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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Cellular Aspects of Diabetes and Obesity Study Section (CADO)
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Appel, Michael C
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Washington University
Schools of Medicine
Saint Louis
United States
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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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