Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion.

Abstract Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 μm 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 μm l-ascorbic acid (56%) or 300 μm iron (Fe2+ or Fe3+ (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 theK 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 μm propyl gallate. The metabolism of [5-3H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 μm 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.