All ETDs from UAB

Advisory Committee Chair

Kathleen H Berecek

Advisory Committee Members

Juan L Conreras

Stuart J Frank

Lori L McMahon

Erik M Schwiebert

Document Type

Dissertation

Date of Award

2008

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Insulin secretory defects within pancreatic beta-cells (β-cells) of islets of Langerhans play a role in the pathogenesis of diabetes mellitus. Furthermore, it is also well established that ATP and zinc are co-secreted with insulin in response to elevated extracellular glucose. Despite this knowledge, the physiological roles of extracellular secreted ATP and zinc within islets are ill-defined. We hypothesized that secreted ATP and zinc are autocrine purinergic signaling molecules that activate P2X purinergic receptor (P2XR) channels expressed by β-cells to enhance glucose-stimulated insulin secretion (GSIS). To test this postulate, ELISA, luciferin/luciferase-based bioluminescence and zinquin-based fluorescence assays were utilized to examine insulin, ATP and zinc secretion, respectively, from β-cells of primary islets. Additionally, reverse-transcription polymerase chain reaction (RT-PCR), Western blot analysis and immunofluorescence were employed to investigate P2XR channel subtype expression within immortalized β-cells and β-cells of primary islets. Findings from this study confirmed that the physiological insulin secretagogue, glucose, also stimulated secretion of the ligands for P2XR channels, ATP and zinc. Importantly, when these secreted purinergic signaling molecules were scavenged (apyrase) or chelated (DTPA), respectively, GSIS was significantly attenuated. In support of these findings, the application of exogenous ATP and zinc induced insulin secretion. Furthermore, iv antagonism of the sensors and intracellular transducers of extracellular ATP and zinc - P2XR channels, resulted in a significant attenuation of GSIS. The activation of these P2XR channels stimulated insulin secretion in a glucose-independent but Ca2+-dependent manner. In support of these findings, the expression of ATP-gated and zinc-modulated P2XR (P2X2,3,4,6) channels was observed within both immortalized β-cells and β-cells of primary islets. Additionally, immunohistochemistry of pancreatic tissue sections confirmed the expression of these P2XR channel subtypes within β-cells of islets. On the basis of these results, we conclude that 1) secreted ATP and zinc have profound autocrine regulatory influence on insulin secretion that is mediated via ATP-gated and zincmodulated P2XR channel signaling cascades 2) these signaling cascades play an integral and underlying role in GSIS and 3) the augmentation of ATP and zinc secretory pathways, oral nutritional zinc supplementation and stimulation of the P2XR channeldriven signaling pathway may be therapeutically beneficial for the treatment of diabetes.

Share

COinS