Immp2l Enhances the Structure and Function of Mitochondrial Gpd2 Dehydrogenase.

'Inner mitochondrial membrane peptidase 2 like' (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, 'mitochondrial glycerol phosphate dehydrogenase 2' (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD-/- KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD-/- KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD-/- KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD-/- KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD-/- KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD-/- KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD-/- KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD-/- KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD-/- KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD-/- KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

GlcA-mediated glycerol-3-phosphate synthesis contributes to the oxidation resistance of Aspergillus fumigatus via decreasing the cellular ROS.

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.

[Transient infantile hypertriglyceridemia caused by GPD1 deficiency: report of two cases and literature review].

Objective: To investigate the clinical phenotype and genotype of transient infantile hypertriglyceridemia (HTGTI). Methods: The clinical data of two HTGTI children, diagnosed at Children's Hospital of Fudan University from July 2019 to January 2020, were collected and analyzed retrospectively. The literature up to 25th January 2020 were searched in PubMed, CNKI and Wanfang databases with the key words of "hypertriglyceridemia" and "glycerol phosphate dehydrogenase-1 (GPD1)". Results: Two children, including a 5-month-old female and a 13-month-old male, who presented with hepatomegaly, hypertriglyceridemia, transaminase elevation and hepatic steatosis, were admitted to the hospital. Gene detection found compound heterozygous variation of GPD1. After a low-fat diet with enriched medium-chain fatty acids, their plasma triglyceride level were significantly decreased, and finally normalized in case 2. Literature review found 17 patients with GPD1 gene variation reported in 5 papers, including 16 HTGTI cases and one case of different phenotype. Most of the cases presented with hepatomegaly, hypertriglyceridemia and transaminase elevation, while some had developmental retardation, splenomegaly, hypoglycemia, obesity and insulin resistance. The c.361-1G>C was the most common variation of GPD1. Conclusions: HTGTI caused by GPD1 deficiency is mainly manifested with hepatomegaly, hypertriglyceridemia, transaminase elevation as well as hepatic steatosis and fibrosis. The most common variation of GPD1 is c.361-1G>C.

GPD1 Enhances the Anticancer Effects of Metformin by Synergistically Increasing Total Cellular Glycerol-3-Phosphate.

Metformin is an oral drug widely used for the treatment of type 2 diabetes mellitus. Numerous studies have demonstrated the value of metformin in cancer treatment. However, for metformin to elicit effects on cancer often requires a high dosage, and any underlying mechanism for how to improve its inhibitory effects remains unknown. Here, we found that low mRNA expression of glycerol-3-phosphate dehydrogenase 1 (GPD1) may predict a poor response to metformin treatment in 15 cancer cell lines. In vitro and in vivo, metformin treatment alone significantly suppressed cancer cell proliferation, a phenotype enhanced by GPD1 overexpression. Total cellular glycerol-3-phosphate concentration was significantly increased by the combination of GPD1 overexpression and metformin treatment, which suppressed cancer growth via inhibition of mitochondrial function. Eventually, increased reactive oxygen species and mitochondrial structural damage was observed in GPD1-overexpressing cell lines treated with metformin, which may contribute to cell death. In summary, this study demonstrates that GPD1 overexpression enhances the anticancer activity of metformin and that patients with increased GPD1 expression in tumor cells may respond better to metformin therapy. SIGNIFICANCE: GPD1 overexpression enhances the anticancer effect of metformin through synergistic inhibition of mitochondrial function, thereby providing new insight into metformin-mediated cancer therapy.

Human Glycerol 3-Phosphate Dehydrogenase: X-ray Crystal Structures That Guide the Interpretation of Mutagenesis Studies.

Human liver glycerol 3-phosphate dehydrogenase ( hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzyme-substrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E·NAD, and ternary E·NAD·DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion-activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains were likewise examined by double-mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

Anaerobic glycerol-3-phosphate dehydrogenase complex from hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Glycerol-3-phosphate (G3P) is a key intermediate of glycerol metabolism and is oxidized to dihydroxyacetone phosphate aerobically or anaerobically by appropriate G3P dehydrogenases. A hyperthermophilic archaeon Thermococcus kodakarensis KOD1 has a novel operon consisting of three genes encoding an anaerobic G3P dehydrogenase (G3PDH), an NADH oxidase (NOX), and a molybdopterin oxidoreductase (MOX). Typically, the G3PDH gene (glpA) is included in an operon with genes encoding essential subunits of the G3PDH complex, glpB and glpC. The three genes from T. kodakarensis were cloned and expressed in Escherichia coli, and their recombinant proteins, Tk-G3PDH, Tk-NOX and Tk-MOX, were characterized. The optimal temperature of Tk-G3PDH for activity was 80°C, indicating high thermal stability. Tk-G3PDH has flavin adenine dinucleotide as a prosthetic group and catalyzes oxidation of G3P with kcat/Km 1.93 × 103 M-1s-1 at 80°C, compared with 9.83 × 105 M-1s-1 for the E. coli G3PDH complex at 37°C. Interestingly, Tk-G3PDH can catalyze this reaction even as a monomer, whereas GlpA must form a complex with GlpB and GlpC. Tk-G3PDH also forms a putative heteropentamer with Tk-NOX and Tk-MOX (G3PDH:NOX:MOX = 2:2:1). This complex may form an electron transfer pathway to a final electron acceptor in the cell membrane, as is the case for the typical G3PDH complex GlpABC.

Modulation of the Glycerol Phosphate availability led to concomitant reduction in the citric acid excretion and increase in lipid content and yield in Yarrowia lipolytica.

In order to improve TriAcylGycerol (TAG) lipids accumulation in the yeast Yarrowia lipolytica on glucose, double over-expression of the major acyl-CoA:diacylglycerol acyltransferase encoding gene (ylDGA2) and of the glycerol-phosphate dehydrogenase encoding gene (ylGPD1) was carried out. The genes were over-expressed in a strain impaired for the mobilization of the accumulated lipids, through the deletion of the genes encoding acyl-coenzyme A oxidases (POX1-6 genes) and the deletion of the very efficient lipase attached to the lipid bodies, encoded by ylTGL4. This metabolic engineering strategy had the objective of pulling the C-flow into the TAG synthesis by increasing the availability of glycerol-3-phosphate and its binding to fatty acids for the TAG synthesis. This strain showed a strong improvement in production performances on glucose in terms of lipid content (increase from 18 to 55%), lipid yield (increase from 0,035 to 0.14gg -1) and by-product formation (decrease in citric acid yield from 0.68 to 0.4gg -1). For developing bioprocess for the production of triacylglycerol from renewable carbon sources as glucose it is of first importance to control the C/N ratio in order to avoid citric acid excretion during lipid accumulation. Our engineered strain showed a delay in the onset of citric acid excretion as suggested by the 15% modulation of the critical C/N ratio.

Mitochondrial glycerol 3-phosphate facilitates bumblebee pre-flight thermogenesis.

Bumblebees (Bombus terrestris) fly at low ambient temperatures where other insects cannot, and to do so they must pre-warm their flight muscles. While some have proposed mechanisms, none fully explain how pre-flight thermogenesis occurs. Here, we present a novel hypothesis based on the less studied mitochondrial glycerol 3-phosphate dehydrogenase pathway (mGPDH). Using calorimetry, and high resolution respirometry coupled with fluorimetry, we report substrate oxidation by mGPDH in permeabilised flight muscles operates, in vitro, at a high flux, even in the absence of ADP. This may be facilitated by an endogenous, mGPDH-mediated uncoupling of mitochondria. This uncoupling increases ETS activity, which results in increased heat release. Furthermore, passive regulation of this mechanism is achieved via dampened temperature sensitivity of mGPDH relative to other respiratory pathways, and subsequent consumption of its substrate, glycerol 3-phosphate (G3P), at low temperatures. Mitochondrial GPDH may therefore facilitate pre-flight thermogenesis through poor mitochondrial coupling. We calculate this can occur at a sufficient rate to warm flight muscles until shivering commences, and until flight muscle function is adequate for bumblebees to fly in the cold.

Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay.

The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Glycerol-3-phosphate dehydrogenase 1 deficiency induces compensatory amino acid metabolism during fasting in mice.

BACKGROUND: Glucose is used as an energy source in many organs and obtained from dietary carbohydrates. However, when the external energy supply is interrupted, e.g., during fasting, carbohydrates preserved in the liver and glycogenic precursors derived from other organs are used to maintain blood glucose levels. Glycerol and glycogenic amino acids derived from adipocytes and skeletal muscles are utilized as glycogenic precursors. Glycerol-3-phosphate dehydrogenase 1 (GPD1), an NAD+/NADH-dependent enzyme present in the cytosol, catalyzes the reversible conversion of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP). Since G3P is one of the substrates utilized for gluconeogenesis in the liver, the conversion of G3P to DHAP by GPD1 is essential for maintaining blood glucose levels during fasting. We focused on GPD1 and examined its roles in gluconeogenesis during fasting. METHODS: Using GPD1 null model BALB/cHeA mice (HeA mice), we measured gluconeogenesis from glycerol and the change of blood glucose levels under fasting conditions. We also measured gene expression related to gluconeogenesis in the liver and protein metabolism in skeletal muscle. BALB/cBy mice (By mice) were used as a control. RESULTS: The blood glucose levels in the HeA mice were lower than that in the By mice after glycerol administration. Although lack of GPD1 inhibited gluconeogenesis from glycerol, blood glucose levels in the HeA mice after 1-4h of fasting were significantly higher than that in the By mice. Muscle protein synthesis in HeA mice was significantly lower than that in the By mice. Moreover, blood alanine levels and usage of alanine for gluconeogenesis in the liver were significantly higher in the HeA mice than that in the By mice. CONCLUSIONS: Although these data indicate that a lack of GPD1 inhibits gluconeogenesis from glycerol, chronic GPD1 deficiency may induce an adaptation that enhances gluconeogenesis from glycogenic amino acids.

Two glycerol 3-phosphate dehydrogenase isogenes from Candida versatilis SN-18 play an important role in glycerol biosynthesis under osmotic stress.

Two isogenes of glycerol 3-phosphate dehydrogenase (GPD) from Candida versatilis SN-18 were cloned and sequenced. These intronless genes (Cagpd1 and Cagpd2) were both predicted to encode a 378 amino acid polypeptide, and the deduced amino acid sequences mutually showed 76% identity. Interestingly, Cagpd1 and Cagpd2 were located tandemly in a locus of genomic DNA within a 262 bp interval. To our knowledge, this represents a novel instance of isogenic genes relating to glucose metabolism. The stress response element (STRE) was found respectively at -93 to -89 bp upstream of the 5'end of Cagpd1 and -707 to -703 bp upstream of Cagpd2, indicating that these genes are involved in osmotic stress response. In heterologous expression using a gpd1Δgpd2Δ double deletion mutant of Saccharomyces cerevisiae, Cagpd1 and Cagpd2 transformants complemented the function of GPD, with Cagpd2 being much more effective than Cagpd1 in promoting growth and glycerol synthesis. Phylogenetic analysis of the amino acid sequences suggested that Cagpd1p and Cagpd2p are NADP(+)-dependent GPDs (EC 1.1.1.94). However, crude enzyme extract from Cagpd1 and Cagpd2 transformants showed GPD activity with only NAD(+) as cofactor. Hence, both Cagpd1p and Cagpd2p are likely NAD(+)-dependent GPDs (EC 1.1.1.8), similar to GPDs from S. cerevisiae and Candida magnoliae.

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates.

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.

Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants.

Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement.

Production of polyhydroxybutyrate and alginate from glycerol by Azotobacter vinelandii under nitrogen-free conditions.

Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source.

Inborn errors of cytoplasmic triglyceride metabolism.

Triglyceride (TG) synthesis, storage, and degradation together constitute cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds sequentially, producing FAs and glycerol. Several reactions of CTGM can be catalyzed by more than one enzyme, creating great potential for complex tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic energy source during fasting and is related to obesity. Inborn errors and mouse models have demonstrated the importance of CTGM for non-adipose tissues, including skeletal muscle, myocardium and liver, because steatosis and dysfunction can occur. We discuss known inborn errors of CTGM, including deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1 (partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease (NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy, cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE, hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia) and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis). Mouse models often resemble human phenotypes but may diverge markedly. Inborn errors have been described for less than one-third of CTGM enzymes, and new phenotypes may yet be identified.

Functional characterization of Yersinia pestis aerobic glycerol metabolism.

Yersinia pestis biovar Orientalis isolates have lost the capacity to ferment glycerol. Herein we provide experimental validation that a 93 bp in-frame deletion within the glpD gene encoding the glycerol-3-phosphate dehydrogenase present in all biovar Orientalis strains is sufficient to disrupt aerobic glycerol fermentation. Furthermore, the inability to ferment glycerol is often insured by a variety of additional mutations within the glpFKX operon which prevents glycerol internalization and conversion to glycerol-3-phosphate. The physiological impact of functional glpFKX in the presence of dysfunctional glpD was assessed. Results demonstrate no change in growth kinetics at 26 °C and 37 °C. Mutants deficient in glpD displayed decreased intracellular accumulation of glycerol-3-phosphate, a characterized inhibitor of cAMP receptor protein (CRP) activation. Since CRP is rigorously involved in global regulation Y. pestis virulence, we tested a possible influence of a single glpD mutation on virulence. Nonetheless, subcutaneous and intranasal murine challenge was not impacted by glycerol metabolism. As quantified by crystal violet assay, biofilm formation of the glpD-deficient KIM6+ mutant was mildly repressed; whereas, chromosomal restoration of glpD in CO92 resulted in a significant increase in biofilm formation.

Enzyme architecture: the effect of replacement and deletion mutations of loop 6 on catalysis by triosephosphate isomerase.

Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170-173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186-3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12-13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-(13)C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-(13)C,2-(2)H]GA.

Glycerol-3-phosphate metabolism in wheat contributes to systemic acquired resistance against Puccinia striiformis f. sp. tritici.

Glycerol-3-phosphate (G3P) is a proposed regulator of plant defense signaling in basal resistance and systemic acquired resistance (SAR). The GLY1-encoded glycerol-3-phosphate dehydrogenase (G3PDH) and GLI1-encoded glycerol kinase (GK) are two key enzymes involved in the G3P biosynthesis in plants. However, their physiological importance in wheat defense against pathogens remains unclear. In this study, quantification analysis revealed that G3P levels were significantly induced in wheat leaves challenged by the avirulent Puccinia striiformis f. sp. tritici (Pst) race CYR23. The transcriptional levels of TaGLY1 and TaGLI1 were likewise significantly induced by avirulent Pst infection. Furthermore, knocking down TaGLY1 and TaGLI1 individually or simultaneously with barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) inhibited G3P accumulation and compromised the resistance in the wheat cultivar Suwon 11, whereas the accumulation of salicylic acid (SA) and the expression of the SA-induced marker gene TaPR1 in plant leaves were altered significantly after gene silencing. These results suggested that G3P contributes to wheat systemic acquired resistance (SAR) against stripe rust, and provided evidence that the G3P function as a signaling molecule is conserved in dicots and monocots. Meanwhile, the simultaneous co-silencing of multiple genes by the VIGS system proved to be a powerful tool for multi-gene functional analysis in plants.

Association between cytosolic glycerol 3-phosphate dehydrogenase gene expression in human subcutaneous adipose tissue and BMI.

BACKGROUND/AIMS: Cytosolic glycerol 3-phosphate dehydrogenase (cGPDH) is a key enzyme providing glycerol 3-phosphate for triacylglycerol synthesis in adipose tissue and is regarded as a marker for adipocyte differentiation. The aim of this study was to test the hypothesis that an increase in cGPDH gene expression in subcutaneous adipose tissue is associated with obesity. METHODS: mRNA levels in human subcutaneous adipose tissue were analysed by Real-Time PCR. RESULTS: We found that human subcutaneous adipose tissue cGPDH activity and cGPDH mRNA level were greater in obese patients than in lean subjects and were positively correlated with BMI and fat mass. Moreover, a strong positive correlation between subcutaneous adipose tissue cGPDH mRNA level and cGPDH activity was found. The data presented here indicates also that PPARγ mRNA level is positively correlated with body mass index and fat mass as well as with adipose tissue cGPDH mRNA level. Moreover, the association between subcutaneous adipose tissue cGPDH mRNA level and fatty acid translocase (FAT/CD36) mRNA level was also observed. CONCLUSION: The obtained results suggest that in comparison to lean subjects the increase in subcutaneous adipose tissue cGPDH gene expression in the obese, is probably the result of adipose tissue expansion during obesity.