Glycerol-3-phosphate Dehydrogenase Research Articles

Engineering Glucose-to-Glycerol Pathway in Klebsiella pneumoniae and Boosting 3-Hydroxypropionic Acid Production Through CRISPR Interference.

The recent decline of the international biodiesel industry has led to decreased production and therefore increased the price of glycerol, which is a major by-product of biodiesel but a substrate for production of 3-hydroxypropionic acid (3-HP), that is, glycerol as a feedstock has no advantage over glucose in price. Hence, we engineered glucose to the glycerol pathway and improved 3-HP production by CRISPR interference (CRISPRi). To begin with, we cloned the genes encoding glycerol 3-phosphate dehydrogenase (gpd1) and glycerol 3-phosphatase (gpp2) from Saccharomyces cerevisiae, which jointly catalyze glucose into glycerol. The genes gpd1 and gpp2 were co-expressed in K. pneumoniae with the dCas9 gene integrated in genome, and this recombinant strain produced 2 g/L glycerol in the shake flask. To minimize the glucose consumption by competing pathways including the EMP pathway, glycerol oxidation pathway, and by-products pathways, we developed an CRISPRi system in aforementioned recombinant K. pneumoniae strain to inhibit the expression of the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) and 2,3-butanediol production gene (budA), resulting in a bi-functional strain harboring both glucose-to-glycerol pathway and CRISPRi system. Reverse transcription and quantitative PCR (RT-qPCR) results showed that this engineered CRISPRi system transcriptionally inhibited gapA and budA by 82% and 24%, respectively. In shake flask cultivation, this bi-functional strain produced 2.8 g/L glycerol using glucose as the carbon source, which was 46.6% increase compared to the strain without the engineered CRISPRi system. Moreover, this bi-functional strain produced 0.78 g/L 3-HP using glucose as the sole carbon source. In fed-batch cultivation, this bi-functional strain produced 1.77 g/L 3-HP. This study provides insights for co-utilization of distinct carbon sources.

Mitochondrial GCN5L1 regulates cytosolic redox state and hepatic gluconeogenesis via glycerol phosphate shuttle GPD2

Hepatic gluconeogenesis is crucial for maintaining blood glucose during starvation, and a major contributor for hyperglycemia. Cellular redox state is related to mitochondrial biology and regulates conversion of specific metabolites to glucose. General control of amino acid synthesis 5 (GCN5) like-1 (GCN5L1) is a mitochondria-enriched protein which modulates glucose and amino acid metabolism. Here we show a new regulatory mode of GCN5L1 on gluconeogenesis using lactate and glycerol. We observed GCN5L1 deletion dramatically inhibited glucose production derived from glycerol and lactate, due to increased cytosolic redox state. The underlying mechanism is that GCN5L1 directly binds to the key component of mitochondrial shuttle glycerol phosphate dehydrogenase 2 (GPD2) and modulates its activity. These results have significant implications for understanding the physiological role and regulatory mechanism of mitochondrial shuttle in diabetes development and provide a novel therapeutic potential for diabetes.

A ferroptosis defense mechanism mediated by glycerol-3-phosphate dehydrogenase 2 in mitochondria

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis invivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

Characterizing the Interplay of Rubisco and Nitrogenase Enzymes in Anaerobic-Photoheterotrophically Grown Rhodopseudomonas palustris CGA009 through a Genome-Scale Metabolic and Expression Model.

ABSTRACTRhodopseudomonas palustris CGA009 is a Gram-negative purple nonsulfur bacterium that grows phototrophically by fixing carbon dioxide and nitrogen or chemotrophically by fixing or catabolizing a wide array of substrates, including lignin breakdown products for its carbon and fixing nitrogen for its nitrogen requirements. It can grow aerobically or anaerobically and can use light, inorganic, and organic compounds for energy production. Due to its ability to convert different carbon sources into useful products during anaerobic growth, this study reconstructed a metabolic and expression (ME) model of R. palustris to investigate its anaerobic-photoheterotrophic growth. Unlike metabolic (M) models, ME models include transcription and translation reactions along with macromolecules synthesis and couple these reactions with growth rate. This unique feature of the ME model led to nonlinear growth curve predictions, which matched closely with experimental growth rate data. At the theoretical maximum growth rate, the ME model suggested a diminishing rate of carbon fixation and predicted malate dehydrogenase and glycerol-3 phosphate dehydrogenase as alternate electron sinks. Moreover, the ME model also identified ferredoxin as a key regulator in distributing electrons between major redox balancing pathways. Because ME models include the turnover rate for each metabolic reaction, it was used to successfully capture experimentally observed temperature regulation of different nitrogenases. Overall, these unique features of the ME model demonstrated the influence of nitrogenases and rubiscos on R. palustris growth and predicted a key regulator in distributing electrons between major redox balancing pathways, thus establishing a platform for in silico investigation of R. palustris metabolism from a multiomics perspective.IMPORTANCE In this work, we reconstructed the first ME model for a purple nonsulfur bacterium (PNSB). Using the ME model, different aspects of R. palustris metabolism were examined. First, the ME model was used to analyze how reducing power entering the R. palustris cell through organic carbon sources gets partitioned into biomass, carbon dioxide fixation, and nitrogen fixation. Furthermore, the ME model predicted electron flux through ferredoxin as a major bottleneck in distributing electrons to nitrogenase enzymes. Next, the ME model characterized different nitrogenase enzymes and successfully recapitulated experimentally observed temperature regulations of those enzymes. Identifying the bottleneck responsible for transferring an electron to nitrogenase enzymes and recapitulating the temperature regulation of different nitrogenase enzymes can have profound implications in metabolic engineering, such as hydrogen production from R. palustris. Another interesting application of this ME model can be to take advantage of its redox balancing strategy to gain an understanding of the regulatory mechanism of biodegradable plastic production precursors, such as polyhydroxybutyrate (PHB).

The Drosophila melanogaster enzyme glycerol-3-phosphate dehydrogenase 1 is required for oogenesis, embryonic development, and amino acid homeostasis

As the fruit fly, Drosophila melanogaster, progresses from one life stage to the next, many of the enzymes that compose intermediary metabolism undergo substantial changes in both expression and activity. These predictable shifts in metabolic flux allow the fly meet stage-specific requirements for energy production and biosynthesis. In this regard, the enzyme glycerol-3-phosphate dehydrogenase 1 (GPDH1) has been the focus of biochemical genetics studies for several decades and, as a result, is one of the most well-characterized Drosophila enzymes. Among the findings of these earlier studies is that GPDH1 acts throughout the fly lifecycle to promote mitochondrial energy production and triglyceride accumulation while also serving a key role in maintaining redox balance. Here, we expand upon the known roles of GPDH1 during fly development by examining how depletion of both the maternal and zygotic pools of this enzyme influences development, metabolism, and viability. Our findings not only confirm previous observations that Gpdh1 mutants exhibit defects in larval development, lifespan, and fat storage but also reveal that GPDH1 serves essential roles in oogenesis and embryogenesis. Moreover, metabolomics analysis reveals that a Gpdh1 mutant stock maintained in a homozygous state exhibits larval metabolic defects that significantly differ from those observed in the F1 mutant generation. Overall, our findings highlight unappreciated roles for GPDH1 in early development and uncover previously undescribed metabolic adaptations that could allow flies to survive the loss of this key enzyme.

Glycerol-3-Phosphate Dehydrogenase: The K120 and K204 Side Chains Define an Oxyanion Hole at the Enzyme Active Site.

The cationic K120 and K204 side chains lie close to the C-2 carbonyl group of substrate dihydroxyacetone phosphate (DHAP) at the active site of glycerol-3-phosphate dehydrogenase (GPDH), and the K120 side chain is also positioned to form a hydrogen bond to the C-1 hydroxyl of DHAP. The kinetic parameters for unactivated and phosphite dianion-activated GPDH-catalyzed reduction of glycolaldehyde and acetaldehyde (AcA) show that the transition state for the former reaction is stabilized by ca 5 kcal/mole by interactions of the C-1 hydroxyl group with the protein catalyst. The K120A and K204A substitutions at wild-type GPDH result in similar decreases in kcat, but Km is only affected by the K120A substitution. These results are consistent with 3 kcal/mol stabilizing interactions between the K120 or K204 side chains and a negative charge at the C-2 oxygen at the transition state for hydride transfer from NADH to DHAP. This stabilization resembles that observed at oxyanion holes for other enzymes. There is no detectable rescue of the K204A variant by ethylammonium cation (EtNH3+), compared with the efficient rescue of the K120A variant. This is consistent with a difference in the accessibility of the variant enzyme active sites to exogenous EtNH3+. The K120A/K204A substitutions cause a (6 × 106)-fold increase in the promiscuity of wild-type hlGPDH for catalysis of the reduction of AcA compared to DHAP. This may reflect conservation of the active site for an ancestral alcohol dehydrogenase, whose relative activity for catalysis of reduction of AcA increases with substitutions that reduce the activity for reduction of the specific substrate DHAP.

Obese Adipose Tissue‐Derived Extracellular Vesicles increase Colorectal Cancer Cell proliferation and glycolysis

Colorectal cancer (CRC) is the second deadliest cancer worldwide and obesity is an established risk factor for both men and women. However, we still have limited knowledge regarding the molecular mechanisms that support obesity‐induced CRC. Obesity substantially modifies the adipose tissue (AT) and increases secretion of various factors including nanometer‐sized extracellular vesicles (EVs). EVs are lipid bilayer vesicles released from most types of cells and contain lipids, proteins, microRNAs and other cytoplasmic components of the parent cell. Considering their ability to travel to distant sites and deliver cargo to recipient cells, EVs could act as intercellular mediators that alter the tumorigenic potential of recipient cancer cells. Therefore, we hypothesized that obese AT secreted EVs carry cargo that enhances tumorigenicity of CRC cells. Here, we aimed to determine the functional and metabolic changes induced by obese human AT‐secreted EVs on human CRC cell lines. Visceral AT tissue from patients with obesity (BMI>35) undergoing bariatric surgery and non‐obese patients (BMI<30) undergoing anti‐reflux surgery was cultured ex vivo in EV‐free DMEM‐H/Ham’s F12 for 48 hrs. Conditioned media was collected and EVs were isolated by ultracentrifugation. EV size and concentration were measured through Nanoparticle Tracking Analysis (NTA). EV protein cargo was characterized by unbiased proteomics analysis using LC‐MS. Human CRC cell lines HCT‐116 and HT‐29 were treated with 10^7 EV/ml for 24 or 48 hrs and proliferation and mitochondrial/glycolytic ATP production were measured by CCK‐8 assay or Seahorse XF ATP Rate Assay, respectively. NTA reported our EV preps were on average 120 nm in size, suggesting we isolated exosomes and/or small microvesicles. Using KEGG Pathway Database and Panther Protein Classification Software, 25% of EV proteins secreted from human AT were associated with metabolism, with 13% of total metabolic proteins in EVs associating with glycolysis. Obese EVs exhibited increased expression of glycolytic enzymes pyruvate kinase, phosphoglycerate kinase 1, and G3P dehydrogenase. Obese EVs increased proliferation and glycolytic ATP production in CRC cell lines, both serving as indicators of cancer aggressiveness. Taken together, our results suggest that obese adipose EVs mediate the tumorigenic potential of CRC cells. Our study is significant since it aims to identify druggable pathways which support tumorigenesis in obese CRC patients.

Capsaicin directly promotes adipocyte browning in the chemical compound-induced brown adipocytes converted from human dermal fibroblasts

Human brown fat is a potential therapeutic target for preventing obesity and related metabolic diseases by dissipating energy as heat through uncoupling protein 1 (UCP1). We have previously reported a method to obtain chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts under serum-free conditions. However, pharmacological responses to bioactive molecules have been poorly characterised in ciBAs. This study showed that the treatment with Capsaicin, an agonist of transient receptor potential vanilloid 1, directly activated adipocyte browning such as UCP1 expression, mitochondrial biogenesis, energy consumption rates, and glycerol recycling in ciBAs. Furthermore, genome-wide transcriptome analysis indicated that Capsaicin activated a broad range of metabolic genes including glycerol kinase and glycerol 3-phosphate dehydrogenase 1, which could be associated with the activation of glycerol recycling and triglyceride synthesis. Capsaicin also activated UCP1 expression in immortalised human brown adipocytes but inhibited its expression in mesenchymal stem cell-derived adipocytes. Altogether, ciBAs successfully reflected the direct effects of Capsaicin on adipocyte browning. These findings suggested that ciBAs could serve as a promising cell model for screening of small molecules and dietary bioactive compounds targeting human brown adipocytes.

Assessing the anticancer effects of metformin

Background: Metformin is an antihyperglycemic biguanide that is used as the first-line treatment for type 2 diabetes mellitus (T2DM). Metformin use helps control T2DM which is one of the many risk factors for cancer and is proposed to have multiple antagonistic actions against cancer cells. Metformin induces adenosine monophosphate-activated protein kinase (AMPK)-driven cascades, involving two pathways, a direct and an indirect one. This leads to a decrease in the mechanistic activity of mammalian target of rapamycin (mTOR), folate level, c-MYC (a family of regulator genes and proto-oncogenes), NF-κB (nuclear factor kappa of B cell), and also increases tumor suppressor P53 phosphorylation. These cascades will also decrease cyclin D1, reactive oxygen species (ROS), and increase mTOR complex1 (mTORC1) concentrations, apoptosis, and autophagy. Metformin also exerts anti-inflammatory effects through reducing interleukins 6 (IL6) and 8 (IL8), inhibition of protein translation via LKB1 (liver kinase B1), increasing expression of GPD1 (glycerol-3-phosphate dehydrogenase 1) which suppresses cancer by inhibiting mitochondria, inducing cell apoptosis, and inhibiting leptin while increasing adiponectin production. We would like to assess the anticancer effect of metformin on various types of malignancies. Methods: The 266 cancer patients diagnosed in 2018–2019 in King Fahd Hospital of the University (KFHU) were divided into non-diabetic and diabetic cancer patients (with or without metformin). The data includes age, gender, body mass index (BMI), type of cancer with grades and stages, T2DM, with or without metformin, complications, and fate etc. of each case. Results: Out of 300, 266 were selected for the analysis. The results showed the statistical significance (p value 0.022) of metformin use on the cancer grade at diagnosis that favorably influences the morbidity/mortality. Conclusions: Metformin might have an anti-cancer effect on the grade of cancer at the time of diagnosis regardless of its dosage, and this might improve the prognosis of malignancy.

Mitochondrial Generated Redox Stress Differently Affects the Endoplasmic Reticulum of Circulating Lymphocytes and Monocytes in Treatment-Naïve Hodgkin’s Lymphoma

Background. The redox stress caused by Hodgkin’s lymphoma (HL) also involves the peripheral blood mononucleated cells (PBMCs) even before chemotherapy. Here, we tested whether lymphocytes and monocytes show a different response to the increased mitochondrial generation of reactive oxygen species (ROS). Methods. PBMCs, isolated from the blood of treatment-naïve HL patients and control subjects, underwent assessment of malondialdehyde content and enzymatic activity of both hexose- and glucose-6P dehydrogenase (H6PD and G6PD) as well as flow cytometric analysis of mitochondrial ROS content. These data were complemented by evaluating the uptake of the fluorescent glucose analogue 2-NBDG that is selectively stored within the endoplasmic reticulum (ER). Results. Malondialdehyde content was increased in the whole population of HL PBMCs. The oxidative damage matched an increased activity of G6PD, and even more of H6PD, that trigger the cytosolic and ER pentose phosphate pathways, respectively. At flow cytometry, the number of recovered viable cells was selectively decreased in HL lymphocytes that also showed a more pronounced increase in mitochondrial ROS generation and 2-NBDG uptake, with respect to monocytes. Conclusions. PBMCs of HL patients display a selective mitochondrial and ER redox stress most evident in lymphocytes already before the exposure to chemotherapy toxicity.

Cold tolerance strategies of the fall armyworm, Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae)

The fall armyworm (FAW), Spodoptera frugiperda, is native to the tropical and subtropical areas of the American continent and is one of the world's most destructive insect pests and invaded Africa and spread to most of Asia in two years. Glycerol is generally used as a cryoprotectant for overwintering insects in cold areas. In many studies, the increase in glycerol as a main rapid cold hardening (RCH) factor and enhancing the supercooling point was revealed at low temperatures. There are two genes, including glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), that were identified as being associated with the glycerol synthesis pathway. In this study, one GPDH and two GK sequences (GK1 and GK2) were extracted from FAW transcriptome analysis. RNA interference (RNAi) specific to GPDH or GK1 and GK2 exhibited a significant down-regulation at the mRNA level as well as a reduction in survival rate when the RNAi-treated of FAW larvae post a RCH treatment. Following a cold period, an increase in glycerol accumulation was detected utilizing high-pressure liquid chromatography and colorimetric analysis of glycerol quantity in RCH treated hemolymph of FAW larvae. This research suggests that GPDH and GK isozymes are linked to the production of a high quantity of glycerol as an RCH factor, and glycerol as main cryoprotectant plays an important role in survival throughout the cold period in this quarantine pest studied.

Genome-Wide Identification of Cotton (Gossypium spp.) Glycerol-3-Phosphate Dehydrogenase (GPDH) Family Members and the Role of GhGPDH5 in Response to Drought Stress.

Glycerol-3-phosphate dehydrogenase (GPDH) is a key enzyme in plant glycerol synthesis and metabolism, and plays an important role in plant resistance to abiotic stress. Here, we identified 6, 7, 14 and 14 GPDH genes derived from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively. Phylogenetic analysis assigned these genes into three classes, and most of the genes within the family were expanded by whole-genome duplication (WGD) and segmental duplications. Moreover, determination of the nonsynonymous substitution rate/synonymous substitution rate (Ka/Ks) ratio showed that the GPDH had an evolutionary preference for purifying selection. Transcriptome data revealed that GPDH genes were more active in the early stages of fiber development. Additionally, numerous stress-related cis-elements were identified in the potential promoter region. Then, a protein–protein-interaction (PPI) network of GPDH5 in G. hirsutum was constructed. In addition, we predicted 30 underlying miRNAs in G. hirsutum. Functional validation results indicated that silencing GhGPDH5 diminished drought tolerance in the upland cotton TM-1 line. In summary, this study provides a fundamental understanding of the GPDH gene family in cotton, GhGPDH5 exerts a positive effect during drought stress and is potentially involved in stomatal closure movements.

LncRNA HOTAIR/MiR-217/GPD2 Axis Medicates Hypoxia Injury of Myocardial Cells

This study examined impacts of lncRNA HOX Transcript Antisense RNA (HOTAIR), miR-217 and Glycerol-3-phosphate dehydrogenase 2 (GPD2) on progressions of cardiomyocytes after hypoxia damage. Hypoxia treatment induced low cell viability and increased apoptosis. RT-qPCR evaluated suppressed expressions of lncRNA HOTAIR. Overexpressed lncRNA HOTAIR accelerated AC16 cell viability but restrained cell apoptosis and proinflammatory protein expressions while the knockdown of HOTAIR caused opposite results. MiR-217 then was examined to be inhibited by HOTAIR overexpression, whose upregulation reduced AC16 cell viability but facilitated apoptosis and pro-inflammatory protein expressions. Luciferase reporter test then verified that GPD2 was bound and decreased by miR-217, which promoted AC16 cell viability but hampered cell apoptosis and pro-inflammatory protein expressions after overexpression. Moreover, PI3K/AKT signaling pathway was activated by overexpression of GPD2

Glycerol 3-phosphate dehydrogenase regulates heat shock response in Saccharomyces cerevisiae

The aim of this work was to identify elements of adaptive regulatory mechanism for basal level of yeast histone deacetylase Sir2. Heat shock response (HSR) was altered in the absence of the NAD-dependent glycerol 3-phosphate dehydrogenase (Gpd1). Increase in HSR was lower in ΔGpd1 cells than wild-type cells. An inverse correlation existed between Gpd1 and Sir2; Sir2-deleted cells showed higher expression of Gpd1 while deletion of Gpd1 led to higher expression of Sir2. In the absence of Gpd1, basal activity of Sir2 promoter was higher and was increased further upon heat shock, suggesting higher Sir2 levels. No interaction between Gpd1 and Sir2 was detected without or with heat shock using immunoprecipitation. The results show that Gpd1 regulates HSR in yeast cells and likely blocks its uncontrolled activation. As uncontrolled stress adversely affects the cellular adaptive response, Gpd1 may be a component of the cell's catalogue to ensure a balanced response to unmitigated thermal stress.

Glycerol biosynthesis plays an essential role in mediating cold tolerance the red imported fire ant, Solenopsis invicta.

The red imported fire ant (RIFA), Solenopsis invicta Buren is native to South America and known as a global problematic invasive species. At low temperatures, several investigations have demonstrated an increase in glycerol as a primary rapid cold hardening (RCH) component and an increase in the supercooling point. Two genes, glycerol-3-phosphate dehydrogenase (GPDH) and glycerol kinase (GK), have been identified as being involved in the glycerol production process. In this study, one GPDH and two GK sequences were extracted from RIFA transcriptome analysis (Si-GPDH, Si-GK1, and Si-GK2). All three genes were expressed in different body parts and different tissues of S. invicta that Si-GK2 showed a higher expression level than the others. According to gene expression levels by qRT-PCR analysis, the highest expression levels of three genes were observed in fat body tissues. After 1 hof exposure to low temperatures (5°C or lower), the mRNA levels of these genes significantly increased, according to expression analyses. RNA interference (RNAi) of Si-GPDH or Si-GK1 and Si-GK2 exhibited a significant downregulation at the mRNA level. The mortality rate of treated RIFA by double-stranded RNA (dsRNA) specific to GPDH and GK2 significantly increased at low temperatures. This study indicates that GPDH and GK2 as glycerol biosynthesis genes in RIFA have a high expression level to synthesize a high level of glycerol as an RCH factor and they play crucial roles in survival during the cold period.

LPL/AQP7/GPD2 promotes glycerol metabolism under hypoxia and prevents cardiac dysfunction during ischemia.

In the heart, fatty acid is a major energy substrate to fuel contraction under aerobic conditions. Ischemia downregulates fatty acid metabolism to adapt to the limited oxygen supply, making glucose the preferred substrate. However, the mechanism underlying the myocardial metabolic shift during ischemia remains unknown. Here, we show that lipoprotein lipase (LPL) expression in cardiomyocytes, a principal enzyme that converts triglycerides to free fatty acids and glycerol, increases during myocardial infarction (MI). Cardiomyocyte-specific LPL deficiency enhanced cardiac dysfunction and apoptosis following MI. Deficiency of aquaporin 7 (AQP7), a glycerol channel in cardiomyocytes, increased the myocardial infarct size and apoptosis in response to ischemia. Ischemic conditions activated glycerol-3-phosphate dehydrogenase 2 (GPD2), which converts glycerol-3-phosphate into dihydroxyacetone phosphate to facilitate adenosine triphosphate (ATP) synthesis from glycerol. Conversely, GPD2 deficiency exacerbated cardiac dysfunction after acute MI. Moreover, cardiomyocyte-specific LPL deficiency suppressed the effectiveness of peroxisome proliferator-activated receptor alpha (PPARα) agonist treatment for MI-induced cardiac dysfunction. These results suggest that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role in preventing myocardial ischemia-related damage.

Anoxia-reoxygenation modulates cadmium-induced liver mitochondrial reactive oxygen species emission during oxidation of glycerol 3-phosphate

Aquatic organisms are frequently exposed to multiple stressors including low dissolved oxygen (O2) and metals such as cadmium (Cd). Reduced O2 concentration and Cd exposure alter cellular function in part by impairing energy metabolism and dysregulating reactive oxygen species (ROS) homeostasis. However, little is known about the role of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in ROS homeostasis in fish and its response to environmental stress. In this study, mGPDH activity and the effects of anoxia-reoxygenation (A-RO) and Cd on ROS (as hydrogen peroxide, H2O2) emission in rainbow trout liver mitochondria during oxidation of glycerol 3-phosphate (G3P) were probed. Trout liver mitochondria exhibited low mGPDH activity that supported a low respiratory rate but substantial H2O2 emission rate. Cd evoked a low concentration stimulatory-high concentration inhibitory H2O2 emission pattern that was blunted by A-RO. At specific redox centers, Cd suppressed H2O2 emission from site IQ, but stimulated emission from sites IIIQo and GQ. In contrast, A-RO stimulated H2O2 emission from site IQ following 15 min exposure and augmented Cd-stimulated emission from site IIF after 30 min exposure but did not alter the rate of H2O2 emission from sites IIIQo and GQ. Additionally, Cd neither altered the activities of catalase, glutathione peroxidase, or thioredoxin reductase nor the concentrations of total glutathione, reduced glutathione, or oxidized glutathione. Overall, this study indicates that oxidation of G3P drives ROS production from mGPDH and complexes I, II and III, whereas Cd directly modulates redox sites but not antioxidant defense systems to alter mitochondrial H2O2 emission.

Glucose 6-P dehydrogenase delays the onset of frailty by protecting against muscle damage.

BackgroundFrailty is a major age‐associated syndrome leading to disability. Oxidative damage plays a significant role in the promotion of frailty. The cellular antioxidant system relies on reduced nicotinamide adenine dinucleotide phosphate (NADPH) that is highly dependent on glucose 6‐P dehydrogenase (G6PD). The G6PD‐overexpressing mouse (G6PD‐Tg) is protected against metabolic stresses. Our aim was to examine whether this protection delays frailty.MethodsOld wild‐type (WT) and G6PD‐Tg mice were evaluated longitudinally in terms of frailty. Indirect calorimetry, transcriptomic profile, and different skeletal muscle quality markers and muscle regenerative capacity were also investigated.ResultsThe percentage of frail mice was significantly lower in the G6PD‐Tg than in the WT genotype, especially in 26‐month‐old mice where 50% of the WT were frail vs. only 13% of the Tg ones (P < 0.001). Skeletal muscle transcriptomic analysis showed an up‐regulation of respiratory chain and oxidative phosphorylation (P = 0.009) as well as glutathione metabolism (P = 0.035) pathways in the G6PD‐Tg mice. Accordingly, the Tg animals exhibited an increase in reduced glutathione (34.5%, P < 0.01) and a decrease on its oxidized form (−69%, P < 0.05) and in lipid peroxidation (4‐HNE: −20.5%, P < 0.05). The G6PD‐Tg mice also showed reduced apoptosis (BAX/Bcl2: −25.5%, P < 0.05; and Bcl‐xL: −20.5%, P < 0.05), lower levels of the intramuscular adipocyte marker FABP4 (−54.7%, P < 0.05), and increased markers of mitochondrial content (COX IV: 89.7%, P < 0.05; Grp75: 37.8%, P < 0.05) and mitochondrial OXPHOS complexes (CII: 81.25%, P < 0.01; CIII: 52.5%, P < 0.01; and CV: 37.2%, P < 0.05). Energy expenditure (−4.29%, P < 0.001) and the respiratory exchange ratio were lower (−13.4%, P < 0.0001) while the locomotor activity was higher (43.4%, P < 0.0001) in the 20‐month‐old Tg, indicating a major energetic advantage in these mice. Short‐term exercise training in young C57BL76J mice induced a robust activation of G6PD in skeletal muscle (203.4%, P < 0.05), similar to that achieved in the G6PD‐Tg mice (142.3%, P < 0.01).ConclusionsGlucose 6‐P dehydrogenase deficiency can be an underestimated risk factor for several human pathologies and even frailty. By overexpressing G6PD, we provide the first molecular model of robustness. Because G6PD is regulated by pharmacological and physiological interventions like exercise, our results provide molecular bases for interventions that by increasing G6PD will delay the onset of frailty.

In silico and invivo analyses reveal key metabolic pathways enabling the fermentative utilization of glycerol in Escherichia coli.

SummaryMost microorganisms can metabolize glycerol when external electron acceptors are available (i.e. under respiratory conditions). However, few can do so under fermentative conditions owing to the unique redox constraints imposed by the high degree of reduction of glycerol. Here, we utilize in silico analysis combined with in vivo genetic and biochemical approaches to investigate the fermentative metabolism of glycerol in Escherichia coli. We found that E. coli can achieve redox balance at alkaline pH by reducing protons to H2, complementing the previously reported role of 1,2‐propanediol synthesis under acidic conditions. In this new redox balancing mode, H2 evolution is coupled to a respiratory glycerol dissimilation pathway composed of glycerol kinase (GK) and glycerol‐3‐phosphate (G3P) dehydrogenase (G3PDH). GK activates glycerol to G3P, which is further oxidized by G3PDH to generate reduced quinones that drive hydrogenase‐dependent H2 evolution. Despite the importance of the GK‐G3PDH route under alkaline conditions, we found that the NADH‐generating glycerol dissimilation pathway via glycerol dehydrogenase (GldA) and phosphoenolpyruvate (PEP)‐dependent dihydroxyacetone kinase (DHAK) was essential under both alkaline and acidic conditions. We assessed system‐wide metabolic impacts of the constraints imposed by the PEP dependency of the GldA‐DHAK route. This included the identification of enzymes and pathways that were not previously known to be involved in glycerol metabolisms such as PEP carboxykinase, PEP synthetase, multiple fructose‐1,6‐bisphosphatases and the fructose phosphate bypass.

Chrysanthemum morifolium Flower Extract Ameliorates Obesity-Induced Inflammation and Increases the Muscle Mitochondria Content and AMPK/SIRT1 Activities in Obese Rats.

Decreased energy expenditure and chronically positive energy balance contribute to the prevalence of obesity and associated metabolic dysfunctions, such as dyslipidemia, hepatic fat accumulation, inflammation, and muscle mitochondrial defects. We investigated the effects of Chrysanthemum morifolium Ramat flower extract (CE) on obesity-induced inflammation and muscle mitochondria changes. Sprague–Dawley rats were randomly divided into four groups and fed either a normal diet, 45% high-fat diet (HF), HF containing 0.2% CE, or 0.4% CE for 13 weeks. CE alleviated HF-increased adipose tissue mass and size, dyslipidemia, hepatic fat deposition, and systematic inflammation, and increased energy expenditure. CE significantly decreased gene expression involved in adipogenesis, pro-inflammation, and the M1 macrophage phenotype, as well as glycerol-3-phosphate dehydrogenase (GPDH) and nuclear factor-kappa B (NF-kB) activities in epididymal adipose tissue. Moreover, CE supplementation improved hepatic fat accumulation and modulated gene expression related to fat synthesis and oxidation with an increase in adenosine monophosphate-activated protein kinase (AMPK) activity in the liver. Furthermore, CE increased muscle mitochondrial size, mitochondrial DNA (mtDNA) content, and gene expression related to mitochondrial biogenesis and function, including sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and PGC-1α-target genes, along with AMPK-SIRT1 activities in the skeletal muscle. These results suggest that CE attenuates obesity-associated inflammation by modulating the muscle AMPK-SIRT1 pathway.