Glucose homeostasis is achieved by the coordinated efforts of insulin secretion from pancreatic islets and insulin action-glucose uptake by skeletal muscle and adipose tissues. Both processes are controlled by SNARE-mediated exocytosis events, and when these events go awry, Type 2 diabetes ensues. The t-SNARE protein, Syntaxin 4, and its regulatory binding partner, Munc18c, are now recognized as essential lcomponents of the exocytic mechanisms for both processes, although the detailed molecular mechanisms remain unresolved. The long term goal here is to understand the molecular, cellular and physiological basis for the dysregulation of glucose homeostasis leading to the onset of Type 2 diabetes. Towards this goal, published and preliminary data described herein reveal the beginnings of a detailed 'switch mechanism'conserved in beta cells and adipocytes involving the stimulus-induced phosphorylation of Munc18c and its subsequent preference for binding to a new partner, Doc2b. The objective of this application is to delineate the molecular mechanisms by which Munc18 and Doc2b proteins facilitate regulated exocytosis in insulinsecreting and insulin-responsive cell types to control glucose homeostasis. The central hypothesis is that Munc18c functions as the key molecular switch to transiently activate Syntaxin 4 and to promote vesicle delivery events in a stimulus-induced fashion. Supporting this are new findings which show: a) Requirement for Munc18c in a new pre-docking step of exocytosis termed 'syntaxin activation';b) Candidate Munc18c modifying factors to catalyze its tyrosine-phosphorylation present in adipocytes and in beta cells;c) New Munc18c binding factors involved in cytoskeletal remodeling and granule delivery/positioning. This hypothesis will be tested in three specific aims: 1) Determine the requirement for Munc18c-Doc2b interaction in regulating GLUT4 translocation and whole body glucose homeostasis;2) Delineate the role of Munc18-Doc2b protein complexes in biphasic insulin secretion;3) Elucidate the mechanisms by which Munc18c functions in Syntaxin 4-activation and in granule delivery/mobilization processes to facilitate insulin exocytosis. Studies will be accomplished using Doc2b and Munc18 knockout mice for whole body analyses of homeostasis and tissues there from for tissue-specific effects upon biphasic insulin release (islet perifusion) and skeletal muscle glucose uptake (GLUT4 translocation). MIN6 beta cells and 3T3L1 adipocytes will be utilized for molecular/biochemical dissection of spatial and temporal changes in proteinprotein interactions coupled with visualization of dynamic changes in granule/vesicle exocytosis events using microscopy and biochemical subcellular fractionation analyses. Gaining knowledge of how Munc18c regulates these exocytic events will mark progress towards the long-term goal of modulating, perhaps simultaneously, both insulin secretion and insulin-stimulated glucose uptake in order to improve glucose homeostasis in the patient.

Public Health Relevance

Type 2 diabetes has been coined a 'two-hit'disease;one 'hit'is dysfunction of glucose clearance by the skeletal muscle and adipose tissues, and another 'hit'is dysfunction of insulin secretion by the pancreatic islet beta cells. Aberrant abundance and function of the Munc18c protein and its binding partners have been reported in diabetic human muscle and islets and therefore have the potential to mediate cross-talk and underlie both 'hits'. Our research using novel rodent models supports an important and required role for Munc18c, and now it is imperative that the detailed molecular mechanisms involving Munc18c in these processes in skeletal muscle, adipose and islets be elucidated. Conservation of Munc18c-based mechanisms carries great potential for development of novel therapeutic strategies that could simultaneously tackle both 'hits'of this disease to improve the livelihood of people with diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK067912-09
Application #
8449756
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Silva, Corinne M
Project Start
2004-07-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2015-03-31
Support Year
9
Fiscal Year
2013
Total Cost
$305,153
Indirect Cost
$106,918
Name
Indiana University-Purdue University at Indianapolis
Department
Pediatrics
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Blue, Emily K; Ballman, Kimberly; Boyle, Frances et al. (2015) Fetal hyperglycemia and a high-fat diet contribute to aberrant glucose tolerance and hematopoiesis in adult rats. Pediatr Res 77:316-25
Ramalingam, Latha; Yoder, Stephanie M; Oh, Eunjin et al. (2014) Munc18c: a controversial regulator of peripheral insulin action. Trends Endocrinol Metab 25:601-8
Yoder, Stephanie M; Dineen, Stacey L; Wang, Zhanxiang et al. (2014) YES, a Src family kinase, is a proximal glucose-specific activator of cell division cycle control protein 42 (Cdc42) in pancreatic islet ? cells. J Biol Chem 289:11476-87
Ramalingam, Latha; Oh, Eunjin; Thurmond, Debbie C (2014) Doc2b enrichment enhances glucose homeostasis in mice via potentiation of insulin secretion and peripheral insulin sensitivity. Diabetologia 57:1476-84
Ramalingam, Latha; Lu, Jingping; Hudmon, Andy et al. (2014) Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet ?-cells. Biochem J 464:251-8
Kalwat, Michael A; Thurmond, Debbie C (2013) Signaling mechanisms of glucose-induced F-actin remodeling in pancreatic islet * cells. Exp Mol Med 45:e37
Kalwat, Michael A; Yoder, Stephanie M; Wang, Zhanxiang et al. (2013) A p21-activated kinase (PAK1) signaling cascade coordinately regulates F-actin remodeling and insulin granule exocytosis in pancreatic ? cells. Biochem Pharmacol 85:808-16
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
Kepner, Erica M; Yoder, Stephanie M; Oh, Eunjin et al. (2011) Cool-1/?PIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion. Am J Physiol Endocrinol Metab 301:E1072-80

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