Abstract
Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion. We have demonstrated previously that mitochondrially derived glutamate participates directly in the stimulation of insulin exocytosis. The aim of the present study was to impose altered cellular glutamate levels by overexpression of glutamate decarboxylase (GAD) to repress elevation of cytosolic glutamate. INS-1E cells infected with a recombinant adenovirus vector encoding GAD65 showed efficient overexpression of the GAD protein with a parallel increase in enzyme activity. In control cells glutamate levels were slightly increased by 7.5 mm glucose (1.4-fold) compared with the effect at 15 mm (2.3-fold) versus basal 2.5 mm glucose. Upon GAD overexpression, glutamate concentrations were no longer elevated by 15 mm glucose as compared with controls (-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 overexpressing GAD exhibited impaired insulin secretion on stimulation with 15 mm glucose (-37%). The secretory response to 30 mm KCl, used to raise cytosolic Ca(2+) levels, was unaffected. Similar results were obtained in perifused rat pancreatic islets following adenovirus transduction. This GAD65-mediated glutamate decarboxylation correlating with impaired glucose-induced insulin secretion is compatible with a role for glutamate as a glucose-derived factor participating in insulin exocytosis.
Highlights
Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion [1,2,3]
The role of glutamate decarboxylase (GAD) in the beta cell is under intense study because it has been recognized as an autoantigen in insulindependent diabetes [38]
The purpose of the present study was to overexpress GAD65 in beta cells to lower the levels of glutamate through decarboxylation during glucose stimulation
Summary
Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion [1,2,3]. In permeabilized INS-1 cells, mitochondrial activation is associated with a marked stimulation of insulin release, which depends both on activation of the mitochondrial respiratory chain and on provision of carbons for the tricarboxylic acid cycle [13, 14] This secretory response requires a factor generated by mitochondrial metabolism distinct from ATP [13, 15] proposed to be glutamate [14]. Results obtained with a transgenic mouse model have indirectly highlighted the putative role of intracellular glutamate in insulin secretion In these mice, overexpression of the glutamate-decarboxylating enzyme GAD65 in the beta cells resulted in glucose intolerance without any sign of insulitis or loss of beta cells. Overexpression of GAD65 in beta cells resulted in reduced glutamate levels and impaired glucose-stimulated insulin secretion
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