Dataset Information

Global metabolic effects of glycerol kinase overexpression in rat hepatoma cells.

ABSTRACT: 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.

SUBMITTER: Sriram G

PROVIDER: S-EPMC2702542 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Global metabolic effects of glycerol kinase overexpression in rat hepatoma cells.

Sriram Ganesh G Rahib Lola L He Jian-Sen JS Campos Allison E AE Parr Lilly S LS Liao James C JC Dipple Katrina M KM

Molecular genetics and metabolism 20071029 2

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 glyce ...[more]

PMID: 18029214

Similar Datasets

Project description:Glycochenodeoxycholate (GCDC) and taurolithocholate (TLC) are hepatotoxic and cholestatic bile salts, whereas tauroursodeoxycholate (TUDC) is cytoprotective and anticholestatic. Yet they all act, in part, through phosphatidylinositol-3-kinase(PI3K)-dependent mechanisms ("PI3K-paradox"). Hepatocytes express three catalytic PI3K Class I isoforms (p110?/?/?), specific functions of which, in liver, are unclear. In other cell types, p110? is associated with detrimental effects. To shed light on the PI3K enigma, we determined whether hydrophobic and hydrophilic bile salts differentially activate distinct p110 isoforms in hepatocytes, and whether p110? mediates bile salt-induced hepatocyte cell death.Isoform-specific PI3K activity assays were established to determine isoform activation by bile salts in rat hepatocytes. Activation of Akt and JNK was determined by immunoblotting. Following stimulation with hydrophobic bile salts, hepatocellular apoptosis was determined morphologically after Hoechst staining and by analysis of caspase-3/-7 activity or caspase-3 cleavage. Activity or expression of PI3K p110? was inhibited pharmacologically (AS604850) or by knock-down using specific siRNA.All bile salts tested activated p110?, while p110? was activated by TUDC and GCDC. Intriguingly, only hydrophobic bile salts activated p110?. Inhibition of p110? attenuated GCDC-induced Akt- and JNK-activation, but did not alter TUDC- or cAMP-induced Akt-signaling in rat hepatocytes. Inhibition or knock-down of p110? markedly attenuated hydrophobic bile salt-induced apoptosis in rat hepatocytes and human hepatoma cell lines but did not alter Fas-, tumor necrosis factor ?- and etoposide-induced apoptosis. Depletion of Ca(++) prevented GCDC-induced toxicity in rat hepatocytes but did not affect GCDC-induced Akt- and JNK-activation.PI3K p110? is activated by hydrophobic, but not hydrophilic bile salts. Bile salt-induced hepatocyte apoptosis is partly mediated via a PI3K p110? dependent signaling pathway, potentially involving JNK.

Dataset Information

Global metabolic effects of glycerol kinase overexpression in rat hepatoma cells.

Publications

Global metabolic effects of glycerol kinase overexpression in rat hepatoma cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure