GAD65-mediated glutamate decarboxylation reduces glucose-stimulated insulin secretion in pancreatic beta cells

Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell b y generating signals that couple glucose sensing to insulin secretion. We h ave demonstrated previously that mitochondrially derived glutamate particip ates directly in the stimulation of insulin exocytosis. The aim of the pres ent study was to impose altered cellular glutamate levels by overexpression of glutamate decarboxylase (GAD) to repress elevation of cytosolic glutama te. INS-1E cells infected with a recombinant adenovirus vector encoding GAD 65 showed efficient overexpression of the GAD protein with a parallel incre ase in enzyme activity. In control cells glutamate levels were slightly inc reased by 7.5 nim glucose (1.4-fold) compared with the effect at 15 mm (2.3 -fold) versus basal 2.5 mm glucose. Upon GAD overexpression, glutamate conc entrations were no longer elevated by 15 mm glucose as compared with contro ls (-40%). Insulin secretion was stimulated in control cells by glucose at 7.5 mm (2.5-fold) and more efficiently at 15 mm (5.2-fold). INS-1E cells ov erexpressing GAD exhibited impaired insulin secretion on stimulation with 1 5 mm glucose (-37%). The secretory response to 30 mm KCI, used to raise cyt osolic Ca2+ levels, was unaffected. Similar results were obtained in perifu sed rat pancreatic islets following adenovirus transduction. This GAD65-med iated glutamate decarboxylation correlating with impaired glucose-induced i nsulin secretion is compatible with a role for glutamate as a glucose-deriv ed factor participating in insulin exocytosis.