Abstract
Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.
Highlights
Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 M L-ascorbic acid
The combined data support the conclusion that L-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that inhibit certain classes of di-iron metalloenzymes, and that the enzyme is responsible for the proximal signal events in INS-1 cell glucosestimulated insulin release
Because of the potential role of Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) for shuttling reducing equivalents into the mitochondria during glucoseinduced insulin release from pancreatic  cells, we investigated the effects of a di-iron metalloenzyme inhibitor, propyl gallate, on glucose usage and glucose-induced insulin release from the rat insulinoma cell line, INS-1
Summary
Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 M L-ascorbic acid. The combined data support the conclusion that L-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that inhibit certain classes of di-iron metalloenzymes, and that the enzyme is responsible for the proximal signal events in INS-1 cell glucosestimulated insulin release. Particular importance has been attributed to the role of NADH in the glucose-induced activation of mitochondrial metabolism and insulin secretion [13, 14] These studies emphasized the essential roles played by both the glycerol-3-phosphate and the malate-aspartate shuttles in modulating the cytosolic NADH pool. Because of the potential role of mGPDH for shuttling reducing equivalents into the mitochondria during glucoseinduced insulin release from pancreatic  cells, we investigated the effects of a di-iron metalloenzyme inhibitor, propyl gallate, on glucose usage and glucose-induced insulin release from the rat insulinoma cell line, INS-1
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