Rate-limiting steps of gluconeogenesis in liver cells as determined with the aid of fluoro-dicarboxylic acids.

Abstract

Fluoromalate (an inhibitor of malate dehydrogenase and the malate carrier) or difluorooxaloacetate (an inhibitor of aspartate aminotransferase) inhibited gluconeogenesis from both pyruvate and lactate, but had no effect on glucose formation from fructose or endogenous substrates. When cells were incubated with both fluorocarboxylic acids simultaneously, an additive effect on the inhibition of glucose formation from lactate was observed, but the inhibition was not additive when pyruvate was the glucogenic precursor. Pyruvate removal was reduced 15% by difluorooxaloacetate and 44% by fluoromalate. Lactate production from pyruvate was inhibited 36% by fluoromalate but was almost unaffected by difluorooxaloacetate. The results suggest that both malate-oxaloacetate and glutamate-aspartate shuttles participate, but to different extents, in glucose formation from either pyruvate or lactate. The malateoxaloacetate shuttle predominates during gluconeogenesis from pyurvate whereas the glutamateaspartate shuttle is of greater importance when lactate is the gluconeogenic precursor. However, the inhibition by fluoromalate of glucose formation from lactate indicates that not all the reducing equivalents arising during lactate oxidation are directly available for the reductive step of gluconeogenesis. Neither of the two shuttles seems to compensate for inhibition of the alternative system. This lack of compensation implies that each shuttle is working close to or at its maximal capacity. Hence, transfer processes between mitochondria and cytoplasm must be considered as potential rate-limiting and regulatory sites for gluconeogenesis. The failure of fluoromalate to inhibit ethanol oxidation suggests that alternatives to the malate-oxaloacetate shuttle exist for the transfer to the mitochondria of reducing equivalents generated in the cytoplasmic compartment.