The Experts below are selected from a list of 186 Experts worldwide ranked by ideXlab platform
Katrina M Dipple - One of the best experts on this subject based on the ideXlab platform.
-
Glycerol Hypersensitivity in a Drosophila Model for Glycerol Kinase Deficiency Is Affected by Mutations in Eye Pigmentation Genes
PloS one, 2012Co-Authors: Patrick J. Wightman, George R. Jackson, Katrina M DippleAbstract:Glycerol Kinase plays a critical role in metabolism by converting Glycerol to Glycerol 3-phosphate in an ATP dependent reaction. In humans, Glycerol Kinase deficiency results in a wide range of phenotypic variability; patients can have severe metabolic and CNS abnormalities, while others possess hyperGlycerolemia and Glyceroluria with no other apparent phenotype. In an effort to help understand the pathogenic mechanisms underlying the phenotypic variation, we have created a Drosophila model for Glycerol Kinase deficiency by RNAi targeting of dGyk (CG18374) and dGK (CG7995). As expected, RNAi flies have reduced Glycerol Kinase RNA expression, reduced phosphorylation activity and elevated Glycerol levels. Further investigation revealed these flies to be hypersensitive to fly food supplemented with Glycerol. Due to the hygroscopic nature of Glycerol, we predict Glycerol hypersensitivity is a result of greater susceptibility to desiccation, suggesting Glycerol Kinase to play an important role in desiccation resistance in insects. To evaluate a role for genetic modifier loci in determining severity of the Glycerol hypersensitivity observed in knockdown flies, we performed a preliminary screen of lethal transposon insertion mutant flies using a Glycerol hypersensitive survivorship assay. We demonstrate that this type of screen can identify both enhancer and suppressor genetic loci of Glycerol hypersensitivity. Furthermore, we found that the Glycerol hypersensitivity phenotype can be enhanced or suppressed by null mutations in eye pigmentation genes. Taken together, our data suggest proteins encoded by eye pigmentation genes play an important role in desiccation resistance and that eye pigmentation genes are strong modifiers of the Glycerol hypersensitive phenotype identified in our Drosophila model for Glycerol Kinase deficiency.
-
Global Metabolic Effects of Glycerol Kinase Overexpression in Rat Hepatoma Cells
Molecular genetics and metabolism, 2007Co-Authors: Ganesh Sriram, Lola Rahib, James C. Liao, Allison E. Campos, Lilly S. Parr, Katrina M DippleAbstract: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 13 C 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.
-
Glycerol Kinase deficiency alters expression of genes involved in lipid metabolism, carbohydrate metabolism, and insulin signaling
European Journal of Human Genetics, 2007Co-Authors: Lola Rahib, Nicole K. Maclennan, James C. Liao, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase (GK) is at the interface of fat and carbohydrate metabolism and has been implicated in insulin resistance and type 2 diabetes mellitus. To define GK's role in insulin resistance, we examined gene expression in brown adipose tissue in a Glycerol Kinase knockout (KO) mouse model using microarray analysis. Global gene expression profiles of KO mice were distinct from wild type with 668 differentially expressed genes. These include genes involved in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Real-time polymerase chain reaction analysis confirmed the differential expression of selected genes involved in lipid and carbohydrate metabolism. PathwayAssist analysis confirmed direct and indirect connections between Glycerol Kinase and genes in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Network component analysis (NCA) showed that the transcription factors (TFs) PPAR- γ , SREBP-1, SREBP-2, STAT3, STAT5, SP1, CEBP α , CREB, GR and PPAR- α have altered activity in the KO mice. NCA also revealed the individual contribution of these TFs on the expression of genes altered in the microarray data. This study elucidates the complex network of Glycerol Kinase and further confirms a possible role for Glycerol Kinase deficiency, a simple Mendelian disorder, in insulin resistance, and type 2 diabetes mellitus, a common complex genetic disorder.
-
Targeted disruption of Glycerol Kinase gene in mice: expression analysis in liver shows alterations in network partners related to Glycerol Kinase activity
Human Molecular Genetics, 2005Co-Authors: Nicole K. Maclennan, Lola Rahib, Cynthia Shin, Jason Dean, James C. Liao, Zixing Fang, Edward R B Mccabe, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurologicalcrises. Liver shows the highest level of Glycerol Kinase (GK) activity in humans and mice. Absence of geno-type–phenotype correlations in patients with GKD indicates the involvement of modifier genes, includingother network partners. To understand the molecular pathogenesis of GKD, we performed microarrayanalysis on liver mRNA from neonatal Glycerol Kinase (Gyk) knockout (KO) and wild-type (WT) mice.Unsupervised learning revealed that the overall gene expression profile of the KO mice was different fromthat of WT. Real-time PCR confirmed the differences for selected genes. Functional gene enrichment analysiswas usedto find56 increased and 37decreasedgene functionalcategories. PathwayAssist analysisidentifiedchangesingeneexpressionlevelsofgenesinvolvedinorganicacidmetabolismindicatingthatGKwaspartofthe same metabolic network which correlates well with the patients with GKD having metabolic acidemiaduringtheirepisodiccrises.Networkcomponentanalysis(NCA)showedthattranscriptionfactorssterolregu-latory element-binding protein (SREBP)-1c, carbohydrate response element-binding protein (ChREBP), hep-atocyte nuclear factor-4 alpha (HNF-4a) and peroxisome proliferative-activated receptor-alpha (PPARa)hadincreased activity in the Gyk KO mice compared with WT mice, whereas SREBP-2 was less active in the GykKOmice.ThesestudiesshowthatGykdeletioncausesalterationsinexpressionofgenesinseveralregulatorynetworks and is the first time NCA has been used to expand on microarray data from a mouse KO model of ahuman disease.INTRODUCTIONGlycerol Kinase (GK) phosphorylates Glycerol to Glycerol3-phosphate (G-3P), which is a source for dihydroxyacetonephosphate (DHAP), Glycerolipids, glucose, glycogen andprotein (1). Thus, GK is a critical link to glycolysis, glycogen-esis and gluconeogenesis. Mutations in the human GK gene onXp21 cause Glycerol Kinase deficiency (GKD), an X-linkedinborn error of metabolism with episodes of metabolic andneurological crises that include organic acidemia and hypo-glycemia (1). GKD is characterized by hyperGlycerolemiaand can be subdivided into three different categories: isolatedGKD (iGKD) (symptomatic and asymptomatic GKD) andcomplex GKD (cGKD) (1). iGKD involves the GK locusonly, whereas cGKD involves one or more additional lociaround GK including Duchenne muscular dystrophy (DMD),dosage-sensitive sex reversal-AHC critical region on thechromosome gene 1 (DAX1), interleukin-1 receptor accessoryprotein-like, gene 1 (IL1RAPL1) and aristaless-related homeo-box gene (ARX) (1,2). All patients with GKD evidencehyperGlycerolemia and Glyceroluria. Patients with sympto-matic iGKD or cGKD present with vomiting, lethargy and
-
Targeted disruption of Glycerol Kinase gene in mice: expression analysis in liver shows alterations in network partners related to Glycerol Kinase activity
Human molecular genetics, 2005Co-Authors: Nicole K. Maclennan, Lola Rahib, Cynthia Shin, Jason Dean, James C. Liao, Zixing Fang, Edward R B Mccabe, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurological crises. Liver shows the highest level of Glycerol Kinase (GK) activity in humans and mice. Absence of genotype-phenotype correlations in patients with GKD indicates the involvement of modifier genes, including other network partners. To understand the molecular pathogenesis of GKD, we performed microarray analysis on liver mRNA from neonatal Glycerol Kinase (Gyk) knockout (KO) and wild-type (WT) mice. Unsupervised learning revealed that the overall gene expression profile of the KO mice was different from that of WT. Real-time PCR confirmed the differences for selected genes. Functional gene enrichment analysis was used to find 56 increased and 37 decreased gene functional categories. PathwayAssist analysis identified changes in gene expression levels of genes involved in organic acid metabolism indicating that GK was part of the same metabolic network which correlates well with the patients with GKD having metabolic acidemia during their episodic crises. Network component analysis (NCA) showed that transcription factors sterol regulatory element-binding protein (SREBP)-1c, carbohydrate response element-binding protein (ChREBP), hepatocyte nuclear factor-4 alpha (HNF-4alpha) and peroxisome proliferative-activated receptor-alpha (PPARalpha) had increased activity in the Gyk KO mice compared with WT mice, whereas SREBP-2 was less active in the Gyk KO mice. These studies show that Gyk deletion causes alterations in expression of genes in several regulatory networks and is the first time NCA has been used to expand on microarray data from a mouse KO model of a human disease.
Edward R B Mccabe - One of the best experts on this subject based on the ideXlab platform.
-
Conserved family of Glycerol Kinase loci in Drosophila melanogaster.
Molecular genetics and metabolism, 2006Co-Authors: Julian A. Martinez Agosto, Edward R B MccabeAbstract:Glycerol Kinase (GK) is an enzyme that catalyzes the formation of Glycerol 3-phosphate from ATP and Glycerol, the rate-limiting step in Glycerol utilization. We analyzed the genome of the model organism Drosophila melanogaster and identified five GK orthologs, including two loci with sequence homology to the mammalian Xp21 GK protein. Using a combination of sequence analysis and evolutionary comparisons of orthologs between species, we characterized functional domains in the protein required for GK activity. Our findings include additional conserved domains that suggest novel nuclear and mitochondrial functions for Glycerol Kinase in apoptosis and transcriptional regulation. Investigation of GK function in Drosophila will inform us about the role of this enzyme in development and will provide us with a tool to examine genetic modifiers of human metabolic disorders.
-
Targeted disruption of Glycerol Kinase gene in mice: expression analysis in liver shows alterations in network partners related to Glycerol Kinase activity
Human Molecular Genetics, 2005Co-Authors: Nicole K. Maclennan, Lola Rahib, Cynthia Shin, Jason Dean, James C. Liao, Zixing Fang, Edward R B Mccabe, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurologicalcrises. Liver shows the highest level of Glycerol Kinase (GK) activity in humans and mice. Absence of geno-type–phenotype correlations in patients with GKD indicates the involvement of modifier genes, includingother network partners. To understand the molecular pathogenesis of GKD, we performed microarrayanalysis on liver mRNA from neonatal Glycerol Kinase (Gyk) knockout (KO) and wild-type (WT) mice.Unsupervised learning revealed that the overall gene expression profile of the KO mice was different fromthat of WT. Real-time PCR confirmed the differences for selected genes. Functional gene enrichment analysiswas usedto find56 increased and 37decreasedgene functionalcategories. PathwayAssist analysisidentifiedchangesingeneexpressionlevelsofgenesinvolvedinorganicacidmetabolismindicatingthatGKwaspartofthe same metabolic network which correlates well with the patients with GKD having metabolic acidemiaduringtheirepisodiccrises.Networkcomponentanalysis(NCA)showedthattranscriptionfactorssterolregu-latory element-binding protein (SREBP)-1c, carbohydrate response element-binding protein (ChREBP), hep-atocyte nuclear factor-4 alpha (HNF-4a) and peroxisome proliferative-activated receptor-alpha (PPARa)hadincreased activity in the Gyk KO mice compared with WT mice, whereas SREBP-2 was less active in the GykKOmice.ThesestudiesshowthatGykdeletioncausesalterationsinexpressionofgenesinseveralregulatorynetworks and is the first time NCA has been used to expand on microarray data from a mouse KO model of ahuman disease.INTRODUCTIONGlycerol Kinase (GK) phosphorylates Glycerol to Glycerol3-phosphate (G-3P), which is a source for dihydroxyacetonephosphate (DHAP), Glycerolipids, glucose, glycogen andprotein (1). Thus, GK is a critical link to glycolysis, glycogen-esis and gluconeogenesis. Mutations in the human GK gene onXp21 cause Glycerol Kinase deficiency (GKD), an X-linkedinborn error of metabolism with episodes of metabolic andneurological crises that include organic acidemia and hypo-glycemia (1). GKD is characterized by hyperGlycerolemiaand can be subdivided into three different categories: isolatedGKD (iGKD) (symptomatic and asymptomatic GKD) andcomplex GKD (cGKD) (1). iGKD involves the GK locusonly, whereas cGKD involves one or more additional lociaround GK including Duchenne muscular dystrophy (DMD),dosage-sensitive sex reversal-AHC critical region on thechromosome gene 1 (DAX1), interleukin-1 receptor accessoryprotein-like, gene 1 (IL1RAPL1) and aristaless-related homeo-box gene (ARX) (1,2). All patients with GKD evidencehyperGlycerolemia and Glyceroluria. Patients with sympto-matic iGKD or cGKD present with vomiting, lethargy and
-
Targeted disruption of Glycerol Kinase gene in mice: expression analysis in liver shows alterations in network partners related to Glycerol Kinase activity
Human molecular genetics, 2005Co-Authors: Nicole K. Maclennan, Lola Rahib, Cynthia Shin, Jason Dean, James C. Liao, Zixing Fang, Edward R B Mccabe, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurological crises. Liver shows the highest level of Glycerol Kinase (GK) activity in humans and mice. Absence of genotype-phenotype correlations in patients with GKD indicates the involvement of modifier genes, including other network partners. To understand the molecular pathogenesis of GKD, we performed microarray analysis on liver mRNA from neonatal Glycerol Kinase (Gyk) knockout (KO) and wild-type (WT) mice. Unsupervised learning revealed that the overall gene expression profile of the KO mice was different from that of WT. Real-time PCR confirmed the differences for selected genes. Functional gene enrichment analysis was used to find 56 increased and 37 decreased gene functional categories. PathwayAssist analysis identified changes in gene expression levels of genes involved in organic acid metabolism indicating that GK was part of the same metabolic network which correlates well with the patients with GKD having metabolic acidemia during their episodic crises. Network component analysis (NCA) showed that transcription factors sterol regulatory element-binding protein (SREBP)-1c, carbohydrate response element-binding protein (ChREBP), hepatocyte nuclear factor-4 alpha (HNF-4alpha) and peroxisome proliferative-activated receptor-alpha (PPARalpha) had increased activity in the Gyk KO mice compared with WT mice, whereas SREBP-2 was less active in the Gyk KO mice. These studies show that Gyk deletion causes alterations in expression of genes in several regulatory networks and is the first time NCA has been used to expand on microarray data from a mouse KO model of a human disease.
-
Gene therapy for murine Glycerol Kinase deficiency: importance of murine ortholog.
Biochemical and Biophysical Research Communications, 2005Co-Authors: N. Kuwada, Katrina M Dipple, Nicole K. Maclennan, K. Nagano, J. Havens, M. Kumar, Edward R B MccabeAbstract:A Glycerol Kinase (Gyk) knock-out (KO) mouse model permits improved understanding of Glycerol Kinase (GK) deficiency (GKD) pathogenesis, however, early death of affected mice limits its utility. The purpose of this work was to delay death of affected males to investigate thoroughly their phenotypes. An adenoviral vector carrying the human (Adeno-XGK) or mouse (Adeno-XGyk) GK gene was injected into KO mice within 24 h of birth. Adeno-XGK did not change KO mouse survival time despite liver GK activity greater than 100% of wild type. However, Adeno-XGyk improved KO mouse survival time greater than two-fold. These investigations demonstrate that gene replacement therapy for Gyk KO mice is more efficacious using murine Gyk than human GK. These studies expand our understanding of GKD pathogenesis in the murine model, and show that while murine GKD is more severe than in humans, GKD mice have similar metabolic disturbances to affected humans with hypoglycemia and acidemia.
-
AluY insertion (IVS4-52ins316alu) in the Glycerol Kinase gene from an individual with benign Glycerol Kinase deficiency
Human mutation, 2000Co-Authors: Yao-hua Zhang, Katrina M Dipple, Kim C. Worley, Eric Vilain, Bing-ling Huang, G. Finlayson, B.l. Therrell, Prescott L. Deininger, Edward R B MccabeAbstract:Glycerol Kinase deficiency has three distinct forms: an isolated form which may be benign or symptomatic, and a complex form which is symptomatic and part of an Xp21 contiguous gene syndrome. Here we report the case of a male with benign isolated Glycerol Kinase deficiency who was incidentally identified after observation of pseudohypertriglyceridemia. DNA sequencing of this subject's Glycerol Kinase gene showed the insertion of an AluY sequence in intron 4 of the Glycerol Kinase gene. Although Alu insertions have been implicated in other diseases, and a closely related AluY element is found as an insert in the C1 inhibitor gene in patients with hereditary angioedema, this is the first case of Glycerol Kinase deficiency caused by an Alu insertion. Hum Mutat 15:316–323, 2000. © 2000 Wiley-Liss, Inc.
Tadashi Yoshimoto - One of the best experts on this subject based on the ideXlab platform.
-
Preliminary crystallographic study of Thermus aquaticus Glycerol Kinase
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: Hua-shan Huang, Takahiko Inoue, Tadashi YoshimotoAbstract:Glycerol Kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving Glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A. Native data were collected to 2.87 A resolution on a Cu Kα rotating-anode X-ray source.
-
Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of Glycerol Kinase in Thermus flavus.
Microbiology (Reading England), 1999Co-Authors: Emmanuelle Darbon, Hua-shan Huang, Tadashi Yoshimoto, Kiyoshi Ito, Sandrine Poncet, Josef DeutscherAbstract:The genes glpK and glpF, encoding Glycerol Kinase and the Glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of Glycerol Kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the Glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up Glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus Glycerol Kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in Glycerol Kinase of Enterococcus casseliflavus. Purified Glycerol Kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal Glycerol Kinases, phosphorylated T. flavus Glycerol Kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated Glycerol Kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal Glycerol Kinases, which stimulated Glycerol Kinase activity about 10-fold, phosphorylation of T. flavus Glycerol Kinase caused only a slight increase in enzyme activity.
-
THERMOSTABLE Glycerol Kinase FROM THERMUS FLAVUS : CLONING, SEQUENCING, AND EXPRESSION OF THE ENZYME GENE
Biochimica et biophysica acta, 1998Co-Authors: Hua-shan Huang, Kiyoshi Ito, Tsutomu Kabashima, Chang-hong Yin, Yoshiaki Nishiya, Yoshihisa Kawamura, Tadashi YoshimotoAbstract:Abstract The thermostable Glycerol Kinase (EC 2.7.1.30) gene from Thermus flavus was cloned and expressed in Escherichia coli DH5 α . An open reading frame of 1488 bp for the Glycerol Kinase gene ( glpK ) starting with an ATG methionine codon was found, which encodes a protein of 496 amino acid residues whose calculated molecular weight is 54,835. The amino acid sequence of T. flavus Glycerol Kinase is 80.6% and 64.1% identical with those of Bacillus subtilis and E. coli . Transformants of E. coli DH5 α harboring plasmid pGYK12 with a 1505 bp chromosomal DNA fragment containing the T. flavus Glycerol Kinase gene showed about 23.8-fold higher Glycerol Kinase activity than T. flavus .
Lola Rahib - One of the best experts on this subject based on the ideXlab platform.
-
Global Metabolic Effects of Glycerol Kinase Overexpression in Rat Hepatoma Cells
Molecular genetics and metabolism, 2007Co-Authors: Ganesh Sriram, Lola Rahib, James C. Liao, Allison E. Campos, Lilly S. Parr, Katrina M DippleAbstract: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 13 C 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.
-
Glycerol Kinase deficiency alters expression of genes involved in lipid metabolism, carbohydrate metabolism, and insulin signaling
European Journal of Human Genetics, 2007Co-Authors: Lola Rahib, Nicole K. Maclennan, James C. Liao, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase (GK) is at the interface of fat and carbohydrate metabolism and has been implicated in insulin resistance and type 2 diabetes mellitus. To define GK's role in insulin resistance, we examined gene expression in brown adipose tissue in a Glycerol Kinase knockout (KO) mouse model using microarray analysis. Global gene expression profiles of KO mice were distinct from wild type with 668 differentially expressed genes. These include genes involved in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Real-time polymerase chain reaction analysis confirmed the differential expression of selected genes involved in lipid and carbohydrate metabolism. PathwayAssist analysis confirmed direct and indirect connections between Glycerol Kinase and genes in lipid metabolism, carbohydrate metabolism, insulin signaling, and insulin resistance. Network component analysis (NCA) showed that the transcription factors (TFs) PPAR- γ , SREBP-1, SREBP-2, STAT3, STAT5, SP1, CEBP α , CREB, GR and PPAR- α have altered activity in the KO mice. NCA also revealed the individual contribution of these TFs on the expression of genes altered in the microarray data. This study elucidates the complex network of Glycerol Kinase and further confirms a possible role for Glycerol Kinase deficiency, a simple Mendelian disorder, in insulin resistance, and type 2 diabetes mellitus, a common complex genetic disorder.
-
Targeted disruption of Glycerol Kinase gene in mice: expression analysis in liver shows alterations in network partners related to Glycerol Kinase activity
Human Molecular Genetics, 2005Co-Authors: Nicole K. Maclennan, Lola Rahib, Cynthia Shin, Jason Dean, James C. Liao, Zixing Fang, Edward R B Mccabe, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurologicalcrises. Liver shows the highest level of Glycerol Kinase (GK) activity in humans and mice. Absence of geno-type–phenotype correlations in patients with GKD indicates the involvement of modifier genes, includingother network partners. To understand the molecular pathogenesis of GKD, we performed microarrayanalysis on liver mRNA from neonatal Glycerol Kinase (Gyk) knockout (KO) and wild-type (WT) mice.Unsupervised learning revealed that the overall gene expression profile of the KO mice was different fromthat of WT. Real-time PCR confirmed the differences for selected genes. Functional gene enrichment analysiswas usedto find56 increased and 37decreasedgene functionalcategories. PathwayAssist analysisidentifiedchangesingeneexpressionlevelsofgenesinvolvedinorganicacidmetabolismindicatingthatGKwaspartofthe same metabolic network which correlates well with the patients with GKD having metabolic acidemiaduringtheirepisodiccrises.Networkcomponentanalysis(NCA)showedthattranscriptionfactorssterolregu-latory element-binding protein (SREBP)-1c, carbohydrate response element-binding protein (ChREBP), hep-atocyte nuclear factor-4 alpha (HNF-4a) and peroxisome proliferative-activated receptor-alpha (PPARa)hadincreased activity in the Gyk KO mice compared with WT mice, whereas SREBP-2 was less active in the GykKOmice.ThesestudiesshowthatGykdeletioncausesalterationsinexpressionofgenesinseveralregulatorynetworks and is the first time NCA has been used to expand on microarray data from a mouse KO model of ahuman disease.INTRODUCTIONGlycerol Kinase (GK) phosphorylates Glycerol to Glycerol3-phosphate (G-3P), which is a source for dihydroxyacetonephosphate (DHAP), Glycerolipids, glucose, glycogen andprotein (1). Thus, GK is a critical link to glycolysis, glycogen-esis and gluconeogenesis. Mutations in the human GK gene onXp21 cause Glycerol Kinase deficiency (GKD), an X-linkedinborn error of metabolism with episodes of metabolic andneurological crises that include organic acidemia and hypo-glycemia (1). GKD is characterized by hyperGlycerolemiaand can be subdivided into three different categories: isolatedGKD (iGKD) (symptomatic and asymptomatic GKD) andcomplex GKD (cGKD) (1). iGKD involves the GK locusonly, whereas cGKD involves one or more additional lociaround GK including Duchenne muscular dystrophy (DMD),dosage-sensitive sex reversal-AHC critical region on thechromosome gene 1 (DAX1), interleukin-1 receptor accessoryprotein-like, gene 1 (IL1RAPL1) and aristaless-related homeo-box gene (ARX) (1,2). All patients with GKD evidencehyperGlycerolemia and Glyceroluria. Patients with sympto-matic iGKD or cGKD present with vomiting, lethargy and
-
Targeted disruption of Glycerol Kinase gene in mice: expression analysis in liver shows alterations in network partners related to Glycerol Kinase activity
Human molecular genetics, 2005Co-Authors: Nicole K. Maclennan, Lola Rahib, Cynthia Shin, Jason Dean, James C. Liao, Zixing Fang, Edward R B Mccabe, Steve Horvath, Katrina M DippleAbstract:Glycerol Kinase deficiency (GKD) is an X-linked inborn error of metabolism with metabolic and neurological crises. Liver shows the highest level of Glycerol Kinase (GK) activity in humans and mice. Absence of genotype-phenotype correlations in patients with GKD indicates the involvement of modifier genes, including other network partners. To understand the molecular pathogenesis of GKD, we performed microarray analysis on liver mRNA from neonatal Glycerol Kinase (Gyk) knockout (KO) and wild-type (WT) mice. Unsupervised learning revealed that the overall gene expression profile of the KO mice was different from that of WT. Real-time PCR confirmed the differences for selected genes. Functional gene enrichment analysis was used to find 56 increased and 37 decreased gene functional categories. PathwayAssist analysis identified changes in gene expression levels of genes involved in organic acid metabolism indicating that GK was part of the same metabolic network which correlates well with the patients with GKD having metabolic acidemia during their episodic crises. Network component analysis (NCA) showed that transcription factors sterol regulatory element-binding protein (SREBP)-1c, carbohydrate response element-binding protein (ChREBP), hepatocyte nuclear factor-4 alpha (HNF-4alpha) and peroxisome proliferative-activated receptor-alpha (PPARalpha) had increased activity in the Gyk KO mice compared with WT mice, whereas SREBP-2 was less active in the Gyk KO mice. These studies show that Gyk deletion causes alterations in expression of genes in several regulatory networks and is the first time NCA has been used to expand on microarray data from a mouse KO model of a human disease.
Hua-shan Huang - One of the best experts on this subject based on the ideXlab platform.
-
Preliminary crystallographic study of Thermus aquaticus Glycerol Kinase
Acta Crystallographica Section D-biological Crystallography, 2001Co-Authors: Hua-shan Huang, Takahiko Inoue, Tadashi YoshimotoAbstract:Glycerol Kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving Glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A. Native data were collected to 2.87 A resolution on a Cu Kα rotating-anode X-ray source.
-
Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of Glycerol Kinase in Thermus flavus.
Microbiology (Reading England), 1999Co-Authors: Emmanuelle Darbon, Hua-shan Huang, Tadashi Yoshimoto, Kiyoshi Ito, Sandrine Poncet, Josef DeutscherAbstract:The genes glpK and glpF, encoding Glycerol Kinase and the Glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of Glycerol Kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the Glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up Glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus Glycerol Kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in Glycerol Kinase of Enterococcus casseliflavus. Purified Glycerol Kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal Glycerol Kinases, phosphorylated T. flavus Glycerol Kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated Glycerol Kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal Glycerol Kinases, which stimulated Glycerol Kinase activity about 10-fold, phosphorylation of T. flavus Glycerol Kinase caused only a slight increase in enzyme activity.
-
THERMOSTABLE Glycerol Kinase FROM THERMUS FLAVUS : CLONING, SEQUENCING, AND EXPRESSION OF THE ENZYME GENE
Biochimica et biophysica acta, 1998Co-Authors: Hua-shan Huang, Kiyoshi Ito, Tsutomu Kabashima, Chang-hong Yin, Yoshiaki Nishiya, Yoshihisa Kawamura, Tadashi YoshimotoAbstract:Abstract The thermostable Glycerol Kinase (EC 2.7.1.30) gene from Thermus flavus was cloned and expressed in Escherichia coli DH5 α . An open reading frame of 1488 bp for the Glycerol Kinase gene ( glpK ) starting with an ATG methionine codon was found, which encodes a protein of 496 amino acid residues whose calculated molecular weight is 54,835. The amino acid sequence of T. flavus Glycerol Kinase is 80.6% and 64.1% identical with those of Bacillus subtilis and E. coli . Transformants of E. coli DH5 α harboring plasmid pGYK12 with a 1505 bp chromosomal DNA fragment containing the T. flavus Glycerol Kinase gene showed about 23.8-fold higher Glycerol Kinase activity than T. flavus .