regulate insulin exocytosis. The central hypothesis for the proposed research is that two key exocytic proteins within the pancreatic beta cell are selectively modified by RNS: Syntaxin 4 by nitrosylation, and Munc18c by nitration.
Two Specific Aims are proposed to test this hypothesis:
Aim 1 will investigate the role of Syntaxin 4 nitrosylation in insulin secretion, while Aim 2 seeks to define the role of Munc18c nitration under normal and aberrant RNS conditions upon insulin secretion. Studies will involve use of three experimental paradigms: normal glucose conditions, hyperglycemia/chronic glucose stimulation, and severe nitrosative. Mechanisms will be resolved using protein-protein interaction studies in vitro and in cells, SNARE assembly and stability, and functional requirement for nitrosylation and nitration of these SNARE proteins in biphasic insulin release using isolated islets expressing mutants insensitive- or nitrosomimetic. Gaining knowledge of how Syntaxin 4 and Munc18c function are affected by nitrosative post-translational modification will mark important progress towards the long-term goal of preserving beta cell function and viability in order to improve glucose homeostasis in the patient.
Diabetes mellitus affects nearly 21 million people in the U.S., and the number is growing. One key cause of diabetes is loss of proper beta cell insulin secretion, which is linked to increased nitrosative stress in beta cells, and we have identified nitrosative modifications of Syntaxin 4 and Munc18c-two key proteins in beta cell insulin secretion function. Understanding how Syntaxin 4 and Munc18c are affected by nitrosative modification will mark important progress to the overall goal of protecting insulin homeostasis and improving the quality of life for people with diabetes.
|Kalwat, Michael A; Wiseman, Dean A; Luo, Wei et al. (2012) Gelsolin associates with the N terminus of syntaxin 4 to regulate insulin granule exocytosis. Mol Endocrinol 26:128-41|
|Wiseman, Dean A; Kalwat, Michael A; Thurmond, Debbie C (2011) Stimulus-induced S-nitrosylation of Syntaxin 4 impacts insulin granule exocytosis. J Biol Chem 286:16344-54|