DIFFERENTIAL-EFFECTS OF OVEREXPRESSED GLUCOKINASE AND HEXOKINASE-I INISOLATED ISLETS - EVIDENCE FOR FUNCTIONAL SEGREGATION OF THE HIGH ANDLOW 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 adenovir uses to overexpress the liver and islet isoforms of glucokinase as wel l as low K-m hexokinase I in isolated rat islets of Langerhans. Glucos e 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 i n 2- or 5-[H-3]glucose usage, lactate production, glycogen content, or glucose oxidation, Furthermore, glucokinase overexpression enhanced i nsulin secretion in response to stimulatory glucose or glucose plus ar ginine by only 36-53% relative to control. islets, In contrast to the minimal effects of overexpresed glucokinases, overexpression of hexoki nase I caused a 2.5-4-fold enhancement in all metabolic parameters exc ept glycogen content when measured at a basal glucose concentration (3 mM). Eased 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 evide nce for functional partitioning of glucokinase and hexokinase and sugg est 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.