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Volume 272, Number 40, Issue of October 3, 1997 pp. 24837-24842
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Adenovirus-mediated Expression of the Catalytic Subunit of Glucose-6-phosphatase in INS-1 Cells
EFFECTS ON GLUCOSE CYCLING, GLUCOSE USAGE, AND INSULIN SECRETION

(Received for publication, May 29, 1997, and in revised form, June 27, 1997)

Khiet Trinh , Carol Minassian , Alex J. Lange ^§ , Robert M. O'Doherty and Christopher B. Newgard

From  Gifford Laboratories for Diabetes Research and the Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and the ^§ Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota 55455

Glucose-6-phosphatase (Glu-6-Pase) catalyzes the terminal step of gluconeogenesis, the conversion of glucose 6-phosphate (Glu-6-P) to free glucose. This enzyme activity is thought to be conferred by a complex of proteins residing in the endoplasmic reticulum (ER), including a Glu-6-P translocase that transports Glu-6-P into the lumen of the ER, a phosphohydrolase catalytic subunit residing in the lumen, and putative glucose and inorganic phosphate transporters that allow exit of the products of the reaction. In this study, we have investigated the effect of adenovirus-mediated overexpression of the Glu-6-Pase catalytic subunit on glucose metabolism and insulin secretion, using a well differentiated insulinoma cell line, INS-1. We found that the overexpressed Glu-6-Pase catalytic subunit was normally glycosylated, correctly sorted to the ER, and caused a 10-fold increase in Glu-6-Pase enzymatic activity in in vitro assays. Consistent with these findings, a 4.2-fold increase in ³H₂O incorporation into glucose was observed in INS-1 cells treated with the recombinant adenovirus containing the Glu-6-Pase catalytic subunit cDNA (AdCMV-Glu-6-Pase). 3-[³H]Glucose usage was decreased by 32% in AdCMV-Glu-6-Pase-treated cells relative to controls, resulting in a proportional 30% decrease in glucose-stimulated insulin secretion. Our findings indicate that overexpression of the Glu-6-Pase catalytic subunit significantly impacts glucose metabolism and insulin secretion in islet -cells. However, INS-1 cells treated with AdCMV-Glu-6-Pase do not exhibit the severe alterations of -cell function and metabolism associated with islets from rodent models of obesity and non-insulin-dependent diabetes mellitus, suggesting the involvement of genes in addition to the catalytic subunit of Glu-6-Pase in the etiology of such -cell dysfunction.

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