Mitochondrial glycerol kinase activity in rat brain.
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ABSTRACT:
SUBMITTER: Tildon JT
PROVIDER: S-EPMC1163884 | biostudies-other | 1976 Aug
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:Glycerol kinase (GK) is an enzyme with diverse (moonlighting) cellular functions. GK overexpression affects central metabolic fluxes substantially; therefore, to elucidate the mechanism underlying these changes, we employed a systems-level evaluation of GK overexpression in H4IIE rat hepatoma cells. Microarray analysis revealed altered expression of genes in metabolism (central carbon and lipid), which correlated with previous flux analysis, and of genes regulated by the glucocorticoid receptor (GR). Oil Red O staining showed that GK overexpression leads to increased fat storage in H4IIE cells. Network component analysis revealed that activities of peroxisome proliferator-activated receptor alpha, GR, and seven other transcription factors were altered by GK overexpression. The increased activity of GR was experimentally verified by quantitative RT-PCR of GR-responsive genes in the presence and absence of the glucocorticoid agonist, dexamethasone. This systems biology approach further emphasizes GK's essential role in central and lipid metabolism and experimentally verifies GK's alternative (moonlighting) function of affecting GR transcription factor activity.
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Project description:Sequence analysis using the Promoser program predicted two promoter-like regions for rat mtGPAT: a distal promoter approximately 30kb upstream and a proximal promoter near the first translational codon. Rat liver cells transfected with pGL3-basic vector containing the distal and proximal promoter resulted in 10.8- and 4.8-fold increase in the luciferase activity, respectively. Results of electromobility shift assay and chromatin immunoprecipitation suggested binding of transcription factors to the distal and proximal promoter regions. 5' RACE PCR showed two transcripts with different transcriptional start sites. When transfected rat liver cells were starved and refed, there was about 2.7-fold increase in the luciferase activity with cells transfected with the distal promoter while the proximal promoter showed no change. Thus, the two promoters could be functionally distinguished. Taken together, the results suggest that there are two promoters for rat mtGPAT gene and that the transcriptional regulation is mediated through the distal promoter.
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Project description:The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.
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Project description:Analogues of glycerol in which each of the three hydroxy groups is successively replaced by fluorine or hydrogen have been examined as substrates or inhibitors of glycerol kinase (Candida mycoderma) to assess the ability of fluorine to mimic a substrate hydroxy group in enzyme-analogue interactions. The four diols resulting from replacement of the hydroxy groups at C-1 or C-2 of sn-glycerol by fluorine or hydrogen are weak substrates. Similar substitution of the C-3 hydroxy group gives compounds which act as competitive inhibitors of glycerol or dihydroxyacetone phosphorylation but show no activity as substrates. Comparison of the steady-state kinetic parameters of the corresponding analogues shows that replacement of a hydroxy group by either fluorine or hydrogen leads to compounds with similar activity in this system. A convenient synthesis of (+)-propane-1,2-diol is described.
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Project description:The rate of conversion of [1,3-14C]glycerol into 14CO2 was measured in the presence and absence of unlabelled alternative substrates in whole homogenates from the brains of young (4-6 and 18-20 days old) and adult rats. Unlabelled glucose decreased 14CO2 production from [1,3-14C]glycerol by about 40% at all ages studied. Unlabelled 3-hydroxybutyrate significantly decreased the 14CO2 production from both low (0.2 mM) and high (2.0 mM) concentrations of glycerol in 4-6- and 18-20-day-old rat pups. However, the addition of 3-hydroxybutyrate had no effect on the rate of 14CO2 production from 2.0 mM-glycerol in adult rats, suggesting that the interaction of 3-hydroxybutyrate with glycerol in adult rat brain is complex and may be related to the biphasic kinetics previously reported for glycerol oxidation. Unlabelled glutamine decreased the production of 14CO2 by brain homogenates from 18-20-day-old and adult rats, but not in 4-6-day-old rat pups. In the converse situation, the addition of unlabelled glycerol to whole brain homogenates had little effect on the rate of 14CO2 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [U-14C]glutamine, although some significant differences were noted. Collectively these results suggest that glycerol and these other substrates may be metabolized in separate subcellular compartments in brain such that the products of glucose, 3-hydroxybutyrate and glutamine metabolism can dilute the oxidation of glycerol, but the converse cannot occur. The data also demonstrate that there are complex age-related changes in the interaction of glycerol with 3-hydroxybutyrate and glutamine. The fact that glycerol oxidation was only partially suppressed by the addition of 1-5 mM-glucose, -3-hydroxybutyrate or -glutamine could also suggest that glycerol may be selectively utilized as an energy substrate in some discrete brain region.
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