Glycerol kinase has several diverse activities in mammalian cells.
Glycerol kinase deficiency is a complex, single-gene, inborn error of metabolism wherein no genotype-phenotype correlation has been established. Since
glycerol kinase has been suggested to exhibit additional activities than
glycerol phosphorylation, expression level perturbation in this
enzyme may affect cellular physiology globally. To investigate this possibility, we conducted metabolic investigations of wild-type and two
glycerol kinase-overexpressing H4IIE rat
hepatoma cell lines constructed in this study. The
glycerol kinase-overexpressing cell lines exhibited a significantly higher consumption of
carbon sources per cell, suggesting excess
carbon expenditure. Furthermore, we quantified intracellular metabolic fluxes by employing stable
isotope 13C labeling with a mathematically designed substrate mixture, gas chromatography-mass spectrometry, and comprehensive isotopomer balancing. This flux analysis revealed that the pentose phosphate pathway flux in the
glycerol kinase-overexpressing cell lines was 2-fold higher than that in the wild-type, in addition to subtler flux changes in other pathways of carbohydrate metabolism. Furthermore, the activity and transcript level of the lipogenic
enzyme glucose-6-phosphate dehydrogenase, the rate-limiting
enzyme of the pentose phosphate pathway, were also about 2-fold higher than that of the wild-type; these data corroborate the flux analysis results. This study shows that
glycerol kinase affects
carbon metabolism globally, possibly through its additional functions, and highlights
glycerol kinase's multifaceted role in cellular physiology.