Differential effects of overexpressed glucokinase and hexokinase I in isolated islets: Evidence for functional segregation of the high and low K_m enzymes

Glucose-stimulated insulin secretion is believed to require metabolism of the sugar via a high K_m pathway in which glucokinase (hexokinase IV) is rate-limiting. In this study, we have used recombinant adenoviruses to overexpress the liver and islet isoforms of glucokinase as well as low K_m hexokinase I in isolated rat islets of Langerhans. Glucose phosphorylating activity increased by up to 20-fold in extracts from islets treated with adenoviruses containing the cDNAs encoding either tissue isoform of glucokinase, but such cells exhibited no increase in 2- or 5-[³H]glucose usage, lactate production, glycogen content, or glucose oxidation. Furthermore, glucokinase overexpression enhanced insulin secretion in response to stimulatory glucose or glucose plus arginine by only 36-53% relative to control islets. In contrast to the minimal effects of overexpressed glucokinases, overexpression of hexokinase I caused a 2.5-4-fold enhancement in all metabolic parameters except glycogen content when measured at a basal glucose concentration (3 mM). Based on measurement of glucose phosphorylation in intact cells, overexpressed glucokinase is clearly active in a non-islet cell line (CV-1) but not within islet cells. That this result cannot be ascribed to the levels of glucokinase regulatory protein in islets is shown by direct measurement of its activity and mRNA. These data provide evidence for functional partitioning of glucokinase and hexokinase and suggest that overexpressed glucokinase must interact with factors found in limiting concentration in the islet cell in order to become activated and engage in productive metabolic signaling.