Access Type

Open Access Dissertation

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmaceutical Sciences

First Advisor

ANJANEYULU KOWLURU

Abstract

Type 2 diabetes mellitus is marked by a substantial beta-cell failure which is characterized by defective insulin secretion and resistance to insulin. Understanding the molecular events leading to Glucose-stimulated insulin secretion [GSIS] might serve as therapeutic potential towards diabetes. GSIS involves interplay between small G-proteins and their regulatory factors. Herein, I tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide exchange factor [GEF] for the small G-protein Arf6, mediates the activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of downstream effectors. Salient features of my study are: [i] ARNO/Arf6 is expressed in clonal β-cells, rodent islets and human islets; [ii] overexpression of inactive mutants of ARNO or Arf6 or siRNAs of Arf6 or ARNO reduces both GSIS and membrane depolarization induced insulin release in clonal β-cell line; [iii] secinH3, a selective inhibitor of ARNO/Arf6 signaling pathway, also inhibits GSIS in INS 832/13 cells and rodent islets; [iv] insulinotropic concentration of glucose stimulates Arf6 activation; [v] glucose-induced Arf6 activation is inhibited by secinH3 or siRNA-ARNO, suggesting a critical involvement of ARNO/Arf6 in insulin secretion; and [vi] glucose promotes association between ARNO and Arf6 as evidenced by co-immunoprecipitation and confocal microscopic studies. These findings provide the first evidence to implicate novel roles for ARNO/Arf6 in insulin secretion.

There are many factors that contribute to GSIS including various enzymes, small G-proteins and actin remodelers. As a step towards elucidating the ARNO/Arf6 signaling cascade, I identified potential downstream effectors that were regulated by ARNO/Arf6 upon glucose stimulation. I identified potential effectors using an ARNO-selective inhibitor [e.g., secinH3] and determined regulatory roles for Arf6/ARNO in promoting phospholipase D [PLD], extracellular-regulated kinases [ERK 1/2], Rac1/Cdc42, NADPH oxidase [Nox], reactive oxygen species [ROS], dynamin-1 and cofilin [actin-severing protein] signaling steps in isolated beta-cells.

Lastly, this work demonstrates dysfunction of Arf6 and Rac1 in beta-cells exposed to glucotoxicity and their abnormal response to physiological concentrations of glucose. This evidence indicates that defective insulin secretion seen in progressive beta-cell failure even after normalization might be at the level of abnormal functioning of small G-proteins. Together my data suggest Arf6/ARNO → PLD →Rac1 → Nox → cofilin signaling cascade regulate the exo-endocytotic pathway leading to GSIS.

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