ADENOVIRUS-MEDIATED EXPRESSION OF THE CATALYTIC SUBUNIT OF GLUCOSE-6-PHOSPHATASE IN INS-1 CELLS - EFFECTS ON GLUCOSE CYCLING, GLUCOSE USAGE, AND INSULIN-SECRETION

Glucose-6-phosphatase (Glu-6-Pase) catalyzes the terminal step of gluc oneogenesis, the conversion of glucose 6-phosphate (Glu-6-P) to free g lucose, This enzyme activity is thought to be conferred by a complex o f 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 p hosphohydrolase catalytic subunit residing in the lumen, and putative glucose and inorganic phosphate transporters that allow exit of the pr oducts of the reaction, In this study, we have investigated the effect of adenovirus-mediated overexpression of the Glu-6-Pase catalytic sub unit on glucose metabolism and insulin secretion, using a well differe ntiated insulinoma cell line, INS-1, We found that the overexpressed G lu-6-Pase catalytic subunit was normally glycosylated, correctly sorte d to the ER, and caused a 10-fold increase in Glu-6-Pase enzymatic act ivity in in vitro assays, Consistent with these findings, a 4.2-fold i ncrease in (H2O)-H-3 incorporation into glucose was observed in INS-1 cells treated with the recombinant adenovirus containing the Glu-6-Pas e catalytic subunit cDNA (AdCMV-Glu-6-Pase). 3-[H-3]Glucose usage was decreased by 32% in AdCMV-Glu-6-Pase-treated cells relative to control s, resulting in a proportional 30% decrease in glucose-stimulated insu lin secretion, Our findings indicate that overexpression of the Glu-6- Pase catalytic subunit significantly impacts glucose metabolism and in sulin secretion in islet beta-cells. However, INS-1 cells treated with AdCMV-Glu-6-Pase do not exhibit the severe alterations of beta-cell f unction and metabolism associated with islets from rodent models of ob esity and non-insulin-dependent diabetes mellitus, suggesting the invo lvement of genes in addition to the catalytic subunit of Glu-6-Pase in the etiology of such beta-cell dysfunction.