Oxidative Pentose Phosphate Pathway Research Articles

6-Phosphogluconate dehydrogenase inhibition arrests growth and induces apoptosis in gastric cancer via AMPK activation and oxidative stress.

Poor outcomes in advanced gastric cancer necessitate alternative therapeutic strategies. 6-Phosphogluconate dehydrogenase (6-PGDH), an enzyme that catalyzes the decarboxylation step in the oxidative pentose phosphate pathway, has been identified as a promising therapeutic target in many cancers. In this study, we systematically investigated the expression and function of 6-PGDH in gastric cancer. We found that 6-PGDH expression and activity were aberrantly elevated in gastric cancer tissues compared to their adjacent normal tissues. 6-PGDH knockdown using two independent shRNAs resulted in minimal 6-PGDH levels and activity, decreased growth, and enhanced gastric cancer cell sensitivity to 5-flurorouracil. However, 6-PGDH knockdown did not affect the cancer cells. Mechanistic studies showed that 6-PGDH inhibition disrupted lipid biosynthesis and redox homeostasis in gastric cancer, inhibited growth, and induced apoptosis. Notably, the in vitro findings were validated using an in vivo gastric cancer xenograft mouse model. This study established that 6-PGDH is broadly elevated in gastric cancer patients and that 6-PGDH inhibition can sensitize gastric cancer cells in response to chemotherapy.

Newcastle Disease Virus Manipulates Mitochondrial MTHFD2-Mediated Nucleotide Metabolism for Virus Replication.

Viruses require host cell metabolic reprogramming to satisfy their replication demands; however, the mechanism by which the Newcastle disease virus (NDV) remodels nucleotide metabolism to support self-replication remains unknown. In this study, we demonstrate that NDV relies on the oxidative pentose phosphate pathway (oxPPP) and the folate-mediated one-carbon metabolic pathway to support replication. In concert with [1,2-13C2] glucose metabolic flow, NDV used oxPPP to promote pentose phosphate synthesis and to increase antioxidant NADPH production. Metabolic flux experiments using [2,3,3-2H] serine revealed that NDV increased one-carbon (1C) unit synthesis flux through the mitochondrial 1C pathway. Interestingly, methylenetetrahydrofolate dehydrogenase (MTHFD2) was upregulated as a compensatory mechanism for insufficient serine availability. Unexpectedly, direct knockdown of enzymes in the one-carbon metabolic pathway, except for cytosolic MTHFD1, significantly inhibited NDV replication. Specific complementation rescue experiments on small interfering RNA (siRNA)-mediated knockdown further revealed that only a knockdown of MTHFD2 strongly restrained NDV replication and was rescued by formate and extracellular nucleotides. These findings indicated that NDV replication relies on MTHFD2 to maintain nucleotide availability. Notably, nuclear MTHFD2 expression was increased during NDV infection and could represent a pathway by which NDV steals nucleotides from the nucleus. Collectively, these data reveal that NDV replication is regulated by the c-Myc-mediated 1C metabolic pathway and that the mechanism of nucleotide synthesis for viral replication is regulated by MTHFD2. IMPORTANCE Newcastle disease virus (NDV) is a dominant vector for vaccine and gene therapy that accommodates foreign genes well but can only infect mammalian cells that have undergone cancerous transformation. Understanding the remodeling of nucleotide metabolic pathways in host cells by NDV proliferation provides a new perspective for the precise use of NDV as a vector or in antiviral research. In this study, we demonstrated that NDV replication is strictly dependent on pathways involved in redox homeostasis in the nucleotide synthesis pathway, including the oxPPP and the mitochondrial one-carbon pathway. Further investigation revealed the potential involvement of NDV replication-dependent nucleotide availability in promoting MTHFD2 nuclear localization. Our findings highlight the differential dependence of NDV on enzymes for one-carbon metabolism, and the unique mechanism of action of MTHFD2 in viral replication, thereby providing a novel target for antiviral or oncolytic virus therapy.

Transcriptome dynamics provides insights into divergences of the photosynthesis pathway between Saccharum officinarum and Saccharum spontaneum.

Saccharum spontaneum and Saccharum officinarum contributed to the genetic background of modern sugarcane cultivars. Saccharum spontaneum has shown a higher net photosynthetic rate and lower soluble sugar than S. officinarum. Here, we analyzed 198 RNA-sequencing samples to investigate the molecular mechanisms for the divergences of photosynthesis and sugar accumulation between the two Saccharum species. We constructed gene co-expression networks based on differentially expressed genes (DEGs) both for leaf developmental gradients and diurnal rhythm. Our results suggested that the divergence of sugar accumulation may be attributed to the enrichment of major carbohydrate metabolism and the oxidative pentose phosphate pathway. Compared with S. officinarum, S. spontaneum DEGs showed a high enrichment ofphotosynthesis and contained more complex regulation of photosynthesis-related genes. Noticeably, S. spontaneum lacked gene interactions with sulfur assimilation stimulated by photorespiration. In S. spontaneum, core genes related to clock and photorespiration displayed a sensitive regulation by the diurnal rhythm and phase-shift. Small subunit of Rubisco (RBCS) displayed higher expression in the source tissues of S. spontaneum. Additionally, it was more sensitive under a diurnal rhythm, and had more complex gene networks than that in S. officinarum. This indicates that the differential regulation of RBCS Rubisco contributed to photosynthesis capacity divergence in both Saccharum species.

COMPARATIVE FLUX BALANCE ANALYSES OF SERINE ALKALINE PROTEASE OVERPRODUCTION IN Bacillus subtilis AT GENOME AND SMALL SCALE

This work aims to conduct flux balance analysis of serine alkaline protease overproduction in Bacillus subtilis using enzyme-constrained genome scale model and to compare the results with fluxes obtained from a smaller, bioreaction-based model. Fluxes of the enzyme constraint genome scale model were calculated using CobraToolbox v3.0 and compared with those of bioreaction-based model for the specific growth rate of zero. The active reaction number first increased and then remained constant with specific growth rate for enzyme constrained genome scale model. The SAP synthesis flux increased with a decrease in specific growth rate for both models. The TCA cycle was active for both models, but with lower fluxes for enzyme-constrained genome scale model. Anaplerotic reactions were active only for bioreaction-based model. Glycolysis pathway fluxes were active for enzyme-constrained genome scale model, meanwhile gluconeogenesis pathway fluxes were active for bioreaction-based model. Oxidative pentose phosphate pathway was inactive for both models and generally higher pentose phosphate pathway fluxes were obtained using bioreaction-based model. The fluxes toward amino acid synthesis pathways and serine alkaline protease synthesis were higher with bioreaction-based model. Since TCA cycle fluxes were lower with enzyme constrained genome scale model, ATP synthesis was lower with enzyme constrained genome scale model compared to bioreaction-based model. For both models, active pathways were the same for TCA cycle, pentose phosphate pathway, amino acid synthesis pathways except glycolysis pathway. The results showed that bioreaction-based model gave more sound results compared to enzyme constrained genome scale model since gluconeogenesis should be active with the carbon source of citrate.

Dark fermentative hydrogen production and transcriptional analysis of genes involved in the unicellular halotolerant cyanobacterium Aphanothece halophytica under nitrogen and potassium deprivation.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is known as a potential hydrogen (H2) producer. This study aimed to investigate the enhancement of H2 production under nutrient deprivation. The results showed that nitrogen and potassium deprivation induced dark fermentative H2 production by A. halophytica, while no differences in H2 production were found under sulfur and phosphorus deprivation. In addition, deprivation of nitrogen and potassium resulted in the highest H2 production in A. halophytica due to the stimulation of hydrogenase activity. The effect of adaptation time under nitrogen and potassium deprivation on H2 production was investigated. The results showed that the highest H2 accumulation of 1,261.96 ± 96.99µmol H2 g dry wt-1 and maximum hydrogenase activity of 179.39 ± 8.18µmol H2 g dry wt-1 min-1 were obtained from A. halophytica cells adapted in the nitrogen- and potassium-deprived BG11 medium supplemented with Turk Island salt solution (BG110-K) for 48h. An increase in hydrogenase activity was attributed to the decreased O2 concentration in the system, due to a reduction of photosynthetic O2 evolution rate and a promotion of dark respiration rate. Moreover, nitrogen and potassium deprivation stimulated glycogen accumulation and decreased specific activity of pyruvate kinase. Transcriptional analysis of genes involved in H2 metabolism using RNA-seq confirmed the above results. Several genes involved in glycogen biosynthesis (glgA, glgB, and glgP) were upregulated under both nitrogen and potassium deprivation, but genes regulating enzymes in the glycolytic pathway were downregulated, especially pyk encoding pyruvate kinase. Interestingly, genes involved in the oxidative pentose phosphate pathway (OPP) were upregulated. Thus, OPP became the favored pathway for glycogen catabolism and the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which resulted in an increase in H2 production under dark anaerobic condition in both nitrogen- and potassium-deprived cells.

Inhibition of 6-Phosphogluconate Dehydrogenase Reverses Epirubicin Resistance Through Metabolic Reprograming in Triple-Negative Breast Cancer Cells.

At present, chemotherapy is the most effective strategy for treating triple-negative breast cancer (TNBC), but its efficacy was limited by the development of chemo-resistance. The exact mechanism of chemoresistance still remains unclear. This study aims to examine whether 6-phosphogluconate dehydrogenase (6PGD), a key enzyme in the oxidative pentose phosphate pathway (PPP), could promote the resistance of TNBC cells to epirubicin. A TNBC epirubicin-resistant cell line was developed by increasing concentration and the effectiveness was tested. The expression and knockdown efficiency of 6PGD were further validated by performing quantitative real-time PCR (qPCR) and Western blot. The effects of 6PGD on parental and drug-resistant TNBC cell lines were verified based on proliferation and apoptosis experiments. Finally, nicotinamide adenine dinucleotide phosphate (NADPH) and lactate quantitative experiments were performed to examine the mechanism of 6PGD in promoting drug resistance. Epirubicin-resistant cancer cells exhibited a higher level of 6PGD in contrast to epirubicin-sensitive cells. In addition, 6PGD inhibited by genetic and pharmacological approaches significantly suppressed the growth and survival of both epirubicin-sensitive and epirubicin-resisteant TNBC cells. It should be noted that 6PGD inhibition sensitized epirubicin-resistant TNBC cells to epirubicin treatment. Moreover, it was also found that the levels of NADPH and lactate increased in epirubicin-resistant TNBC cells but decreased in response to 6PGD inhibition. The present results indicated that 6PGD inhibition disrupted metabolic reprogramming in epirubicin-resistant TNBC cells. Our work demonstrated that 6PGD inhibition reversed the resistance of TNBC cells to epirubicin, providing an alternative therapeutic choice to tackle the challenge of epirubicin resistance in TNBC treatment.

PRMT6 promotes tumorigenicity and cisplatin response of lung cancer through triggering 6PGD/ENO1 mediated cell metabolism

Metabolic reprogramming is a hallmark of cancer, including lung cancer. However, the exact underlying mechanism and therapeutic potential are largely unknown. Here we report that protein arginine methyltransferase 6 (PRMT6) is highly expressed in lung cancer and is required for cell metabolism, tumorigenicity, and cisplatin response of lung cancer. PRMT6 regulated the oxidative pentose phosphate pathway (PPP) flux and glycolysis pathway in human lung cancer by increasing the activity of 6-phospho-gluconate dehydrogenase (6PGD) and α-enolase (ENO1). Furthermore, PRMT6 methylated R324 of 6PGD to enhancing its activity; while methylation at R9 and R372 of ENO1 promotes formation of active ENO1 dimers and 2-phosphoglycerate (2-PG) binding to ENO1, respectively. Lastly, targeting PRMT6 blocked the oxidative PPP flux, glycolysis pathway, and tumor growth, as well as enhanced the anti-tumor effects of cisplatin in lung cancer. Together, this study demonstrates that PRMT6 acts as a post-translational modification (PTM) regulator of glucose metabolism, which leads to the pathogenesis of lung cancer. It was proven that the PRMT6-6PGD/ENO1 regulatory axis is an important determinant of carcinogenesis and may become a promising cancer therapeutic strategy.

Dark accumulation of downstream glycolytic intermediates initiates robust photosynthesis in cyanobacteria.

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies have shown that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid dynamic changes (on a time scale of seconds) in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enabled estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicated that downstream glycolytic intermediates, including phosphoglycerate and phosphoenolpyruvate, accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant decreases in the initial oxygen evolution rate were observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathways. Accordingly, the degree of decrease in the initial oxygen evolution rate was proportional to the accumulated pool size of glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

Glucose-6-Phosphate Dehydrogenases: The Hidden Players of Plant Physiology.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes a metabolic hub between glycolysis and the pentose phosphate pathway (PPP), which is the oxidation of glucose-6-phosphate (G6P) to 6-phosphogluconolactone concomitantly with the production of nicotinamide adenine dinucleotide phosphate (NADPH), a reducing power. It is considered to be the rate-limiting step that governs carbon flow through the oxidative pentose phosphate pathway (OPPP). The OPPP is the main supplier of reductant (NADPH) for several "reducing" biosynthetic reactions. Although it is involved in multiple physiological processes, current knowledge on its exact role and regulation is still piecemeal. The present review provides a concise and comprehensive picture of the diversity of plant G6PDHs and their role in seed germination, nitrogen assimilation, plant branching, and plant response to abiotic stress. This work will help define future research directions to improve our knowledge of G6PDHs in plant physiology and to integrate this hidden player in plant performance.

Two or more bacterial mixtures increase tea plant growth, yield, nutrient intake and antioxidant enzyme activity

This study was carried out in order to determine bacterial strains that can be used as biological fertilizer in tea cultivation. The present study assessed possible effects of mineral NPK fertilizer (TF: 80 kg ha−1 compound 25: 5:10) and ACC deaminase-containing, N2-fixing, and P-solubilizing 6 bacteria strains as single (T1-T6:Pseudomonas fluorescens RC483, Pseudomonas fluorescens RC77E, Pseudomonas putida RC19b, Bacillus licheniformis RC5B2, Bacillus subtilis RC5A2, Bacillus pumilus RC7A1), binary (T7: RC483 + RC5A2, T8: RC77E + RC7A1), triple (T9: RC77E + RC5A2+ RC5B2, T10: RC483+ RC19b + RC7A1) and quadruple (T11: RC77E + RC5B2 + RC7A1+ RC5A2) inoculations, chemical fertilizer (80 kg ha−1 compound 25: 5: 10) and control treatments on growth, yield, yield, nutrient uptake and enzyme activity of Pazar-20 tea clones manufactured for commercial purposes. Experimental groups determined in the present study were formed by considering the results of our preliminary study and bacteria that do not have antagonistic effects on each other. Results showed that dual, triple and quadruple inoculations stimulated overal second and third leaf area, leaf yield, macro- and micro-nutrient concenrations, chlorophyll and anthocyanin content, and antioxidant, oxidative, catalytic, hydrolytic and quality-related enzymes activity. Tea plant growth responses were dependent on the inoculant strain and formulations used, as well as on the parameters evaluated. In conclusion, bacterial seed inoculations especially with quadruple bacterial formulation determined to be most effective in tea growth can improve tea yield and productivity on sustainable basis. The ability to increase enzyme activity, which may be important in taste and aroma, leaf antioxidant, oxidative pentose phosphate pathway (OPPP) and tea processing technology by using different combinations prepared with bacterial isolates will be able to a significant contribution to tea technology studies.

Glucose-6-Phosphate Dehydrogenase, ZwfA, a Dual Cofactor-Specific Isozyme Is Predominantly Involved in the Glucose Metabolism of Pseudomonas bharatica CSV86T.

Glucose-6-phosphate dehydrogenase (Zwf) is an important enzyme in glucose metabolism via the Entner-Doudoroff pathway and the first enzyme in the oxidative pentose-phosphate pathway. It generates NAD(P)H during the conversion of glucose-6-phosphate (G6P) to 6-phosphogluconolactone, thus aiding in anabolic processes, energy yield, and oxidative stress responses. Pseudomonas bharatica CSV86T preferentially utilized aromatic compounds over glucose and exhibited a significantly lower growth rate on glucose (0.24 h-1) with a prolonged lag phase (~10 h). In strain CSV86T, glucose was metabolized via the intracellular phosphorylative route only because it lacked an oxidative (gluconate and 2-ketogluconate) route. The genome harbored three genes zwfA, zwfB, and zwfC encoding three Zwf isozymes. The present study aimed to understand gene arrangement, gene expression profiling, and molecular and kinetic properties of the purified enzymes to unveil their physiological significance in the strain CSV86T. The zwfA was found to be a part of the zwfA-pgl-eda operon, which was proximal to other glucose transport and metabolic clusters. The zwfB was found to be arranged as a gnd-zwfB operon, while zwfC was present independently. Among the three, zwfA was transcribed maximally, and the purified ZwfA displayed the highest catalytic efficiency, cooperativity with respect to G6P, and dual cofactor specificity. Isozymes ZwfB and ZwfC were NADP+-preferring and NADP+-specific, respectively. Among other functionally characterized Zwfs, ZwfA from strain CSV86T displayed poor catalytic efficiency and the further absence of oxidative routes of glucose metabolism reflected its lower growth rate on glucose compared to P. putida KT2440 and could be probable reasons for the unique carbon source utilization hierarchy. IMPORTANCE Pseudomonas bharatica CSV86T metabolizes glucose exclusively via the intracellular phosphorylative Entner-Doudoroff pathway leading the entire glucose flux through Zwf as the strain lacks oxidative routes. This may lead to limiting the concentration of downstream metabolic intermediates. The strain CSV86T possesses three isoforms of glucose-6-phosphate dehydrogenase, ZwfA, ZwfB, and ZwfC. The expression profile and kinetic properties of purified enzymes will help to understand glucose metabolism. Isozyme ZwfA dominated in terms of expression and displayed cooperativity with dual cofactor specificity. ZwfB preferred NADP+, and ZwfC was NADP+ specific, which may aid in redox cofactor balance. Such beneficial metabolic flexibility facilitated the regulation of metabolic pathways giving survival/fitness advantages in dynamic environments. Additionally, multiple genes allowed the distribution of function among these isoforms where the primary function was allocated to one of the isoforms.

Glycogen metabolism is required for optimal cyanobacterial growth in the rapid light-dark cycle of low-Earth orbit

Some designs for bioregenerative life support systems to enable human space missions incorporate cyanobacteria for removal of carbon dioxide, generation of oxygen, and treatment of wastewater, as well as providing a source of nutrition. In this study, we examined the effects of the short light-dark (LD) cycle of low-Earth orbit on algal and cyanobacterial growth, approximating conditions on the International Space Station, which orbits Earth roughly every 90 min. We found that growth of green algae was similar in both normal 12 h light:12 h dark (12 h:12 h LD) and 45′:45′ LD cycles. Three diverse strains of cyanobacteria were not only capable of growth in short 45′:45′ LD cycles, but actually grew better than in 12 h:12 h LD cycles. We showed that 45′:45′ LD cycles do not affect the endogenous 24 h circadian rhythms of Synechococcus elongatus. Using a dense library of randomly barcoded transposon mutants, we identified genes whose loss is detrimental for the growth of S. elongatus under 45′:45′ LD cycles. These include several genes involved in glycogen metabolism and the oxidative pentose phosphate pathway. Notably, 45′:45′ LD cycles did not affect the fitness of strains that carry mutations in the biological circadian oscillator or the clock input and output regulatory pathways. Overall, this study shows that cultures of cyanobacteria could be grown under natural sunlight of low-Earth orbit and highlights the utility of a functional genomic study in a model organism to better understand key biological processes in conditions that are relevant to space travel.

Reduction in chloroplastic ribulose-5-phosphate-3-epimerase decreases photosynthetic capacity in Arabidopsis

Chloroplast ribulose-5-phosphate-3-epimerase (RPE) is a critical enzyme involved in the Calvin-Benson cycle and oxidative pentose phosphate pathways in higher plants. Three Arabidopsis rpe mutants with reduced level of RPE were identified through their high NPQ (nonphotochemical quenching) phenotype upon illumination, and no significant difference of plant size was found between these rpe mutants and WT (wild type) plants under growth chamber conditions. A decrease in RPE expression to a certain extent leads to a decrease in CO2 fixation, V cmax and J max. Photosynthetic linear electron transport was partially inhibited and activity of ATP synthase was also decreased in the rpe mutants, but the levels of thylakoid protein complexes and other Calvin-Benson cycle enzymes in rpe mutants were not affected. These results demonstrate that some degree of reduction in RPE expression decreases carbon fixation in chloroplasts, which in turn feedback inhibits photosynthetic electron transport and ATP synthase activity due to the photosynthetic control. Taken together, this work provides evidence that RPE plays an important role in the Calvin-Benson cycle and influences the photosynthetic capacity of chloroplasts.

Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity

G6PD (glucose-6-phosphate dehydrogenase) is the rate-limiting enzyme in the oxidative pentose phosphate pathway that can generate cytosolic NADPH for biosynthesis and oxidative defense. Since cytosolic NADPH can be compensatively produced by other sources, the enzymatic activity deficiency alleles of G6PD are well tolerated in somatic cells but the effect of null mutations is unclear. Herein, we show that G6PD KO sensitizes cells to the stresses induced by hydrogen peroxide, superoxide, hypoxia, and the inhibition of the electron transport chain. This effect can be completely reversed by the expressions of natural mutants associated with G6PD deficiency, even without dehydrogenase activity, exactly like the WT G6PD. Furthermore, we demonstrate that G6PD can physically interact with AMPK (AMPK-activated protein kinase) to facilitate its activity and directly bind to NAMPT (nicotinamide phosphoribosyltransferase) to promote its activity and maintain the NAD(P)H/NAD(P)+ homeostasis. These functions are necessary to the antistress ability of cells but independent of the dehydrogenase activity of G6PD. In addition, the WT G6PD and naturally inactive mutant also can similarly regulate the metabolism of glucose, glutamine, fatty acid synthesis, and GSH and interact with the involved enzymes. Therefore, our findings reveal the previously unidentified functions of G6PD that can act as the important physiological neutralizer of stresses independently of its enzymatic activity.

Transcriptome analysis of malate-induced Schizochytrium sp. FJU-512 reveals a novel pathway for biosynthesis of docosahexaenoic acid with enhanced expression of genes responsible for acetyl-CoA and NADPH accumulation.

Schizochytrium is one of the few oleaginous microalgae that produce docosahexaenoic acid (DHA)-rich lipids. In this study, global changes in gene expression levels of Schizochytrium sp. FJU-512 cultured with malate in a 15 l-bioreactor was analyzed using comparative transcriptomics. The changes were found mainly in the genes involved in oxidative phosphorylation, β-oxidation, and pentose phosphate pathways. Consequently, the global changes in genes associated with the pathways could lead to an increase in the influx throughputs of pyruvate, branched-chain amino acids, fatty acids, and vitamin B6. Our transcriptome analysis indicated pyruvate dehydrogenase E2 component and acetolactate synthase I/II/III large subunit as major contributors to acetyl-CoA biosynthesis, whereas glucose-6-phosphate dehydrogenase was indicated as the major contributor to the biosynthesis of NADPH. An increase in DHA titer of up to 22% was achieved with the addition of malate to the fed-batch culture of Schizochytrium sp. FJU-512. This study provides an alternate method to enhance DHA production in Schizochytrium sp. FJU-512 through malate induced upregulation of genes responsible for acetyl-CoA and NADPH biosynthesis.

Erythritol synthesis is elevated in response to oxidative stress and regulated by the non-oxidative pentose phosphate pathway in A549 cells

BackgroundErythritol is a predictive biomarker of cardiometabolic diseases and is produced from glucose metabolism through the pentose phosphate pathway (PPP). Little is known regarding the regulation of endogenous erythritol synthesis in humans.ObjectiveIn the present study, we investigated the stimuli that promote erythritol synthesis in human lung carcinoma cells and characterized potential points of regulation along the PPP.MethodsHuman A549 lung carcinoma cells were chosen for their known ability to synthesize erythritol. A549 cells were treated with potential substrates for erythritol production, including glucose, fructose, and glycerol. Using siRNA knockdown, we assessed the necessity of enzymes G6PD, TKT, TALDO, and SORD for erythritol synthesis. We also used position-specific 13C-glucose tracers to determine whether the carbons for erythritol synthesis are derived directly from glycolysis or through the oxidative PPP. Finally, we assessed if erythritol synthesis responds to oxidative stress using chemical and genetic models.ResultsIntracellular erythritol was directly associated with media glucose concentration. In addition, siRNA knockdown of TKT or SORD inhibited erythritol synthesis, whereas siG6PD did not. Both chemically induced oxidative stress and constitutive activation of the antioxidant response transcription factor NRF2 elevated intracellular erythritol.ConclusionOur findings indicate that in A549 cells, erythritol synthesis is proportional to flux through the PPP and is regulated by non-oxidative PPP enzymes.

Ascorbate and Hydrogen Peroxide Modify Metabolite Profile of Wheat Differently

Effects of ascorbate (ASA) and hydrogen peroxide (H2O2) on metabolite profile was compared in wheat. Interestingly, the redox environment became more oxidized after ASA treatment and more reduced after H2O2 addition based on the ratios of oxidised and reduced ascorbate and glutathione. The excess of ASA could inhibit, while H2O2 could induce the oxidative pentose phosphate pathway producing reducing power as shown by the unchanged and decreased glucose-6-phosphate content, respectively. This different effect on glucose-6-phosphate content can also explain the reduced formation of several amino acids from the intermediate products of glycolysis after ASA treatment and their constant or greater levels after H2O2 addition. In contrast to most amino acids, the accumulation of Pro was greatly induced by ASA, and this change was fivefold greater than after H2O2 addition. This difference could also contribute to the distinct redox shifts after the two treatments, since NADPH is oxidised during Pro synthesis. The more oxidising environment after ASA treatment activated several transcripts related to the ascorbate–glutathione cycle and the pentose phosphate pathway. Our results indicate the overcompensating effect of ASA and H2O2 on the redox environment in leaf tissues and the subsequent different adjustment of metabolite profile and the related transcript levels.

Alternative splicing of Arabidopsis G6PD5 recruits NADPH-producing OPPP reactions to the endoplasmic reticulum.

Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of the oxidative pentose-phosphate pathway (OPPP). The OPPP mainly provides NADPH and sugar-phosphate building blocks for anabolic pathways and is present in all eukaryotes. In plant cells, the irreversible part of the OPPP is found in several compartments. Among the isoforms catalyzing the first OPPP step in Arabidopsis, G6PD1 to G6PD4 target plastids (with G6PD1 being also directed to peroxisomes), whereas G6PD5 and G6PD6 operate in the cytosol. We noticed that alternative splice forms G6PD5.4 and G6PD5.5 encode N-terminally extended proteoforms. Compared to G6PD5.1, RT-PCR signals differed and fluorescent reporter fusions expressed in Arabidopsis protoplasts accumulated in distinct intracellular sites. Co-expression with organelle-specific markers revealed that the G6PD5.4 and G6PD5.5 proteoforms label different subdomains of the endoplasmic reticulum (ER), and analysis of C-terminal roGFP fusions showed that their catalytic domains face the cytosol. In g6pd5-1 g6pd6-2 mutant protoplasts lacking cytosolic G6PDH activity, the ER-bound proteoforms were both active and thus able to form homomers. Among the Arabidopsis 6-phosphogluconolactonases (catalyzing the second OPPP step), we noticed that isoform PGL2 carries a C-terminal CaaX motif that may be prenylated for membrane attachment. Reporter-PGL2 fusions co-localized with G6PD5.4 in ER subdomains, which was abolished by Cys-to-Ser exchange in the 256CSIL motif. Among the Arabidopsis 6-phosphogluconate dehydrogenases (catalyzing the third OPPP step), S-acylated peptides were detected for all three isoforms in a recent palmitoylome, with dual cytosolic/peroxisomal PGD2 displaying three sites. Co-expression of GFP-PGD2 diminished crowding of OFP-G6PD5.4 at the ER, independent of PGL2's presence. Upon pull-down of GFP-G6PD5.4, not only unlabeled PGD2 and PGL2 were enriched, but also enzymes that depend on NADPH provision at the ER, indicative of physical interaction with the OPPP enzymes. When membrane-bound G6PD5.5 and 5.4 variants were co-expressed with KCR1 (ketoacyl-CoA reductase, involved in fatty acid elongation), ATR1 (NADPH:cytochrome-P450 oxidoreductase), or pulled C4H/CYP73A5 (cinnamate 4-hydroxylase) as indirectly (via ATR) NADPH-dependent cytochrome P450 enzyme, co-localization in ER subdomains was observed. Thus, alternative splicing of G6PD5 can direct the NADPH-producing OPPP reactions to the cytosolic face of the ER, where they may operate as membrane-bound metabolon to support several important biosynthetic pathways of plant cells.

Endurance Exercise-mediated Metabolic Reprogramming Reverses High-caloric Diet-induced Hepatic Steatosis

PURPOSE: Metabolic distress caused by excess caloric intake (e.g., high-fat and high-carbohydrate diet) contributes to non-alcoholic fatty liver disease (NAFLD), one of the most common diseases in the United States, affecting almost 25% of the U.S. population. While there is no cure for NAFLD, growing evidence has emerged that endurance exercise protects the liver against NAFLD by restoring liver function. However, mechanisms of exercise-induced hepatic protection remain an unresolved topic. The present study investigated if endurance exercise (EXE)-mediated alterations of lipid and carbohydrate metabolism (e.g., lipogenesis, lipolysis, mitochondria biosynthesis, and insulin signaling) and cell turnover (e.g., senescence and apoptosis) were associated with protection against NAFLD. METHODS: nine-week-old female C57BL/6 J mice were randomly divided into three groups: normal diet group (CON, n = 11); high-fat diet/high-fructose group (HFD/HF, n = 11); and HFD/HF + EXE group (HFD/HF + EXE, n = 11). The mice assigned to HFD/HF and HFD/HF + EXE groups were fed with HFD/HF for 12 weeks, after which the mice assigned to the EXE group began treadmill running exercise for 12 weeks (60 min a day, five days a week), with HFD/HF diet continued. RESULTS: Our study showed that EXE attenuated hepatic steatosis, reduced de novo lipogenesis (e.g., reduction in ACLY and DGAT1), and enhanced mitochondrial biogenesis and fatty-acid activation and transport proteins to the mitochondria (e.g., OXPHOS, ACSL1, and CPT-1A). Also, EXE improved hepatic glucose regulation (e.g., upregulation of glycogenic signaling cascades: p-IRβ, p-AKT, p-GSK3β, and GLUT2; and downregulation of gluconeogenic proteins: GAPDH, G6PC, PCK1) and prevented hepatic senescence (e.g., suppression of senescence-related proteins p53, p22, and p16), pro-inflammatory cytokines (TNF-α and IL-1β), and oxidative stress (NOX2 downregulation and GPX upregulation). Finally, EXE improved cell turnover via apoptosis (e.g., activation of CASPASE 3 and PARP1 cleavage). CONCLUSIONS: Our study suggests that EXE-mediated metabolic reprogramming (inhibiting lipogenesis, enhancing lipid oxidation, and preventing gluconeogenesis but promoting oxidative pentose phosphate pathway) and anti-senescence may be a crucial protective mechanism against NAFLD.

Cytosolic glucose-6-phosphate dehydrogenases play a pivotal role in Arabidopsis seed development

Embryo development is essential for seed yield and post-germination growth. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in oxidative pentose phosphate pathway (OPPP), is widely involved in plant development and stress tolerance by providing nicotinamide adenine dinucleotide phosphate (NADPH). In this study, the double mutant (g6pd5/6), overexpression line (G6PD5/6OE) and complementation line (g6pd5/6Comp) of cytosolic glucose-6-phosphate dehydrogenases (Cyt-G6PD) were used to investigate Cyt-G6PD roles in embryo development of Arabidopsis. The results showed that the germination rate of g6pd5/6 seeds was delayed in comparison with that of Col-0; moreover, 11.5% of g6pd5/6 seeds did not germinate. The dysfunction of Cyt-G6PD resulted in decreased fresh weight and primary root length of g6pd5/6 seedlings. The height and silique length of g6pd5/6 plants were also decreased. Moreover, the abortion rate of siliques and seeds of g6pd5/6 plants were increased compared with those of Col-0, G6PD5/6OE and g6pd5/6Comp lines. However, the dysfunction of Cyt-G6PD did not affect pollen activity; but in g6pd5/6, the embryo development was partially delayed or inhibited. The contents of fatty acids and storage proteins, two main storage materials in Arabidopsis seeds, were decreased in g6pd5/6 seeds. Exogenous application of fatty acids (C18:2; C18:3) alleviated the delayed germination of g6pd5/6 seeds. RT-qPCR results further demonstrated that the early embryo development genes were down-regulated in g6pd5/6. Taken together, Cyt-G6PD plays a pivotal role in plant seed development by regulating the transcriptions of early embryo development genes and the accumulation of storage materials (especially fatty acids).