The molecular basis for primary hereditary hypertriglyceridemia has been identified in fewer than 5% of cases. Investigation of monogenic dyslipidemias has the potential to expose key metabolic ...pathways. We describe a hitherto unreported disease in ten individuals manifesting as moderate to severe transient childhood hypertriglyceridemia and fatty liver followed by hepatic fibrosis and the identification of the mutated gene responsible for this condition. We performed SNP array-based homozygosity mapping and found a single large continuous segment of homozygosity on chromosomal region 12q13.12. The candidate region contained 35 genes that are listed in Online Mendelian Inheritance in Man (OMIM) and 27 other genes. We performed candidate gene sequencing and screened both clinically affected individuals (children and adults with hypertriglyceridemia) and also a healthy cohort for mutations in GPD1, which encodes glycerol-3-phosphate dehydrogenase 1. Mutation analysis revealed a homozygous splicing mutation, c.361−1G>C, which resulted in an aberrantly spliced mRNA in the ten affected individuals. This mutation is predicted to result in a truncated protein lacking essential conserved residues, including a functional site responsible for initial substrate recognition. Functional consequences of the mutation were evaluated by measuring intracellular concentrations of cholesterol and triglyceride as well as triglyceride secretion in HepG2 (hepatocellular carcinoma) human cells lines overexpressing normal and mutant GPD1 cDNA. Overexpression of mutant GPD1 in HepG2 cells, in comparison to overexpression of wild-type GPD1, resulted in increased secretion of triglycerides (p = 0.01). This finding supports the pathogenicity of the identified mutation.
This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride ...accumulation by affecting early (days 0-2) and late (days 4-8), but not middle (days 2-4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein β (C/EBPβ; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPβ expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process.
Dekkera bruxellensis is mainly associated with lambic beer fermentation and wine production and may contribute in a positive or negative manner to the flavor development. This yeast is able to ...produce phenolic compounds, such as 4-ethylguaiacol and 4-ethylphenol which could spoil the wine, depending on their concentration. In this work we have investigated how this yeast responds when exposed to conditions causing osmotic stress, as high sorbitol or salt concentrations. We observed that osmotic stress determined the production and accumulation of intracellular glycerol, and the expression of NADH-dependent glycerol-3-phosphate dehydrogenase (GPD) activity was elevated. The involvement of the HOG MAPK pathway in response to this stress condition was also investigated. We show that in D. bruxellensis Hog1 protein is activated by phosphorylation under hyperosmotic conditions, highlighting the conserved role of HOG MAP kinase signaling pathway in the osmotic stress response.
Gene Accession numbers in GenBank: DbHOG1: JX65361, DbSTL1: JX965362.
•We examine the osmotic stress response in the wine yeast Dekkera bruxellensis.•We observed that osmotic stress determined the accumulation of intracellular glycerol.•The expression of glycerol-3-phosphate dehydrogenase activity was increased.•The Hog1 protein is activated by phosphorylation under hyperosmotic conditions.
Background: NAD-dependent glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the interconversion of dihydroxyacetone phosphate and
l-glycerol-3-phosphate. Although the enzyme has been characterized ...and cloned from a number of sources, until now no three-dimensional structure has been determined for this enzyme. Although the utility of this enzyme as a drug target against
Leishmania mexicana is yet to be established, the critical role played by GPDH in the long slender bloodstream form of the related kinetoplastid
Trypanosoma brucei makes it a viable drug target against sleeping sickness.
Results: The 1.75Å crystal structure of apo GPDH from
L. mexicana was determined by multiwavelength anomalous diffraction (MAD) techniques, and used to solve the 2.8 Å holo structure in complex with NADH. Each 39 kDa subunit of the dimeric enzyme contains a 189-residue N-terminal NAD-binding domain and a 156-residue C-terminal substrate-binding domain. Significant parts of both domains share structural similarity with plant acetohydroxyacid isomeroreductase. The discovery of extra, fatty-acid like, density buried inside the C-terminal domain indicates a possible post-translational modification with an associated biological function.
Conclusions: The crystal structure of GPDH from
L. mexicana is the first structure of this enzyme from any source and, in view of the sequence identity of 63%, serves as a valid model for the
T. brucei enzyme. The differences between the human and trypanosomal enzymes are extensive, with only 29% sequence identity between the parasite and host enzyme, and support the feasibility of exploiting the NADH-binding site to develop selective inhibitors against trypanosomal GPDH. The structure also offers a plausible explanation for the observed inhibition of the
T. brucei enzyme by melarsen oxide, the active form of the trypanocidal drugs melarsoprol and cymelarsan.
OBJECTIVE: To find out whether lipid stores are influenced by phenolic compounds in wine. DESIGN: Differentiated 3T3-L1 cells were treated with catechin, epicatechin or procyanidin extracts with ...different degrees of polymerization at 150 micromolar for different periods of time (0.5-24 h). SUBJECTS: Cell line 3T3-L1. MEASUREMENTS: Cellular viability, glycerol-3-phosphate dehydrogenase activity, glycerol release in the medium, HSL mRNA levels, triacylglycerols and protein. RESULTS: Catechin, epicatechin and procyanidin extracts were not toxic for the 3T3-L1 cells in the conditions assayed. Glycerol-3-phosphate dehydrogenase activity was markedly decreased by 150 micromolar procyanidin extracts. The release of glycerol into the medium was increased in 150 micromolar procyanidin extract-treated cells and reached a plateau after 15 h exposure. Procyanidins caused a time-dependent reduction in the HSL mRNA levels. CONCLUSIONS: These results suggest that procyanidins from grape and wine affect lipid metabolism whilst their monomers (catechin and epicatechin) do not. This effect is more pronounced when the degree of polymerization is higher. Procyanidin extracts cause a time-dependent reduction in the HSL mRNA levels, inhibit triacylglycerol synthesis and also favour triacylglycerol hydrolysis until the HSL mRNA had reached very low levels.
The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is ...uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.
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•Glycerol-3-phosphate shuttle is uncoupled in some cancers such as kidney cancers•High flux through cytosolic GPD supports lipid synthesis and cancer proliferation•Loss of mitochondrial GPD upregulates the expression of cytosolic GPD•Low-GPD2 expressing tumors are more sensitive to lipid synthesis inhibition
Yao et al. demonstrate that the glycerol-3-phosphate shuttle is uncoupled in kidney cancers where the cytosolic reaction is faster than the mitochondrial reaction. High flux through cytosolic GPD is important for redox homeostasis and lipid synthesis. Loss of mitochondrial GPD promotes tumor growth and sensitizes tumors to lipid synthesis inhibitors.
Glycerol 3‐phosphate dehydrogenase (Gpd1 isoform) catalyzes the rate limiting step of glycerol synthesis and is a critical component of the osmo‐responsive machinery in yeast. The three‐dimensional ...structure of the enzyme is similar to the enzyme from many other organisms, including humans. A recent study with the human enzyme has proposed K120 (K152 in yeast) to be in the correct orientation for catalysis; K204 (K245 in yeast) is out of plane and is not a participant in the catalytic cycle. The current work was carried out to establish the role of K245 in the catalytic cycle of yeast Gpd1. K245A mutant was found to possess lower catalytic activity. Osmotically stressed cells expressing Gpd1 (K245A) showed no change in intracellular glycerol as compared with wild‐type cells which showed ~60% increase. Fluorescence microscopy, native polyacrylamide gel electrophoresis (PAGE) analysis, fluorescence spectroscopy, and Thioflavin T spectrofluorimetry showed a relatively unstable, aggregation‐ and degradation‐prone conformation for the mutant. In silico studies showed an aggregation “hotspot” around K245. This study establishes the requirement of K245 for conformational stability and functional adaptation of Gpd1 in Saccharomyces cerevisiae.
Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone ...phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.
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