The dense core transmembrane vesicle protein IA-2 is a regulator of vesicle number and insulin secretion

doi:10.1073/pnas.0408887102

The dense core transmembrane vesicle protein IA-2 is a regulator of vesicle number and insulin secretion

^*Experimental Medicine Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892; ^†Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Churchill Hospital, Oxford OX3 7LJ, United Kingdom; and ^‡Department of Medicine, Guy's, King's College, and St Thomas' School of Medicine, King's College London, London SE5 9PJ, United Kingdom

Edited by Hugh O. McDevitt, Stanford University School of Medicine, Stanford, CA, and approved April 22, 2005 (received for review November 30, 2004)

Abstract

IA-2 is an enzymatically inactive member of the transmembrane protein tyrosine phosphate family located in dense core secretory vesicles and a major autoantigen in type 1 diabetes. Recent studies showed that targeted disruption of the IA-2 gene in mice resulted in impairment of insulin secretion and glucose intolerance. Insulin homeostasis, however, is a complex process involving a cascade of regulatory factors, and IA-2 is widely expressed in neuroendocrine cells throughout the body. Consequently, it is uncertain whether the impairment of insulin secretion in IA-2 knockout mice is a direct result of the knockout of IA-2 in beta cells or to counter regulatory alterations resulting from IA-2 knockout in other neuroendocrine cells. To define the function of IA-2, we studied the secretion of insulin in a single cell type, MIN-6, by overexpressing and knocking down IA-2. Our experiments showed that overexpression of IA-2 resulted in a 6-fold increase in glucose- or K⁺-induced insulin secretion and a ≈3-fold increase in the number of secretory vesicles and the insulin content of cells. In contrast, knockdown of endogenous IA-2 by short interfering RNA resulted in nearly a complete loss of glucose-induced insulin secretion and a 50% decrease in basal insulin release. The half-life of insulin in cells overexpressing IA-2 was nearly twice as great as that in mock-transfected cells, suggesting that IA-2 was stabilizing the insulin-containing vesicles. From these results we conclude that in beta cells, IA-2 is an important regulator of dense core vesicle number and glucose-induced and basal insulin secretion.

Footnotes

↵ § To whom correspondence should be addressed. E-mail: anotkins{at}mail.nih.gov.
Author contributions: S.-i.H. and A.L.N. designed research; S.-i.H., A.C., and M.R.C. performed research; S.-i.H., A.C., and M.R.C. analyzed data; and S.-i.H. and A.L.N. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: PTP, protein tyrosine phosphatase; siRNA, short interfering RNA; KRBH, Krebs-Ringer's solution bicarbonate Hepes.