Assay of Insulin Mediator Activity with Soluble Pyruvate Dehydrogenase Phosphatase

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Abstract
The putative mediator of intracellular insulin action was assayed quantitatively by its ability to increase the activity of solubilized pyruvate dehydrogenase (PDH) phosphatase. Conversion of soluble beef heart PDH b to PDH a by PDH phosphatase increased when incubation was carried out in the presence of a curve insulin mediator fraction generated from insulin-treated adipose tissue or liver plasma membranes. Increased PDH phosphatase activity was proportional to the concentration of added insulin mediator. Mediator generation was rapid, with a half-time of .apprx. 45 sec and was insulin dose dependent. Half-maximal mediator activity was produced at 0.3 nM added insulin, with maximal activity being generated at .apprx. 3 nM insulin. Mediator activity was significantly decreased at 7 nM insulin, but was increased 4-fold after ethanol extraction. Mediator behaved as an activator of PDH phosphatase, apparently by abolishing the inhibitory effects of ATP on phosphatase activity, but had no effect on PDH kinase activity. The assay of insulin mediator activity described here can be carried out under standardized conditions, in contrast to previously described methods using particulate mitochondrial preparations.

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