Insulin regulation of pyruvate kinase activity in isolated adipocytes. Crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action.

We recently identified glutamine:fructose-6-phosphate amidotransferase (GFAT) as an insulin-regulated enzyme in adipocytes. Moreover, we found that loss of GFAT activity is not due to a direct action of insulin but rather is mediated by enhanced glucose uptake and the subsequent routing of glucose through the hexosamine biosynthesis pathway. To assess whether other cytosolic enzymes are controlled through formation of hexosamine products, we treated adipocytes for 5 h with physiological concentrations of insulin (ED50 = 0.33 ng/ml), glucose (ED50 = 4.5 mM), and glutamine (ED50 = 4.4 mM) and then measured pyruvate kinase (PK) activity. Combined treatment resulted in a progressive (t 1/2 of 2.5 h) and marked (3-fold) increase in PK activity, whereas omission of one or more of these components failed to alter enzyme activity. Several lines of additional evidence implicated the hexosamine biosynthesis pathway in PK regulation; therefore, it appears that the M2 isoform of pyruvate kinase represents another enzyme regulated by insulin through stimulation of glucose uptake and formation of hexosamine products. Related studies revealed that enhancement of PK activity is dependent upon ongoing mRNA synthesis and de novo protein synthesis and is mediated by an increase in enzyme content. Considered together, these findings provide new insights into the cascade of metabolic events triggered by insulin and implicated a novel metabolic pathway in the pretranslational control of enzyme function.