GLUTAMATE PRODUCTION IN ISLETS OF LANGERHANS - PROPERTIES OF PHOSPHATE-ACTIVATED GLUTAMINASE

Homogenates of rat pancreas, pancreatic islets, and HIT-T15 cells (a clonal line derived from B cells) catalyzed the breakdown of glutamine to glutamate. This activity was markedly stimulated by the addition of orthophosphate and was much greater in homogenates from islets and the B-cell-derived clonal cell line than in those from whole pancreas. Islet glutaminase was half-maximally stimulated with 40 mmol/L phosphate. Kinetic analyses of the rates of glutamine hydrolysis showed that the Vmax for the reaction increased with the increase in phosphate concentration, whereas the Km for glutamine (2.6 ± 0.2 mmol/L) was unaltered. The pH optimum for enzyme activity was 8.0 to 8.5 at all phosphate concentrations studied. Glutamine breakdown was enhanced by adenosine triphosphate ([ATP] ∼100% at 10 mmol/L) and citrate (∼30% at 10 mmol/L), but it was unaffected by malate, 2-oxoglutarate, lactate, and ammonia. Glutamate significantly inhibited glutamine hydrolysis. Freshly isolated islets had a low content of both glutamate and glutamine. After culturing for 1 hour in an amino acid-containing medium, the concentrations of glutamine and glutamate increased. Subsequent perifusion without amino acids caused a loss of glutamine and a concomitant increase in glutamate level. Perifusion with 1 mmol/L glutamine led to an increase in both internal glutamine and glutamate. The addition to the perifusion medium of either 10 mmol/L glutamine, 10 mmol/L orthophosphate, or both substantially enhanced insulin release evoked by 10 mmol/L leucine. It is concluded that (1) pancreatic islets contain a phosphate-activated glutaminase, which is most likely located within B cells; (2) ATP and citrate may act as physiologic stimulators of glutaminase, whereas glutamate might function as its inhibitor; (3) glutamine serves as an efficient source of intracellular glutamate in perifused islets and, perhaps, in islets in pancreas in vivo; and (4) high glutaminase activity may be of importance for optimal insulin secretion elicited by amino acid secretagogues. © 1992.