Inositol biosynthesis is inversely regulated by glycolytic activity

Inositol is an essential metabolite required for membrane biogenesis, cell signaling and trafficking, and gene expression. Its importance is underscored by the fact that inositol depletion leads to death in eukaryotic cells ranging from yeast to human. Perturbation of inositol homeostasis is implicated in numerous human disorders. In yeast, inositol is synthesized de novo from glucose-6-phosphate (G-6-P) through a two-step pathway controlled by the rate-limiting enzyme myo-inositol phosphate synthase (MIPS), encoded by INO1. INO1 expression is tightly controlled by the Henry regulatory circuit in response to inositol and by the Reg1-Snf1 pathway in response to glucose. As both inositol synthesis and glycolysis utilize the same precursor, G-6-P, the current study tested the hypothesis that inositol synthesis is regulated by glycolytic activity. Genetic and pharmacological approaches were used to alter glycolysis. Inositol synthesis was determined by liquid chromatography mass spectrometry analysis of [U-¹³C]-glucose incorporation into inositol and by assaying INO1 mRNA and MIPS protein expression. In rho0 cells and wild type cells treated with the electron transport inhibitor potassium cyanide, both of which exhibit increased glycolytic activity, inositol levels were reduced by 47.5% and 57.9%, respectively, compared to controls. INO1 mRNA and MIPS protein levels were also decreased in these cells. The effect of downregulation of glycolysis was determined using the humanized yeast strain HHK2, which expresses a decreased rate of glycolysis due to the less active human hexokinase. Consistent with our hypothesis, HHK2 cells exhibited a 41.5% increase in inositol synthesis. Dysregulation of inositol synthesis did not affect glycolysis. These results identify altered glycolytic activity as a mechanism of inositol regulation and demonstrate the role of metabolic crosstalk in controlling these biochemical pathways.