Access Type

Open Access Thesis

Date of Award

January 2017

Degree Type

Thesis

Degree Name

M.S.

Department

Pharmaceutical Sciences

First Advisor

Anjaneyulu Kowluru

Abstract

METFROMIN, GLUCOTOXICITY AND ISLET DYSFUNCTION

by

SARTAJ BAIDWAN

MAY 2017

Advisor: Dr. Anjaneyulu Kowluru

Major: Pharmaceutical Sciences

Degree: Master of Science

Glucotoxicity is the leading cause for β-cell dysfunction [e.g., defective glucose-stimulated insulin secretion] in Type 2 Diabetes [T2DM]. Recent studies from our lab have shown sustained Rac1 activation leading to the activation of downstream signaling steps including stress kinase [p53, p38MAPK] activation and mitochondrial dysregulation [caspase-3 activation] in pancreatic islet beta-cells exposed to glucotoxic [HG] conditions [20 mM; 24 hrs]. Metformin [MF] is an oral anti-diabetic drug that is being widely prescribed to T2DM. MF works by suppressing hepatic glucose production and increasing glucose uptake by the target tissues. However, potential beneficial effects of MF on pancreatic beta-cell dysfunction under HG conditions have not been studied to date. Therefore, in the current studies, we asked if MF [0-30 μM; clinically relevant concentrations] affords protective effects against HG-induced metabolic dysfunction of the pancreatic beta [INS-1 832/13] cells. Since recent studies from our laboratory have demonstrated activation of Rac1, a small G-protein, as an upstream signaling event to stress kinase activation, we asked if protective effects of MF may, in part, be due to inhibition of HG-induced Rac1 activation in INS-1 832/13 cells. Data from these studies have suggested nearly 40% inhibition in HG-induced Rac1 activation [3.43±0.57 fold over basal; n=4; p<0.05] by MF. Evidence is also presented to highlight novel roles for sustained activation of Rac1 in HG-induced expression of Cluster of Differentiation 36 [CD36], a fatty acid transporter protein, which is implicated in cell apoptosis. Western blot analysis indicated a significant increase in the phosphorylation of p38MAPK [2.31±0.21 fold over basal; n=5; p<0.05], JNK1/2 and phosphorylation of p53 [4.42±1.20 fold over basal; n=3; p<0.05] in INS-1 832/13 cells. MF [15µM] markedly attenuated HG-induced p38MAPK [74.8%], JNK 1 and p53 [55.7%] activation under these experimental conditions. Our data from Bax phosphorylation [an indicator of cell dysregulation] studies demonstrated an increase in the phosphorylation of two Bax isoforms [Baxα by 1.63± 0.04 fold over basal; n=3; p<0.05; and Baxβ by 1.32±0.11 over basal; n=3; p<0.05]. MF [30µM] attenuated the phosphorylation of only Baxα isoform [by 77.3%]. Lastly, our data also suggested that co-provision of MF significantly reduced [72.4%] HG-induced caspase-3 activation. Together, these findings suggest significant protection by MF against HG-induced metabolic defects [activation of Rac1-stress kinase-caspase-3 signaling module] in the islet beta-cell. Potential implications of these findings in the context of novel and direct regulation of islet β-cell function by metformin are discussed.

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