Glycerol 3-phosphate Levels Research Articles

Dose-dependent effect of Dendrobium officinale polysaccharide on anti-aging in Caenorhabditis elegans: A metabolomics analysis focused on lipid and nucleotide metabolism regulation

Dendrobium officinale polysaccharide (DOP) has garnered attention for its potential anti-aging effects, yet the dose-dependent impact on aging, especially from a metabolomics perspective, remains less explored. Here, we investigated the dose-dependent effect of DOP on anti-aging in Caenorhabditis elegans, examining underlying mechanisms through metabolomics analysis. Based on food clearance assays, doses of 62.5, 125.0, and 250.0 mg/L of DOP were selected for lifespan experiments in C. elegans. Results indicated that both 125 and 250 mg/L DOP significantly extended lifespan, enhanced stress resistance, and preserved locomotion behavior, with superior effects observed at 250 mg/L. Metabolomics analysis revealed a dose-effect relationship between DOP concentration and 14 metabolites, primarily associated with lipid and nucleotide metabolism. Notably, metabolites including docosapentaenoic acid (DPA), arachidonic acid, uric acid, uridine 5′-diphosphogalactose (UDP-galactose), UDP-N-acetylglucosamine (UDP-GlcNAc), citric acid, l-Saccharopine, L-2-Aminoadipic acid, 5′-S-Methyl-5′-thioadenosine, 1-Linoleoyl glycerol, cyclic ADP-ribose, glycerol 3-phosphate (G3P), and adenosine exhibited increased levels with higher DOP doses, while palmitic acid levels declined with escalating DOP dose in the range from 62.5 to 250.0 mg/L. Furthermore, Pearson correlation analysis revealed strong associations between mean lifespan and levels of DPA, palmitic acid, G3P, UDP-galactose and UDP-GlcNAc. These findings underscore a significant relationship between lifespan extension and metabolic products of lipid and nucleotide metabolism in DOP-treated C. elegans, shedding light on potential mechanisms underlying anti-aging effects of DOP.

Riboflavin salvage by Borrelia burgdorferi supports carbon metabolism and is essential for survival in the tick vector.

The Lyme disease agent, Borrelia burgdorferi, harbors a significantly reduced genome and relies on the scavenging of critical nutrients from its tick and mammalian hosts for survival. Riboflavin salvage has been shown to be important for B. burgdorferi infection of mice, yet the contributions of riboflavin to B. burgdorferi metabolism and survival in the tick remain unknown. Using a targeted mass spectrometry approach, we confirmed the importance of bb0318, the putative ATPase component of an ABC-type riboflavin transporter, for riboflavin salvage and the production of FMN and FAD. This analysis further revealed that Δbb0318 B. burgdorferi displayed increased levels of glycerol 3-phosphate compared to the wild-type. The glycerol 3-phosphate dehydrogenase activity of GlpD was found to be FAD-dependent and the transcription and translation of glpD were significantly decreased in Δbb0318 B. burgdorferi. Finally, gene bb0318 was found to be important for maximal spirochete burden in unfed larvae and essential for survival in feeding ticks. Together, these data demonstrate the importance of riboflavin salvage for B. burgdorferi carbon metabolism and survival in ticks.

Enhanced production of seed oil with improved fatty acid composition by overexpressing NAD+ -dependent glycerol-3-phosphate dehydrogenase in soybean.

There is growing interest in expanding the production of soybean oils (mainly triacylglycerol, or TAG) to meet rising feed demand and address global energy concerns. We report that a plastid-localized glycerol-3-phosphate dehydrogenase (GPDH), encoded by GmGPDHp1 gene, catalyzes the formation of glycerol-3-phosphate (G3P), an obligate substrate required for TAG biosynthesis. Overexpression of GmGPDHp1 increases soybean seed oil content with high levels of unsaturated fatty acids (FAs), especially oleic acid (C18:1), without detectably affecting growth or seed protein content or seed weight. Based on the lipidomic analyses, we found that the increase in G3P content led to an elevated diacylglycerol (DAG) pool, in which the Kennedy pathway-derived DAG was mostly increased, followed by PC-derived DAG, thereby promoting the synthesis of TAG containing relatively high proportion of C18:1. The increased G3P levels induced several transcriptional alterations of genes involved in the glycerolipid pathways. In particular, genes encoding the enzymes responsible for de novo glycerolipid synthesis were largely upregulated in the transgenic lines, in-line with the identified biochemical phenotype. These results reveal a key role for GmGPDHp1-mediated G3P metabolism in enhancing TAG synthesis and demonstrate a strategy to modify the FA compositions of soybean oils for improved nutrition and biofuel.

Abstract B107: Metabolic reprogramming enhances the efficacy of mTOR inhibition in colorectal cancer

Abstract Background: PI3K/mTOR pathway is mutated in 10-20% of colorectal cancer (CRC) specimens and has been associated with poor survival. Phosphatidic acid (PA) is a central lipid membrane metabolite and lipid second messenger which has been shown to target mTOR. It is thought that PA lipid signaling to mTOR in part promotes mTOR mediated cancer cell growth, proliferation and survival. Diacylglycerol kinases (DGKs) are one of several mechanisms of PA generation. In this study, we found diacylglycerol kinases to be synthetically lethal in mTOR inhibitor resistant CRC. We evaluated the anti-proliferative, pharmacodynamic and metabolic effects of dual inhibition with mTOR (TAK-228) and DGK (ritanserin and R59022) inhibitors. Methods: A synthetic lethal screen was performed with two TAK-228 resistant colorectal cancer cell lines (HCT116 and SW620). Subsequent experiments were performed with one TAK-228 sensitive (DLD1) and one resistant (HCT116) CRC cell lines. Efficacy of TAK-228/ritanserin and TAK-228/R59022 combination therapy was evaluated with CellTiter-Glo cell viability and clonogenic colony formation assays. Global metabolomics profiling of DLD1 and HCT116 cells upon treatment with TAK-228, R59022, and in combination was performed using ultra high pressure liquid chromatography coupled to mass spectrometry. Pharmacologic DGK inhibition was phenocopied using lentiviral shRNA knockdown of DGKα. Immunoblotting was performed to evaluate mechanism of action of TAK-228 combination therapy. Results: TAK-228 combined with ritanserin and R59022 demonstrated decreased cell viability and colony formation as compared to either single agent. Immunoblotting confirmed TAK-228 abrogates PI3K/mTOR pathway activity. DGK inhibition alone resulted in a compensatory activation of mTOR signaling. DGK inhibition disrupted the phosphatidic acid pathway in DLD1 and HCT116 as evidenced by a decrease in PA synthesis and elevation of glycerol 3-phosphate levels, respectively: altering energy metabolism. Specifically, in HCT116, glucose utilization, glutaminolysis, and Krebs cycle anaplerosis were elevated; while one carbon metabolism was decreased. Lentiviral shRNA transduction resulted in DGKα knockdown as evaluated by RT-PCR and immunoblot. Phenocopy combination therapy with TAK-228 and DGKα knockdown resulted in an increased sensitivity to mTOR inhibition compared to mock transduced control. Conclusions: Pharmacologic and shRNA knockdown inhibition of DGK in combination with mTOR inhibition resulted in decreased cancer cell viability and decreased colony formation. Pharmacologic inhibition of mTOR and DGK, alone or in combination, alter metabolic wiring in crucial pathways such as energy metabolism, nucleotide biosynthesis, and the generation of lipid precursors. Impaired phosphatidic acid production may sensitize cells to mTOR inhibition. These results suggest a therapeutic anti-cancer advantage of simultaneously targeting lipid signaling/metabolism via diacylglycerol kinases and mTOR. Citation Format: Peter J Klauck, Hayley J Hawkins, Madison Weber, Stacey M Bagby, Christopher H Lieu, Sarah J Hartman, Betelehem W Yacob, Kelly D Sullivan, Monica Brown, Julie A Reisz, Angelo D’Alessandro, Wells A Messersmith, S Gail Eckhardt, Todd M Pitts. Metabolic reprogramming enhances the efficacy of mTOR inhibition in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B107. doi:10.1158/1535-7163.TARG-19-B107

Identification of GPAT1 As a Novel Therapeutic Target for Acute Leukemia By Inhibiting Leukemia Specific Metabolism

With the development of mass spectrometer technology, recent studies revealed the critical roles of cancer-specific metabolism for tumor propagation in several types of cancers. In leukemia, many studies have been conducted to elucidate a leukemia-specific metabolism, and several effective treatments such as IDH1/2 inhibitors targeting acute myeloid leukemia (AML) with IDH1/2 mutation have been developed. To identify the new metabolic pathways on which acute leukemia cells depend, we purified water-soluble metabolites from CD34+ hematopoietic stem and progenitor cells (HSPCs) of healthy donors, AML and acute lymphoblastic leukemia (ALL) patients, and we comprehensively measured 116 metabolites using mass spectrometer analysis. From this experiment, we found that the cellular content of glycerol 3-phosphate (G3P) in CD34+ AML and ALL cells was lower than that of normal CD34+ HSPCs. G3P is an intermediate metabolite in the glycolysis metabolic pathway and is utilized as a substrate for phospholipids synthesis. The initial and rate-limiting step of phospholipids synthesis is the synthesis of lysophosphatidic acid (LPA) from G3P and acyl-CoA mediated by glycerol 3-phosphate acyltransferases (GPATs). Since CD34+ acute leukemia cells contained significantly lower level of G3P, we hypothesized that leukemia cells actively consumed G3P and synthesized LPA by GPATs. GPATs are classified into four isoforms based on intracellular localization and substrate preference. GPAT1 and GPAT2 are mitochondrial GPATs that are localized to the mitochondrial outer membrane, but on the other hand, GPAT3 and GPAT4 are microsomal GPATs that are localized to the endoplasmic reticulum membrane, each encoded by independent genes. GPAT1 is identified as an essential gene for the growth of leukemia cells by RNAi screen analysis in the public database (DepMap). We found that CD34+ immature AML cells exhibited higher GPAT1 expression as compared to CD34- more differentiated AML cells and normal T cells. GPAT1 knockdown inhibited the proliferation of several acute leukemia cell lines including THP-1 and Kasumi-1 in vitro and in vivo. Moreover, a mitochondrial GPATs specific inhibitor (FSG67), which was originally developed as a drug to treat obesity and diabetes, suppressed the growth of the leukemia cell lines through the induction of G1 cell cycle arrest. Growth inhibition was rescued by exogenous administration of LPA, suggesting that the synthetic activity mediated by mitochondrial GPATs should be required for acute leukemia growth. Furthermore, FSG67 induced the apoptosis of leukemia cells derived from AML and ALL patients without affecting normal CD34+ HSPCs at least in vitro. We also confirmed that the injection of FSG67 resulted in the suppression of AML and ALL propagation in vivo using patient-derived xenograft models (see figure). GPAT1 regulates the mitochondrial function by producing LPA which is an essential metabolite for maintaining mitochondrial fusion. Actually, the amount of LPA was decreased in GPAT1 knockdown acute leukemia cells. We next examined mitochondrial energy production by extracellular flux assay, and found that GPAT1 knockdown as well as FSG67 significantly suppressed oxygen consumption rate of acute leukemia cells. Consistent with the impaired mitochondrial function, FSG67 suppressed the mitochondrial membrane potential, indicating that GPAT1 should play a pivotal role in maintaining leukemia-specific mitochondrial function. These results collectively suggest that the synthesis of LPA from G3P catalyzed by GPAT1 has a critical role in propagation of acute leukemia cells irrespective of their lineage origin. Thus, GPAT1 is a novel and common therapeutic target for human acute leukemia through suppressing leukemia-specific mitochondrial function. Figure Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Abstract 2429: HIF-1α repressed glycerol-3-phosphate shuttle to reduce oxidative stress in liver cancer

Abstract Liver cancer is the fifth most prevalent and third most common cancer globally. Hepatocellular carcinoma (HCC), arises from hepatocytes, accounts for more than 80% of liver cancer. HCC cells undergo extensive metabolic rewiring to support their uncontrolled growth. HCC cells encounter extremely high level of oxidative stress which is mostly generated in the electron transport chain. Reactive oxygen species (ROS) rapidly accumulate in the electron transport chain (ETC) during hypoxia due to inadequate electron transfer to O2 in the ETC. The glycerol phosphate (GP) shuttle plays an important role to transfer electron source (NADH) from cytoplasm to mitochondria to initiate the ETC. The GP shuttle is mediated by glycerol 3 phosphate dehydrogenase (GPD) which converts dihydroxyacetone phosphate to glycerol 3 phosphate (G3P) through the oxidation of NADH to NAD+. This contributes to ATP production in the mitochondria. Here, we reported that hypoxia-inducible factor 1 (HIF-1) acts as a transcriptional repressor instead of an activator to suppress transcription of an important ETC component of the glycerol phosphate shuttle (GPD) to reduce activity of the GP shuttle, thereby reducing oxidative stress. GPD expression was significantly reduced under hypoxic condition. Surprisingly, knockout, knockdown, or inhibition of HIF-1 restored GPD expression in hypoxia. GPD expression was significantly under-expressed in tumorous as compared to non-tumorous liver tissues in 91 HCC patients of our center by 2.67 fold. TCGA database containing 49 HCC patients echoed with our in-house data. Downregulation of GPD was associated with poor cellular differentiation and overall survival in HCC patients. Stable overexpression of GPD1 in multiple HCC cell lines increased the levels of G3P as well as NAD+, which is crucial to lactate dehydrogenase (LDHA) activity. GPD overexpression altered mitochondrial activity, ROS levels, and suppressed HCC cell proliferation. Taken together, this study showed that the HIF-1 negatively controls key component of the GP shuttle to maximize HCC growth. Citation Format: Shuo ZHANG, Robin Kit-Ho LAI, David Kung-Chun CHIU, Iris Mingjing XU, Aki Pui Wah TSE, Chun-Ming WONG, Irene Oi Lin NG, Carmen Chak Lui WONG. HIF-1α repressed glycerol-3-phosphate shuttle to reduce oxidative stress in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2429.

Application of Glycerol for Induced Powdery Mildew Resistance in Triticum aestivum L.

Previous work has demonstrated that glycerol-3-phosphate (G3P) and oleic acid (18:1) are two important signal molecules associated with plant resistance to fungi. In this article, we provide evidence that a 3% glycerol spray application 1–2 days before powdery mildew infection and subsequent applications once every 4 days was sufficient to stimulate the plant defense responses without causing any significant damage to wheat leaves. We found that G3P and oleic acid levels were markedly induced by powdery mildew infection. In addition, TaGLI1 (encoding a glycerol kinase) and TaSSI2 (encoding a stearoylacyl carrier protein fatty acid desaturase), two genes associated with the glycerol and fatty acid (FA) pathways, respectively, were induced by powdery mildew infection, and their promoter regions contain some fungal response elements. Moreover, exogenous application of glycerol increased the G3P level and decreased the level of oleic acid (18:1). Glycerol application induced the expression of pathogenesis-related (PR) genes (TaPR-1, TaPR-2, TaPR-3, TaPR-4, and TaPR-5), induced the generation of reactive oxygen species (ROS) before powdery mildew infection, and induced salicylic acid (SA) accumulation in wheat leaves. Further, we sprayed glycerol in a wheat field and found that it significantly (p < 0.05) reduced the severity of powdery mildew disease and lessened disease-associated kernel weight loss, all without causing any noticeable degradation in wheat seed quality.

Inhibition of the malate-aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion.

Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from α-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from αTC1-6 (αTC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and αTC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in αTC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from αTC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in αTC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not αTC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary β- than in α-cells. Thus, suppressed glycerol phosphate shuttle activity in the α-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate- and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.

Glycerol-3-phosphate metabolism plays a role in stress response in the red alga Pyropia haitanensis.

Glycerol-3-phosphate (G3P) has been suggested as a novel regulator of plant defense signaling, however, its role in algal resistance remains largely unknown. The glycerol kinase (also designated as NHO1) and NAD-dependent G3P dehydrogenase (GPDH) are two key enzymes involved in the G3P biosynthesis. In our study, we cloned the full-length cDNA of NHO1 (NHO1Ph ) and GPDH (GPDHP h ) from the red alga Pyropia haitanensis (denoted as NHO1Ph and GPDHP h ) and examined their expression level under flagellin peptide 22 (flg22) stimulation or heat stress. We also measured the level of G3P and floridoside (a downstream product of G3P in P.haitanensis) under flg22 stimulation or heat stress. Both NHO1Ph and GPDHP h shared high sequence identity and structural conservation with their orthologs from different species, especially from red algae. Phylogenetic analysis showed that NHO1s and GPDHs from red algae were closely related to those from animals. Under flg22 stimulation or heat stress, the expression levels of NHO1Ph and GPDHP h were up-regulated, G3P levels increased, and the contents of floridoside decreased. But the floridoside level increased in the recovery period after heat stress. Taken together, we found that G3P metabolism was associated with the flg22-induced defense response and heat stress response in P.haitanensis, indicating the general conservation of defense response in angiosperms and algae. Furthermore, floridoside might also participate in the stress resistance of P.haitanensis.

Application of glycerol as a foliar spray activates the defence response and enhances disease resistance of Theobroma cacao.

Previous work has implicated glycerol-3-phosphate (G3P) as a mobile inducer of systemic immunity in plants. We tested the hypothesis that the exogenous application of glycerol as a foliar spray might enhance the disease resistance of Theobroma cacao through the modulation of endogenous G3P levels. We found that exogenous application of glycerol to cacao leaves over a period of 4 days increased the endogenous level of G3P and decreased the level of oleic acid (18:1). Reactive oxygen species (ROS) were produced (a marker of defence activation) and the expression of many pathogenesis-related genes was induced. Notably, the effects of glycerol application on G3P and 18:1 fatty acid content, and gene expression levels, in cacao leaves were dosage dependent. A 100 mm glycerol spray application was sufficient to stimulate the defence response without causing any observable damage, and resulted in a significantly decreased lesion formation by the cacao pathogen Phytophthora capsici; however, a 500 mm glycerol treatment led to chlorosis and cell death. The effects of glycerol treatment on the level of 18:1 and ROS were constrained to the locally treated leaves without affecting distal tissues. The mechanism of the glycerol-mediated defence response in cacao and its potential use as part of a sustainable farming system are discussed.

Glycerol Affects Root Development through Regulation of Multiple Pathways in Arabidopsis

Glycerol metabolism has been well studied biochemically. However, the means by which glycerol functions in plant development is not well understood. This study aimed to investigate the mechanism underlying the effects of glycerol on root development in Arabidopsis thaliana. Exogenous glycerol inhibited primary root growth and altered lateral root development in wild-type plants. These phenotypes appeared concurrently with increased endogenous glycerol-3-phosphate (G3P) and H2O2 contents in seedlings, and decreased phosphate levels in roots. Upon glycerol treatment, G3P level and root development did not change in glycerol kinase mutant gli1, but G3P level increased in gpdhc1 and fad-gpdh mutants, which resulted in more severely impaired root development. Overexpression of the FAD-GPDH gene attenuated the alterations in G3P, phosphate and H2O2 levels, leading to increased tolerance to exogenous glycerol, which suggested that FAD-GPDH plays an important role in modulating this response. Free indole-3-acetic acid (IAA) content increased by 46%, and DR5pro::GUS staining increased in the stele cells of the root meristem under glycerol treatment, suggesting that glycerol likely alters normal auxin distribution. Decreases in PIN1 and PIN7 expression, β-glucuronidase (GUS) staining in plants expressing PIN7pro::GUS and green fluorescent protein (GFP) fluorescence in plants expressing PIN7pro::PIN7-GFP were observed, indicating that polar auxin transport in the root was downregulated under glycerol treatment. Analyses with auxin-related mutants showed that TIR1 and ARF7 were involved in regulating root growth under glycerol treatment. Glycerol-treated plants showed significant reductions in root meristem size and cell number as revealed by CYCB1;1pro::GUS staining. Furthermore, the expression of CDKA and CYCB1 decreased significantly in treated plants compared with control plants, implying possible alterations in cell cycle progression. Our data demonstrated that glycerol treatment altered endogenous levels of G3P, phosphate and ROS, affected auxin distribution and cell division in the root meristem, and eventually resulted in modifications of root development.

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.

A Feedback Regulatory Loop between G3P and Lipid Transfer Proteins DIR1 and AZI1 Mediates Azelaic-Acid-Induced Systemic Immunity

Systemic acquired resistance (SAR), a highly desirable form of plant defense, provides broad-spectrum immunity against diverse pathogens. The recent identification of seemingly unrelated chemical inducers of SAR warrants an investigation of their mutual interrelationships. We show that SAR induced by the dicarboxylic acid azelaic acid (AA) requires the phosphorylated sugar derivative glycerol-3-phosphate (G3P). Pathogen inoculation induced the release of free unsaturated fatty acids (FAs) and thereby triggered AA accumulation, because these FAs serve as precursors for AA. AA accumulation in turn increased the levels of G3P, which is required for AA-conferred SAR. The lipid transfer proteins DIR1 and AZI1, both of which are required for G3P- and AA-induced SAR, were essential for G3P accumulation. Conversely, reduced G3P resulted in decreased AZI1 and DIR1 transcription. Our results demonstrate that an intricate feedback regulatory loop among G3P, DIR1, and AZI1 regulates SAR and that AA functions upstream of G3P in this pathway.

Glycerol-3-phosphate and systemic immunity

Glycerol-3-phosphate (G3P), a conserved three-carbon sugar, is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis. G3P can be derived via the glycerol kinase-mediated phosphorylation of glycerol or G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate. Previously, we showed G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in GLY1 encoded G3Pdh accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Recently, we showed that G3P is also a potent inducer of systemic acquired resistance (SAR) in plants. SAR is initiated after a localized infection and confers whole-plant immunity to secondary infections. SAR involves generation of a signal at the site of primary infection, which travels throughout the plants and alerts the un-infected distal portions of the plant against secondary infections. Plants unable to synthesize G3P are defective in SAR and exogenous G3P complements this defect. Exogenous G3P also induces SAR in the absence of a primary pathogen. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues. Together, these observations suggest that the cooperative interaction of DIR1 and G3P mediates the induction of SAR in plants.

Involvement of the G3P shuttle and β-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica can accumulate up to 38% of its dry weight (DW) as lipids. Factors involved in lipid accumulation, particularly triglycerides, are not well identified. Using different mutations in the glycerol-3-phosphate (G3P) shuttle pathway ( Δgut2 affecting the anabolic dehydrogenase or overexpressing GPD1 affecting the catabolic dehydrogenase), we were able to modulate G3P concentration. We show that in a Po1d genetic background, GPD1 overexpression, GUT2 inactivation or both mutations together result in 1.5, 2.9, and 5.6-fold respective increases in the level of G3P leading to an increase of triacylglyceride (TAG) accumulation. Moreover, our results indicate that each strain with an increased concentration of G3P, also presented a decreased concentration of glycerol. Analysis of the different genes involved in glycerol metabolism indicated that Y. lipolytica does not possess a gene for glycerol-3-phosphatase. These findings suggest that Y. lipolytica has a modified and unique metabolism of glycerol that is dedicated to G3P synthesis (and also to TAG synthesis) which may contribute to its oleaginous character. Furthermore, coupling the G3P shuttle disorders to a deficient β-oxidation pathway (by inactiving POX1-6 or MFE1 genes) increased TAG and free fatty acids content. Finally, we obtained strains that accumulated up to 65–75% of their DW as lipid. Transcriptional analysis in these strains, revealed that the high levels of lipids resulted from over-expression of genes involved in TAG synthesis ( SCT1, encoding a sn-1 acyltransferase; and DGA1, encoding an acylCoA diacylglycerol acyltransferase) and the repression of genes involved in the degradation of TAG ( TGL3 and TGL4, encoding triacylglycerol lipases). These findings indicate that TAG synthesis is limited by the availability of G3P and fatty acids, and that the expression of genes involved in TAG homeostasis is regulated by the G3P shuttle and the β-oxidation pathway. Finally, the synergistic contribution of acyltransferase gene expression to G3P synthesis is required for high levels of TAG synthesis and lipid accumulation in Y. lipolytica.

Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants

Glycerol-3-phosphate (G3P) is an important metabolite that contributes to the growth and disease-related physiologies of prokaryotes, plants, animals and humans alike. Here we show that G3P serves as the inducer of an important form of broad-spectrum immunity in plants, termed systemic acquired resistance (SAR). SAR is induced upon primary infection and protects distal tissues from secondary infections. Genetic mutants defective in G3P biosynthesis cannot induce SAR but can be rescued when G3P is supplied exogenously. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues, and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues, which occurs through the symplast. These observations, along with the fact that dir1 plants accumulate reduced levels of G3P in their petiole exudates, suggest that the cooperative interaction of DIR1 and G3P orchestrates the induction of SAR in plants.

GPD1L Mutations and SCN5A Phosphorylation in Brugada Syndrome

The gene GPD1L encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1). Prior to the discovery of mutations in GPD1L in patients with Brugada Syndrome and Sudden Infant Death Syndrome (SIDS), the importance and function of this enzyme was unknown. GPD1 catalyzes the reversible conversion of glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP). We hypothesized that like its namesake GPD1, GPD1L catalyzed the reaction of G3P to DHAP. A loss-of-function of GPD1L’s enzymatic activity would be expected to increase levels of G3P, which ultimately serves as a substrate for PKC-mediated phosphorylation of SCN5A, a known down-regulator of cardiac sodium channel (SCN5A) function. We found that mutations in GPD1L caused a loss of enzymatic function, increased phosphorylation of SCN5A, and decreased peak sodium current consistent with the molecular phenotype of Brugada Syndrome. The G3P to DHAP conversion also causes reduction of NAD+ to NADH, and loss of GPD1L function may also increase reactive oxygen species producing additional effects on SCN5A and other ion channels. The “natural experiment” of GPD1L mutations found in patients has given insight into basic regulatory mechanisms of cardiac excitability.

Isolation of Novel Animal Cell Lines Defective in Glycerolipid Biosynthesis Reveals Mutations in Glucose-6-phosphate Isomerase

Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 --> Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.

The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling

Conversion of glycerol to glycerol-3-phosphate (G3P) is one of the highly conserved steps of glycerol metabolism in evolutionary diverse organisms. In plants, G3P is produced either via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or via G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP). We have recently shown that G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Since a mutation in the GLY1-encoded G3Pdh conferred more susceptibility compared to a mutation in the GLI1-encoded GK, we proposed that GLY1 is the major contributor of the total G3P pool that participates in defense against C. higginsianum.

Evidence for Impaired Gluconeogenesis in Very Long-chain Acyl-CoA Dehydrogenase-deficient Mice

Hypoketotic hypoglycaemia is a characteristic feature of fatty acid oxidation (FAO) defects. Although the underlying pathogenic mechanism is unknown, one hypothesis points to an impairment in gluconeogenesis. To study hepatic glucose production in FAO defects, we used the knockout mouse model of very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency presenting with stress-induced hypoglycaemia. We analysed metabolites of hepatic glucose production under non-stressed conditions and after stress in comparison to wildtype controls. Analysis included glycogen, glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), glycerol-3-phosphate (G3P) and dihydroxyacetone-phosphate (DHAP). We also measured the activity of the key enzyme glucose-6-phosphatase. Blood and liver glucose were found to be low after stress, and liver glycogen was depleted. In addition, hepatic G6P and F6P were significantly reduced, especially during hypoglycaemia. Importantly, the activity of the enzyme converting G6P into glucose was not impaired. These data indicate a reduced rate of gluconeogenesis. The levels of DHAP and G3P were significantly lower suggesting decreased availability of glucose precursors from glycerol. This study gives biochemical evidence of impaired gluconeogenesis as one of the causes for hypoglycaemia observed in VLCAD deficiency. Whether this is due to lack of a substrate, inhibitory effects on other gluconeogenic enzymes or impaired transcription of gluconeogenic enzymes needs to be resolved in the future.