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.
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