Current treatments of Type 2 diabetic patients for hyperglycemia result in constitutive and non-regulated insulin delivery, and this failure to mimic endogenous insulin secretion leads to hypoglycemia problems and eventual beta cell failure. Thus the long term goal here is to decipher the mechanisms used by pancreatic islet beta cells to meter insulin release, and then devise ways to recapitulate this metering mechanism pharmacologically in the patient. Thousands of insulin granules exist behind a filamentous actin (F-actin) barrier in the beta cell and F-actin remodeling is known to mobilize granules to the t-SNARE proteins at the cell surface, yet the mechanisms involved in remodeling and granule mobilization are largely unknown and untested. Published and preliminary work presented here suggests that the key to actin remodeling lies in the glucose-specific activation of the small Rho family GTPase protein Cdc42, and that Cdc42 is essential for second-phase insulin release from islets. New data also reveal that the Cdc42 guanine dissociation inhibitor (GDI) is required to keep Cdc42 inactive, and its mutation/depletion leads to inappropriate constitutive insulin secretion. The Cdc42-GDI complex binds directly to the v-SNARE on the insulin granules, and disruption of this binding attenuates glucose but not KCl-stimulated insulin secretion. Moreover, this glucose-specific Cdc42 activation is coupled to interactions between t-SNARE proteins and F-actin. Thus, the objective of this application is to delineate the physiologic, cellular and molecular mechanisms by which glucose activates Cdc42 to promote second-phase insulin release, and to determine how Cdc42 controls second phase through Cdc42-v-SNARE interactions and by impacting F-actin-t-SNARE associations to regulate insulin granule targeting and actin reorganization. The central hypothesis for the proposed research is that that Cdc42 becomes activated specifically in response to glucose to coordinate the second phase of insulin release by selectively remodeling F-actin to mobilize and target granules towards SNARE sites at the plasma membrane for exocytosis. This will be tested in three Specific Aims: 1) Elucidate how Cdc42 activation is regulated by glucose in pancreatic beta cells;2) Identify how Cdc42-v-SNARE interactions regulate insulin exocytosis;3) Determine the functional role of actin-t-SNARE interactions in Cdc42-mediated actin remodeling and the regulation of insulin exocytosis. Studies will be accomplished using siRNA-mediated knockdown in islet beta cells with 'rescue'strategies, and by corroborating quantitation of biphasic insulin release (islet perifusion) with visualization of spatial changes in Cdc42 interactions using microscopy and biochemical subcellular fractionation analyses. Gaining knowledge of how Cdc42 functions in second-phase secretion will mark progress towards the long-term goal of modulating actin remodeling in the beta cell to recapitulate regulated insulin secretion and prevent beta cell failure. Project?Narrative? ? ? Although?diabetic?patients?use?sulfonylureas?or?insulin?injection?to?survive,?these?treatments?fail?to? simulate?the?carefully?metered?release?of?insulin?that?would?otherwise?come?from?their?own?pancreatic?islet? cells.??Our?preliminary?data?have?revealed?that?the?metered?release?of?insulin?is?controlled?by?a?protein?named? Cdc42,?and?further?investigation?of?this?protein's?function?in?insulin?secretion?will?hopefully?lead?to?discovery? of?new?therapeutic?strategies?for?insulin?delivery?that?better?simulate?natural?insulin?release?from?a?healthy? human?pancreas?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 #
5R01DK076614-05
Application #
8238394
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Appel, Michael C
Project Start
2008-04-01
Project End
2013-12-28
Budget Start
2012-03-01
Budget End
2013-12-28
Support Year
5
Fiscal Year
2012
Total Cost
$312,029
Indirect Cost
$103,758
Name
Indiana University-Purdue University at Indianapolis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Thurmond, Debbie C; Oh, Eunjin; Miller, Richard A (2015) Potential Site Effects and Transgene Expression Discrepancies in Mouse Lifespan Studies. Cell Metab 22:346-7
Oh, Eunjin; Miller, Richard A; Thurmond, Debbie C (2015) Syntaxin 4 Overexpression Ameliorates Effects of Aging and High-Fat Diet on Glucose Control and Extends Lifespan. Cell Metab 22:499-507
Oh, Eunjin; Stull, Natalie D; Mirmira, Raghavendra G et al. (2014) Syntaxin 4 up-regulation increases efficiency of insulin release in pancreatic islets from humans with and without type 2 diabetes mellitus. J Clin Endocrinol Metab 99:E866-70
Ramalingam, Latha; Yoder, Stephanie M; Oh, Eunjin et al. (2014) Munc18c: a controversial regulator of peripheral insulin action. Trends Endocrinol Metab 25:601-8
Tunduguru, Ragadeepthi; Chiu, Tim T; Ramalingam, Latha et al. (2014) Signaling of the p21-activated kinase (PAK1) coordinates insulin-stimulated actin remodeling and glucose uptake in skeletal muscle cells. Biochem Pharmacol 92:380-8
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 β cells. J Biol Chem 289:11476-87
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 β-cells. Biochem J 464:251-8
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
Kalwat, Michael A; Thurmond, Debbie C (2013) Signaling mechanisms of glucose-induced F-actin remodeling in pancreatic islet β 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 β cells. Biochem Pharmacol 85:808-16

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