Both types 1 &2 diabetes (T1DM, T2DM) result from the inability of ?-cells to secrete sufficient insulin to maintain normal glucose homeostasis due to an acquired secretory defect and/or inadequate ?-cell mass. Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from growth factors (GFs) &nutrients to regulate cell growth &proliferation. Recent studies have determined that inhibition of glycogen synthase kinase-3 (GSK-3) &tuberous sclerosis complex 2 (TSC2) are required for mTOR activation. Our studies have shown that GSK-3 inhibition stimulates mTOR-mediated DNA synthesis &cell cycle progression in rodent &human islets. The highly reproducible stimulation of mTOR-mediated DNA synthesis by lithium, a GSK-3 inhibitor, in human islets is remarkable since nutrients are rarely effective. Since GSK-3 inhibition is also integral to Wnt/2-catenin transcriptional regulation, we propose to evaluate possible interactions of this pathway with mTOR signaling. Our goal is to enhance the growth &proliferative capacity of adult ?-cells by appropriately regulating both mTOR &the canonical Wnt signaling pathways using GSK-3 inhibitors, Wnt agonists &nutrients.
Specific aim 1 is to establish the functions of the GSK-3/TSC/mTOR &the canonical Wnt/GSK-3/2-catenin pathways in primary adult rodent &human islets by A) identifying the molecular &cellular targets &the ability of GSK-3 inhibitors, Wnt agonists, nutrients &rapamycin to modulate these pathways, B) elucidating the role of these pathways in the growth, proliferation &function of ?-cells &C) determining if a differential capacity to affect feedback inhibition of growth factor (GF) signaling distinguishes nutrients from GSK-3 inhibitors. Using isolated islets, we will utilize siRNA of GSK-3, ?-catenin &mTOR, study Wnt- dependent TCF/LEF1 transcription using transgenic TOPGAL mice &evaluate GF signaling and GSK-3 activity after chronic nutrient or GSK-3 inhibitor treatment.
Specific aim 2 is to determine if the mitochondrial transition pore (MTP) &nitric oxide (NO) are required for GSK-3 &mTOR signaling by A) examining if RNA interference of nitric oxide synthase (NOS) isoforms or MTP components, VDAC &ANT, alter the physiological responses of rodent &human islets to nutrients or GSK-3 inhibition, B) evaluating the ability of GSK-3 inhibitors, Wnt agonists or nutrients to regulate NOS expression &NO production &C) investigating how exogenous NO affects the MTP &?-cell growth, proliferation &function.
Specific aim 3 is to determine whether manipulation of GSK-3 and mTOR signaling will promote ?-cell function, growth &proliferation in a T1DM islet transplantation model by pre-culture of human or rodent islets with GSK-3 inhibitors or Wnt agonists 1 rapamycin prior to transplanting a "marginal dose" of islets into STZ-diabetic SCID mice. Understanding the co-regulation of the GSK-3/TSC/mTOR &Wnt/GSK-3/?-catenin pathways will provide new strategies to enhance growth &proliferation of adult ?-cells, serve important unmet medical needs of T1DM &T2DM &provide basic mechanistic knowledge of how nutrients affect these pathways.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK006181-49
Application #
8253735
Study Section
Cellular Aspects of Diabetes and Obesity Study Section (CADO)
Program Officer
Appel, Michael C
Project Start
1977-05-01
Project End
2013-06-30
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
49
Fiscal Year
2012
Total Cost
$342,643
Indirect Cost
$117,220
Name
Washington University
Department
Pathology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Kwon, Guim; Marshall, Connie A; Liu, Hui et al. (2006) Glucose-stimulated DNA synthesis through mammalian target of rapamycin (mTOR) is regulated by KATP channels: effects on cell cycle progression in rodent islets. J Biol Chem 281:3261-7
Pappan, Kirk L; Pan, Zhijun; Kwon, Guim et al. (2005) Pancreatic beta-cell lipoprotein lipase independently regulates islet glucose metabolism and normal insulin secretion. J Biol Chem 280:9023-9
Kwon, Guim; Pappan, Kirk L; Marshall, Connie A et al. (2004) cAMP Dose-dependently prevents palmitate-induced apoptosis by both protein kinase A- and cAMP-guanine nucleotide exchange factor-dependent pathways in beta-cells. J Biol Chem 279:8938-45
Cruz, W S; Kwon, G; Marshall, C A et al. (2001) Glucose and insulin stimulate heparin-releasable lipoprotein lipase activity in mouse islets and INS-1 cells. A potential link between insulin resistance and beta-cell dysfunction. J Biol Chem 276:12162-8
Hill, J R; Kwon, G; Marshall, C A et al. (1998) Hyperglycemic levels of glucose inhibit interleukin 1 release from RAW 264.7 murine macrophages by activation of protein kinase C. J Biol Chem 273:3308-13
Kwon, G; Corbett, J A; Hauser, S et al. (1998) Evidence for involvement of the proteasome complex (26S) and NFkappaB in IL-1beta-induced nitric oxide and prostaglandin production by rat islets and RINm5F cells. Diabetes 47:583-91
Scarim, A L; Arnush, M; Hill, J R et al. (1997) Evidence for the presence of type I IL-1 receptors on beta-cells of islets of Langerhans. Biochim Biophys Acta 1361:313-20
Kwon, G; Hill, J R; Corbett, J A et al. (1997) Effects of aspirin on nitric oxide formation and de novo protein synthesis by RINm5F cells and rat islets. Mol Pharmacol 52:398-405
Kwon, G; Corbett, J A; McDaniel, M L (1996) Interleukin 1-induced Fos and Jun do not regulate inducible nitric oxide synthase in rat islets of Langerhans and RINm5F cells. Endocrinology 137:825-30
Hill, J R; Corbett, J A; Baldwin, A C et al. (1996) Nitric oxide production by the rat insulinoma cell line, RINm5F, Is specific for IL-1: a spectrophotometric IL-1 bioassay. Anal Biochem 236:14-9

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