Access Type

Open Access Dissertation

Date of Award

January 2022

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmaceutical Sciences

First Advisor

Anjaneyulu Kowluru

Abstract

Defects in insulin secretion from pancreatic islet beta- cells leads to long term complications such as type 2 diabetes mellitus. The mechanism and regulation of glucose stimulated insulin secretion (GSIS) as well as factors contributing to pancreatic islet beta-cell demise are yet poorly understood. Previous work from our laboratory demonstrated that Ras-related C3 botulinum toxin substrate 1 (Rac1), a small G- protein is involved in GSIS as well as in culminating dysfunction under metabolic stress. Cycling of Rac1 between its inactive and active form is considered a vital event in glucose stimulated insulin secretion. This conversion is made possible by at least three types of regulatory proteins/ factors. [Guanine nucleotide exchange factors (GEFs)- that activates Rac1 by assisting in conversion of GDP- bound/ inactive Rac1 to GTP-bound/ active Rac1, Guanine nucleotide dissociation inhibitors (GDIs)- that prevents Rac1 activation by preventing GDP dissociation and GTP-ase activating proteins (GAPs)- that converts active Rac1 to its inactive form by completing the hydrolytic cycle.] Metabolic stress on islet beta- cells lead to sustained activation of Rac1, Nox2 mediated oxidative stress and downstream activation of MAPK. My project was aimed on investigating the roles of novel GEF, phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1)- a Rac1 specific GEF, and a novel scaffolding protein, caspase recruitment domain containing protein-9 (CARD9) in insulin secreting clonal pancreatic islet beta- cells. Methodical experimentation using INS-1 832/13 cells, showed that P-Rex1 is a key regulator/GEF for glucose-induced Rac1 activation and GSIS with additional roles of membrane targeting of Rac1 under acute glucose stimulatory conditions. CARD9 regulates GSIS via a Rac1-independent and p38MAPK-dependent signaling module. Under the duress of metabolic stress CARD9 mediates cellular dysfunction via accelerating the Rac- p38MAPK axis, culminating in endoplasmic reticulum stress and oxidative stress. Further, CARD9 is involved activation of NF-kB p65 signaling pathway in islet beta- cells. This study reports original contributory roles of different classes of regulatory proteins on small G- protein mediated islet beta- cell function, that may serve as therapeutic targets to alleviate insulin secretory defects.

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