Abnormal pancreatic ?-cell function can have a profound impact on glucose homeostasis, with insufficient secretion of insulin, coupled with marked resistance to its actions by target tissues, resulting in type-2 diabetes. The overarching goal of this proposal is to develop an understanding of the molecular mechanisms that regulate insulin secretion from ?-cells, such that better therapeutic interventions promoting insulin secretion can be developed. The research is specifically focused on understanding the molecular mechanism by which the GTPase Rab27A exerts such potent regulation over exocytosis of insulin-containing secretory granules. The rate and location of GTP/GDP cycling for a particular Rab imposes precise temporal and spatial regulation to its action. Yet, the extent to which GTP/GDP cycling of Rab27A is regulated, the glucose-induced signaling pathways that govern its rate of cycling, as well as the mechanism by which Rab27A-GTP facilitates secretion of insulin, remain unknown. We hypothesize that the nucleotide state of Rab27A is strictly controlled and that GSIS initiates GTP/GDP cycling of Rab27 on specific identifiable populations of secretory granules. An increase in Rab27-GTP is proposed important to drive granule recruitment/docking via interaction with Slp4a, while GTP hydrolysis on docked granules is proposed to promote priming for fusion. The proposed research is placed into three specific aims. First, we will determine the spatial and temporal properties of Rab27A GTP/GDP cycling and their functional relationships to secretory dynamics during GSIS in cultured mouse ?-cells. Second, we will determine the primary site(s) in the regulated exocytotic pathway at which Rab27A acts to facilitate insulin secretion, define its mechanism of action, and characterize the mechanism of action of specific Rab27A effectors. Lastly, we will define the signaling pathways that regulate the rate of GTP/GDP cycling of Rab27A. Pinpoint the site of action in the secretory pathway regulated by signaling intermediate via effects on Rab27A. The experiments will use mouse ?-cells isolated and cultured from the ashen mouse model, which lack expression of Rab27A protein, and from control C3H/He mice. The experiments incorporate optical (FRET, TIRF-FRET), electrophysiological (patch-clamp, CM- monitoring) and biochemical/ pharmacological approaches to rigorously define mechanisms of Rab27A function.

Public Health Relevance

Insulin secreted by ?-cells of pancreatic islets is essential for maintenance of glucose homeostasis as well as for tissue development and growth. Type-2 diabetes, a disease predicted to affect 250 million people worldwide by the year 2020, occurs when the ability of the ?-cells to release insulin is exceeded by the requirements for the hormone to regulate glucose and lipid metabolism. In diabetic patients, early manifestations of ?-cell dysfunction include delayed and blunted insulin secretory responses to glucose challenges and loss of tight stimulus-secretion coupling. Therefore, there is a critical necessity to understand in full molecular and mechanistic detail the secretory pathway underlying insulin secretion to ultimately develop therapeutic treatments for diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK077050-04
Application #
8228165
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Appel, Michael C
Project Start
2009-03-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
4
Fiscal Year
2012
Total Cost
$337,371
Indirect Cost
$111,726
Name
University of Michigan Ann Arbor
Department
Physiology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Cazares, Victor A; Subramani, Arasakumar; Saldate, Johnny J et al. (2014) Distinct actions of Rab3 and Rab27 GTPases on late stages of exocytosis of insulin. Traffic 15:997-1015
Merrins, Matthew J; Stuenkel, Edward L (2008) Kinetics of Rab27a-dependent actions on vesicle docking and priming in pancreatic beta-cells. J Physiol 586:5367-81