Pyruvate Concentrations Research Articles

Development of novel tetrazole-based pyruvate kinase M2 inhibitors targeting U87MG glioblastoma cells.

Glioblastoma (GB), the most aggressive and life-threatening primary brain tumor in adults, poses significant therapeutic challenges. Tumor pyruvate kinase M2 (PKM2) has been implicated in the proliferation and survival of glioma cells. In this study, we designed and synthesized a series of 23 novel tetrazole-based derivatives. The compounds were thoroughly characterized using 1H, 13C, 19F NMR, along with HRMS analysis. Among them, 1-(imidazo[1,2-a]pyrimidin-3-yl)-2-(5-(naphthalen-2-yl)-2H-tetrazol-2-yl)ethan-1-one (9b) exhibited potent and selective antiproliferative activity against U87MG glioma cells, with minimal effects on bEnd (brain endothelial cell line) non-glioma cells. It emerged as a potent PKM2 inhibitor, with an IC50 of 0.307µM. Apoptosis assays and cell cycle analysis revealed that compound 9b induced early apoptosis and caused G1 phase arrest. A significant decrease in pyruvate concentration further suggested PKM2 inhibition. In silico studies confirmed the binding affinity to the PKM2 inhibitory site, and RT-PCR data demonstrated its inhibitory activity against PKM2. Additionally, it reduced lactate levels, indicating its potential impact on cellular metabolism. Collectively, these findings suggest that the most potent compound holds promise as a therapeutic candidate against GB.

Shaoyao decoction alleviates DSS-induced colitis by inhibiting IL-17a-mediated polarization of M1 macrophages

ETHNOPHARMACOLOGICAL RELEVANCEThe efficacy of Shaoyao decoction (SYD), a traditional Chinese medicine prescription, in alleviating colonic mucosal inflammation in ulcerative colitis (UC) has been established. However, the specific mechanism underlying the therapeutic effects of SYD on UC is not clear. AIM OF STUDYIn this study, we demonstrated the therapeutic effect of SYD on the polarization of M1 macrophages and elucidated the underlying mechanism. MATERIALS AND METHODSUC mice were induced with 3% DSS for one week and subsequently treated with SYD for another week. The composition of SYD was determined by HPLC. To assess the therapeutic efficacy of SYD, key parameters, including body weight changes, DAI scores, colon length, and histological alterations in colonic tissues, were monitored. ELISA was performed to quantify inflammatory cytokines, while Western blotting and immunofluorescence analyses were performed to quantify tight junction protein expression and M1 macrophage infiltration, respectively. Functional assessments focused on lysosomal activity and glucose oxidation in primary macrophages and RAW 264.7 cells exposed to LPS, IL-17a, and SYD using dedicated kits. To elucidate the mechanisms underlying the action of SYD, the data derived from TMT-based proteomics were analyzed to screen and predict its potential targets and regulated pathways. RESULTSAfter treatment for seven days, SYD significantly mitigated colitis symptoms in mice, as determined by a decrease in body weight loss, an increase in colon length, and a decrease in disease activity index (DAI) score. The results of histopathological analysis showed substantial improvements in the integrity of colonic tissue. Additionally, SYD treatment significantly decreased the levels of proinflammatory cytokines, including IL-17a, IL-6, IL-1β, and TNF-α. This effect was accompanied by a reduction in the infiltration of CD86+ macrophages and restoration of occludin and ZO-1 levels, thus improving colonic mucosal permeability. SYD treatment also reversed the upregulation of cathepsin (CTS) E, CTSS, lysosomal-associated membrane protein (LAMP)–1, and LAMP-2 expression observed in CD86+ macrophages and RAW 264.7 cells treated with IL-17a. These changes were accompanied by an increase in lysosomal acidification and the enzymatic activities of CTSE and CTSS, suggesting that SYD can restore lysosomal function. SYD also corrected the metabolic alterations in M1 macrophages, characterized by an increase in the extracellular acidification rate (ECAR) and a decrease in oxygen consumption rate (OCR). This was confirmed by a decrease in the ADP/ATP ratio, downregulation of pyruvate dehydrogenase kinase 4 (PDK4) and lactate dehydrogenase (LDH) expression, and a decrease in the concentrations of intracellular pyruvate and lactate. These findings showed that SYD promotes glucose oxidation in macrophages. The data derived from TMT-based proteomics showed that the PPAR pathway was a key target in regulating the polarization of M1 macrophages. SYD was also found to regulate the expression of the PPAR-α, PPAR-γ, and PPAR-δ proteins. CONCLUSION: SYD inhibited the polarization of M1 macrophages induced by IL-17a. This effect occurred due to the restoration of lysosomal activity and glucose oxidation via activation of the PPAR/NF-κB pathway. Our findings provided novel insights into the mechanisms underlying the therapeutic effects of SYD in UC.

Effects of dechlorane plus on hepatic pathology, metabolic health and gut microbiota in male mice.

Dechlorane plus (DP), a widely used flame retardant, was added to Annex A of the Stockholm Convention on Persistent Organic Pollutants in 2023. This study aimed to investigate the effects of DP on glucose and lipid metabolism by orally exposing eight-week-old male mice to environmentally relevant concentrations of DP (0.5, 1, and 5mg/kg/day) for six weeks. The in vivo effects of DP on liver histomorphology, glucose and lipid metabolism, intestinal microbiota, and the associated molecular mechanisms were assessed. Pathological examination revealed that exposure to 1 and 5mg/kg/day DP induced hepatic damage, characterized by structural disarray of the hepatic cords and vacuolar degeneration of liver cells, while 0.5 and 1mg/kg/day DP exposure led to significant triglycerides (TG) accumulation in the liver. Metabolite analysis showed a marked increase in hepatic pyruvate, glycogen, and TG in mice exposed to 0.5 and 1mg/kg/day DP, while 5mg/kg/day exposure resulted in elevated glycogen levels and reduced pyruvate and glucose concentrations. The underlying mechanisms involved the transcriptional regulation of key enzymes related to glucose and lipid metabolism, as well as the activation of the PI3K/AKT pathway. Exposure to 5mg/kg/day DP upregulated genes associated with glycogenesis (GK), glycolysis (HK1 and PK), and fatty acid synthesis (SREBP1, FAS, and ACC1), while downregulating genes involved in gluconeogenesis (PCK1) and fatty acid β-oxidation (CPT1 and PPARA). The activated PI3K/AKT pathway regulated key proteins (GLUT4, GSK3β, and FoxO1), playing distinct roles in glucose and lipid metabolism. High-throughput 16S rDNA sequencing revealed that 5mg/kg/day DP exposure altered the composition and diversity of intestinal microbiota, reducing the relative abundance of beneficial probiotics at both the phylum and genus levels. These findings offer new insights into the complex mechanisms through which DP affects glucose and lipid metabolism in mammals, contributing to a more comprehensive evaluation of its toxicity.

(General Student Poster Award Winner, 3rd Place) Synthesis of Nylon Resin Precursor Materials from Biomass-Derived Pyruvate with Photo/Biocatalyst Using Visible Light As an Energy Source

Plastics have become extremely indispensable in modern society due to their high durability and ease of processing. They are made from petroleum and emit carbon dioxide, a greenhouse gas, at the time of manufacture and disposal. Because of their high durability, they are difficult to decompose in the natural environment, and the influence of waste plastics on the environment has become a serious problem. Major example of such environmental issues is the soil and ocean pollution. This pollution caused by discarded plastics has increased the significance for biodegradable plastics, which decomposes into low-molecular compounds such as carbon dioxide and water through natural processes. Therefore, systems synthesizing precursor materials for biodegradable plastics from biomass-derived pyruvate with visible light contained in sunlight, a renewable energy source, have been constructed1. However, previous visible-light driven organic synthesis systems have focused on building C-C bonds and have not been able to synthesize nylon precursor materials requiring C-N bonds. Amino acids, organic nitrogen compounds, can be made into biodegradable nylon through polymerization or copolymerization, as typified by silk materials. Among them, L-alanine has a similar structure to L-lactic acid, which is a monomer of poly-L-lactic acid (PLA), a biodegradable plastic, so L-alanine has the potential to become a biodegradable plastic.Based on these backgrounds, in this study, we have developed a visible-light driven L-alanine synthesis system from biomass-derived pyruvate (Figure 1) and attempted to improve the efficiency of the system. The system is composed of a visible-light driven NADH regeneration system consisting of an electron donor (TEOA: triethanolamine), a photosensitizer (water-soluble zinc porphyrin) and an electron mediator (Rh complex)2; and an enzyme, L-alanine dehydrogenase (AlaDH), that uses NADH as a coenzyme. Enzymes have the advantage of being environmentally friendly, as they have high reaction selectivity and the reaction proceeds under mild conditions such as room temperature, normal pressure, and near neutrality.The sample solution (5.0 mL) contained 0.20 M TEOA, 10 μM ZnTPPS: zinc tetraphenylporphyrin tetrasulfonate, 10 μM [Cp*Rh(bpy)(H2O)]2+, 2.0 mM NAD+, 0.10 U AlaDH, 0.50 mM sodium pyruvate and 5.0 mM NH4HCO3 in 500 mM HEPES-NaOH (pH 8.0). The reaction mixture was deaerated by freeze-pump-thaw cycles repeated 6 times, and the gas phase was replaced with argon. Then, the solution was irradiated with 250 W halogen lamp at 30.5 oC. As a control, it was reacted under the dark. The produced L-alanine was qualitatively and quantitatively determined by high-performance liquid chromatograph system. To reduce the amount of expensive coenzyme NAD+ usage, we measured the L-alanine production rate and production concentration at low NAD+ concentrations and investigated the dependence of the reaction rate on pyruvate and ammonia concentrations under these conditions.Figure 2 shows the time course of L-alanine and pyruvate concentrations. The L-alanine concentration increased linearly with the pyruvate concentration decreasing linearly. This means that pyruvate was converted to L-alanine by this system. The conversion yield for pyruvate to L-alanine was ca. 100 % after 24 hours irradiation. In addition, no increase in L-alanine concentration is observed under dark condition, which means that electrons excited by visible light reduced NAD+ to NADH via the electron mediator, and NADH functioned as a coenzyme for AlaDH. Figure 3 shows the L-alanine production concentration after 24 hours irradiation at each initial NAD+ concentration. Even for reducing the initial NAD+ concentration, the L-alanine production concentration remained unchanged, indicating that the regeneration efficiency of the expensive coenzyme NAD+ was improved.We have succeeded in synthesizing L-alanine, a precursor material for biodegradable nylon, from biomass-derived pyruvate with visible-light irradiation using photo/biocatalyst and in improving the efficiency of using the expensive coenzyme NAD+. In the future, ammonia will be required to be derived from environmentally friendly precursor materials in contrast to pyruvate derived from biomass.

The Microbial Metabolite Wuyiencin Potential Targets Threonine dehydratase in Didymella segeticola to Achieve Control of Tea Leaf Spot.

Tea leaf spot caused by Didymella segeticola is a disease that has recently been discovered in the tea plantations of Southwest China, and which has a significant negative impact on the yield and quality of tea leaves. Wuyiencin is a nucleotide antimicrobial that is effective against a range of fungal diseases. However, its mode of action is still unclear. The current study found that wuyiencin inhibited the mycelial growth of D. segeticola in vitro. Meanwhile, in vivo experiments confirmed that wuyiencin had a significant curative effect on tea leaf spot. Microscopic observation represented it damaged the organelles and nucleus in fungal cells. Reverse transcription quantitative PCR assays revealed that mycelium treated with wuyiencin at the half-maximal effective concentration (EC50) dosage for 1 hour exhibited 3.23 times lower expression of Threonine dehydratase (Td) gene, which is responsible for producing pyruvate. The wild type (WT) strain had a 1.77-fold higher pyruvate concentration than that in the td mutant (P < 0.05). The td mutant was more sensitive than the WT to wuyiencin treatment, with the EC50 value in the td mutant being 30.01 μg/ml, compared with 82.34 μg/ml in the WT. Molecular docking demonstrated that wuyiencin bound to Td, with a binding energy of -10.47 kcal/mol. Compared with the WT strain, wuyiencin significantly reduced ATP concentration of the td mutant strain at dosages of 80.0 and 160.0 µg/ml. In total, wuyiencin reduced Td activity, inhibited pyruvate production, and decreased ATP content in the phytopathogenic fungus, ultimately disturbing the growth of the mycelium.

A serum-free culture medium production system by co-culture combining growth factor-secreting cells and l-lactate-assimilating cyanobacteria for sustainable cultured meat production

Large-scale production of cultured meat requires bulk culture medium containing growth-promoting proteins from animal serum. However, animal serum for mammalian cell culture is associated with high costs, ethical concerns, and contamination risks. Owing to its growth factor content, conditioned medium from rat liver epithelial RL34 cells can replace animal serum for myoblast proliferation. More seeded cells and longer culture periods are thought to yield higher growth factor levels, resulting in more effective muscle cell proliferation. However, RL34 cells can deplete nutrients and release harmful metabolites into the culture medium over time, potentially causing growth inhibition and apoptosis. This issue highlights the need for waste clearance during condition medium production. To address this issue, we introduced a lactate permease gene (lldP) and an l-lactate-to-pyruvate conversion enzyme gene (lldD) to generate a recombinant l-lactate-assimilating cyanobacterium Synechococcus sp. KC0110 strain. Transwell co-culture of this strain with RL34 cells exhibited a marked reduction in the levels of harmful metabolites, lactate and ammonium, while maintaining higher concentrations of glucose, pyruvate, and pyruvate-derived amino acids than those seen with RL34 cell monocultures. The co-culture medium supported myoblast proliferation without medium dilution or additional nutrients, which was attributed to the waste clearance and nutrient replenishment effects of the KC0110 strain. This culture system holds potential for the production of low-cost, and animal-free cultured meat.

The importance of acetate, pyruvate, and citrate feeding times in improving xanthan production by Xanthomonas citri.

Xanthan gum is a microbial polysaccharide produced by Xanthomonas and widely used in various industries. To produce xanthan gum, the native Xanthomonas citri-386 was used in a cheese-whey-based culture medium. The culture conditions were investigated in batch experiments based on the response surface methodology to increase xanthan production and viscosity. Three independent variables in this study included feeding times of acetate, pyruvate, and citrate. The maximum xanthan gum production and viscosity within 120 h by X. citri-386 using Box-Behnken design were 25.7g/l and 65 500 cP, respectively, with a 151% and 394% increase as compared to the control sample. Overall, the findings of this study recommend the use of X. citri-386 in the cheese-whey-based medium as an economical medium with optimal amounts of acetate, pyruvate, and citrate for commercial production of xanthan gum on an industrial scale. The adjustment of the pyruvate and acetate concentrations optimized xanthan gum production in the environment.

Exploring ethyl pyruvate as an antifungal and antibacterial agent for food preservation: an in vitro and in silico study

SummaryFood pathogens are one of the microbiological problems that have negative effects on both economic and human health by causing spoilage and decay of important foods such as fruits and vegetables. The adverse effects of chemical residues left by synthetic biocides on human health and the development of resistance mechanisms in microorganisms have directed research towards bio‐fungicide. This study aimed to develop an effective bio‐fungicide using varying concentrations of ethyl pyruvate (1–18 μL disc−1). Additionally, the interaction of EP with several proteins in pathogenic bacteria and fungi was investigated through in silico molecular docking. According to the obtained results, an increase in EP concentration resulted in larger inhibition zone diameters. In this context, at an EP concentration of 18 μL disc−1, the inhibition zones formed against E. coli, S. aureus, L. monocytogenes and S. typhimurium bacteria were measured as 27.50 ± 0.50 mm, 31.00 ± 1.00 mm, 30.00 ± 1.00 mm and 32.66 ± 1.15 mm, respectively. EP at a concentration of 9 μL disc−1 showed a fungicidal effect against B. cinerea and P. expansum and a fungistatic effect against C. gloeosporioides. As a result of in silico tests, the molecular binding energies of EP with E. coli dihydrofolate reductase, S. aureus DNA Gyrase B and B. cinerea 14 alpha‐demethylase were determined to be −4.62, −4.26 and −3.95 kcal mol−1, respectively. Our results holistically revealed that EP has great potential for use in food preservation due to its antifungal and antibacterial properties.

Hyperpolarized Nano-NMR Platform for Quantification of Mass Limited Samples.

Metabolic flux analysis of live cells using NMR enables the study of cancer metabolism and response to treatment. However, conventional NMR platforms require often prohibitively high numbers of cells to achieve significant resolution. In this work, we present a double 1H/13C resonance NMR probe consisting of a solenoid coil with a less than 100 nL sensitive region. In-solution robustness is demonstrated through measurement of decaying hyperpolarized signals. A suspension of live cells and hyperpolarized (HP) [1-13C]pyruvate is loaded in the coil, and dynamic changes in pyruvate and lactate concentrations by fractions of femtomoles are detected from just 2000 live cells at a time, in seconds. Through an integrated microfluidic channel, the probe is used as high-throughput platform to perform nondestructive quantitative analysis of metabolic flux of different leukemia cell lines with sensitivity to detect on target treatment response. This approach platform provides an approach to study mass-limited samples and living cells with dramatically enhanced sensitivity in real time.

Dopamine, an exogenous quorum sensing signaling molecule or a modulating factor in Pseudomonas aeruginosa?

Pseudomonas aeruginosa is recognized globally as an opportunistic pathogen of considerable concern due to its high virulence and pathogenicity, especially in immunocompromised individuals. While research has identified several endogenous quorum sensing (QS) signaling molecules that enhance the virulence and pathogenicity of P. aeruginosa, investigations on exogenous QS signaling molecules or modulating factors remain limited. This study found that dopamine serves as an exogenous QS signaling molecule or modulating factor of P. aeruginosa PAO1, enhancing the production of virulence factors and biofilms. Compared to the control group, treatment with 40 μM dopamine resulted in a 33.1 % increase in biofilm formation, 68.1 % increase in swimming mobility, 63.1 % increase in swarming mobility, 147.2 % increase in the signaling molecule 3-oxo-C12-HSL, and 50.5 %, 28.5 %, 27.0 %, and 33.2 % increases in the virulence factors alginate, rhamnolipids, protease, and pyocyanin, respectively. This study further explored the mechanism of dopamine regulating the biofilm formation and virulence of P. aeruginosa PAO1 through transcriptome and metabolome. Transcriptomic analysis showed that dopamine promoted the expression of virulence genes psl, alg, lasA, rhlABC, rml, and phz in P. aeruginosa PAO1. Metabolomic analysis revealed changes in the concentrations of tryptophan, pyruvate, ethanolamine, glycine, 3-hydroxybutyric acid, and alizarin. Furthermore, KEGG enrichment analysis of altered genes and metabolites indicated that dopamine enhanced phenylalanine, tyrosine, and tryptophan in P. aeruginosa PAO1. The results of this study will contribute to the development of novel exogenous QS signaling molecules or modulating factors and advance our understanding of the interactions between P. aeruginosa and the host environment.

Metabolomic profiling of embryo culture media in patients with repeated implantation failure during assisted reproductive technology cycles.

This study investigated the metabolic status of the spent culture media from embryos of patients with repeated implantation failure (RIF) undergoing in vitro fertilization-intracytoplasmic sperm injection cycles in comparison with the embryos from healthy fertile women. Metabolite levels in spent culture media were assessed and compared between embryos from RIF patients (n=35) and oocyte donors as controls (n=15). Protein levels of insulin-like growth factor 1 (IGF-1) were determined using Western blotting. Concentrations of glucose, pyruvate, and lactate were measured using spectrophotometry. Ionic colorimetric assay kits were utilized to analyze the concentrations of sodium, chloride, calcium, and magnesium ions. High-performance liquid chromatography was employed to measure the concentrations of glutamic acid, aspartic acid, methionine, phenylalanine, and histidine. Glucose consumption and lactate secretion were higher in the control group than in the RIF group. The magnesium concentration was significantly higher in the control group than in the RIF group, but glutamic acid and aspartic acid concentrations were lower in the control group than in the RIF patients (p<0.05). The levels of IGF-1, sodium, calcium, chloride, methionine, histidine, and phenylalanine did not show statistically significant differences between the two groups. The metabolic profile of the culture medium of the embryos in the RIF group differed from that of the control group. These findings suggest potential factors that may affect implantation capacity in RIF patients and provide a new perspective on embryo selection.

Negligible In Vitro Recovery of Macromolecules from Microdialysis Using 100 kDa Probes and Dextran in Perfusion Fluid

Microdialysis is applied in neurointensive care to monitor cerebral glucose metabolism. If recoverable, macromolecules may also serve as biomarkers in brain disease and provide clues to their passage across the blood–brain barrier. Our study aimed to investigate the in vitro recovery of human micro- and macromolecules using microdialysis catheters and perfusion fluids approved for clinical use. In vitro microdialysis of a bulk solution containing physiological or supraphysiological concentrations of glucose, lactate, pyruvate, human IgG, serum albumin, and hemoglobin was performed using two different catheters and perfusion fluids. One had a membrane cut-off of 20 kDa and was used with a standard CNS perfusion fluid, and the other had a membrane cut-off of 100 kDa and was perfused with the same solution supplemented with dextran. The flow rate was 0.3 µl/min. We used both push and push–pull methods. Dialysate samples were collected at 2-h intervals for 6 h and analyzed for relative recovery of each substance. The mean relative recovery of glucose, pyruvate, and lactate was > 90% in all but two sets of experiments. In contrast, the relative recovery of human IgG, serum albumin, and hemoglobin from both bulk solutions was below the lower limit of quantification (LLOQ). Using a push–pull method, recovery of human IgG, serum albumin, and hemoglobin from a bulk solution with supraphysiological concentrations were above LLOQ but with low relative recovery (range 0.9%–1.6%). In summary, exchanging the microdialysis setup from a 20 kDa catheter with a standard perfusion fluid for a 100 kDa catheter with a perfusion solution containing dextran did not affect the relative recovery of glucose and its metabolites. However, it did not result in any useful recovery of the investigated macromolecules at physiological levels, either with or without a push–pull pump system.

Sex differences in kidney metabolism may reflect sex-dependent outcomes in human diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.

9-HODE and 9-HOTrE alter mitochondrial metabolism, increase triglycerides, and perturb fatty acid uptake and synthesis associated gene expression in HepG2 cells

Non-Alcoholic Fatty Liver Disease (NAFLD) prevalence is rising and can lead to detrimental health outcomes such as Non-Alcoholic Steatohepatitis (NASH), cirrhosis, and cancer. Recent studies have indicated that Cytochrome P450 2B6 (CYP2B6) is an anti-obesity CYP in humans and mice. Cyp2b-null mice are diet-induced obese, and human CYP2B6-transgenic (hCYP2B6-Tg) mice reverse the obesity or diabetes progression, but with increased liver triglyceride accumulation in association with an increase of several oxylipins. Notably, 9-hydroxyoctadecadienoic acid (9-HODE) produced from linoleic acid (LA, 18:2, ω-6) is the most prominent of these and 9-hydroxyoctadecatrienoic acid (9-HOTrE) from alpha-linolenic acid (ALA, 18:3, ω-3) is the most preferentially produced when controlling for substrate concentrations in vitro. Transactivation assays indicate that 9-HODE and 9-HOTrE activate PPARα and PPARγ. In Seahorse assays performed in HepG2 cells, 9-HOTrE increased spare respiratory capacity, slightly decreased palmitate metabolism, and increased non-glycolytic acidification in a manner consistent with slightly increased glutamine utilization; however, 9-HODE exhibited no effect on metabolism. Both compounds increased triglyceride and pyruvate concentrations, most strongly by 9-HOTrE, consistent with increased spare respiratory capacity. qPCR analysis revealed several perturbations in fatty acid uptake and metabolism gene expression. 9-HODE increased expression of CD36, FASN, PPARγ, and FoxA2 that are involved in lipid uptake and production. 9-HOTrE decreased ANGPTL4 expression and increased FASN expression consistent with increased fatty acid uptake, fatty acid production, and AMPK activation. Our findings support the hypothesis that 9-HODE and 9-HOTrE promote steatosis, but through different mechanisms as 9-HODE is directly involved in fatty acid uptake and synthesis; 9-HOTrE weakly inhibits mitochondrial fatty acid metabolism while increasing glutamine use.

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures

Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM. During incubations for up to 2 weeks in DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days, while the extracellular lactate concentration increased continuously during the incubation into the millimolar concentration range as long as glucose was present. In an amino acid-free incubation buffer, glucose-fed astrocytes released pyruvate with an initial rate of around 60 nmol/(h × mg) and after around 5 h an almost constant extracellular pyruvate concentration was established that was maintained for several hours. Extracellular pyruvate accumulation was also observed, if glucose had been replaced by mannose, fructose, lactate or alanine. Glucose-fed astrocyte cultures established similar extracellular steady state concentrations of pyruvate by releasing pyruvate into pyruvate-free media or by consuming excess of extracellular pyruvate. Inhibition of the monocarboxylate transporter MCT1 by AR-C155858 lowered extracellular pyruvate accumulation, while inhibition of mitochondrial pyruvate uptake by UK5099 increased the extracellular pyruvate concentration. Finally, the presence of the uncoupler BAM15 or of the respiratory chain inhibitor antimycin A almost completely abolished extracellular pyruvate accumulation. The data presented demonstrate that cultured astrocytes establish a transient extracellular steady state concentration of pyruvate which is strongly affected by modulation of the mitochondrial pyruvate metabolism.

Bicarbonate-Dependent Detoxification by Mitigating Ammonium-Induced Hypoxic Stress in Triticum aestivum Root.

Ammonium (NH4+) toxicity is ubiquitous in plants. To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO3-)-dependent alleviation, wheat plants were hydroponically cultivated in half-strength Hoagland nutrient solution containing 7.5 mM NO3- (CK), 7.5 mM NH4+ (SA), or 7.5 mM NH4+ + 3 mM HCO3- (AC). Transcriptomic analysis revealed that compared to CK, SA treatment at 48 h significantly upregulated the expression of genes encoding fermentation enzymes (pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH)) and oxygen consumption enzymes (respiratory burst oxidase homologs, dioxygenases, and alternative oxidases), downregulated the expression of genes encoding oxygen transporters (PIP-type aquaporins, non-symbiotic hemoglobins), and those involved in energy metabolism, including tricarboxylic acid (TCA) cycle enzymes and ATP synthases, but upregulated the glycolytic enzymes in the roots and downregulated the expression of genes involved in the cell cycle and elongation. The physiological assay showed that SA treatment significantly increased PDC, ADH, and LDH activity by 36.69%, 43.66%, and 61.60%, respectively; root ethanol concentration by 62.95%; and lactate efflux by 23.20%, and significantly decreased the concentrations of pyruvate and most TCA cycle intermediates, the complex V activity, ATP content, and ATP/ADP ratio. As a consequence, SA significantly inhibited root growth. AC treatment reversed the changes caused by SA and alleviated the inhibition of root growth. In conclusion, NH4+ treatment alone may cause hypoxic stress in the roots, inhibit energy generation, suppress cell division and elongation, and ultimately inhibit root growth, and adding HCO3- remarkably alleviates the NH4+-induced inhibitory effects on root growth largely by attenuating the hypoxic stress.

Pyruvate-dependent growth of Methanosarcina acetivorans.

Methanogenesis is a key step during anaerobic biomass degradation. Methanogenic archaea (methanogens) are the only organisms coupling methanogenic substrate conversion to energy conservation. The range of substrates utilized by methanogens is limited, with acetate and H2+CO2 being the ecologically most relevant. The only single methanogenic energy substrate containing more carbon-carbon bonds than acetate is pyruvate. Only the aggregate-forming, freshwater methanogen Methanosarcina barkeri Fusaro was shown to grow on this compound. Here, the pyruvate-utilizing capabilities of the single-celled, marine Methanosarcina acetivorans were addressed. Robust pyruvate-dependent, methanogenic, growth could be established by omitting CO2 from the growth medium. Growth rates which were independent of the pyruvate concentration indicated that M. acetivorans actively translocates pyruvate across the cytoplasmic membrane. When 2-bromoethanesulfonate (BES) inhibited methanogenesis to more than 99%, pyruvate-dependent growth was acetogenic and sustained. However, when methanogenesis was completely inhibited M. acetivorans did not grow on pyruvate. Analysis of metabolites showed that acetogenesis is used by BES-inhibited M. acetivorans as a sink for electrons derived from pyruvate oxidation and that other, thus far unidentified, metabolites are produced.IMPORTANCEThe known range of methanogenic growth substrates is very limited and M. acetivorans is only the second methanogenic species for which growth on pyruvate is demonstrated. Besides some commonalities, analysis of M. acetivorans highlights differences in pyruvate metabolism among Methanosarcina species. The observation that M. acetivorans probably imports pyruvate actively indicates that the capabilities for heterotrophic catabolism in methanogens may be underestimated. The mostly acetogenic growth of M. acetivorans on pyruvate with concomitant inhibition of methanogenesis confirms that energy conservation of methanogenic archaea can be independent of methane formation.

Salivary metabolomics in patients with oral lichen planus: a preliminary study based on NMR spectroscopy

ObjectivesThe present preliminary study aimed to investigate the salivary metabolic profile in patients with asymptomatic oral lichen planus (OLP) using nuclear magnetic resonance (NMR) spectroscopy.Material and methodsStimulated whole mouth saliva (SWMS) samples were collected from 15 reticular OLP female patients and 15 from age- and sex-matched controls (HCs). A total of 23 metabolites were identified and quantified. Mann–Whitney’s U test was used to compare the determined concentration salivary metabolite concentrations between OLP patients and the healthy controls.ResultsThe concentration of acetate, methylamine, and pyruvate was elevated, whereas the concentration of tyrosine was decreased in the saliva of OLP patients compared with HCs. To identify a combination of metabolites, multivariate discrimination function analysis (DFA) was conducted. DFA analysis have shown that the most powerful discrimination between the groups was achieved when methylamine and tyrosine were considered as combined biomarkers.ConclusionsSalivary tyrosine was of particular interest and a promising finding for the screening of OLP and its progression. Further longitudinal studies are required to establish it as a reliable salivary biomarker in OLP.Clinical relevanceThe salivary metabolic profiling can describe the pathologic characteristics of OLP on non-invasive saliva samples and NMR analysis. Salivary metabolites provide details to considered early detectors and to impact oral health of OLP patients.

Transient Synaptic Enhancement Triggered by Exogenously Supplied Monocarboxylate in Drosophila Motoneuron Synapse

Current evidence suggests that glial cells provide C3 carbon sources to fuel neuronal activity; however, this notion has become challenged by biosensor studies carried out in acute brain slices or in vivo, showing that neuronal activity does not rely on the import of astrocyte-produced L-lactate. Rather, stimulated neurons become net lactate exporters, as it was also shown in Drosophila neurons, in which astrocyte-provided lactate returns as lipid droplets to be stored in glial cells. In this view, we investigate whether exogenously supplied monocarboxylates can support Drosophila motoneuron neurotransmitter release (NTR). By assessing the excitatory post-synaptic current (EPSC) amplitude under voltage-clamp as NTR indicative, we found that both pyruvate and L-lactate, as the only carbon sources in the synapses bathing-solution, cause a large transient NTR enhancement, which declines to reach a synaptic depression state, from which the synapses do not recover. The FM1-43 pre-synaptic loading ability, however, is maintained under monocarboxylate, suggesting that SV cycling should not contribute to the synaptic depression state. The NTR recovery was reached by supplementing the monocarboxylate medium with sucrose. However, monocarboxylate addition to sucrose medium does not enhance NTR, but it does when the disaccharide concentration becomes too reduced. Thus, when pyruvate concentrations become too reduced, exogenously supplied L-lactate could be converted to pyruvate and metabolized by the neural mitochondria, triggering the NTR enhancement. Significance statementThe question of whether monocarboxylic acids can fuel the Drosophila motoneuron NTR was challenged. Our findings show that exogenously supplied monocarboxylates trigger a large transient synaptic enhancement just under extreme glycolysis reduction but fail to maintain NTR under sustained synaptic demand, still at low frequency stimulation, driven to the synapses to a synaptic depression state. Glycolysis activation, by adding sucrose to the monocarboxylate bath solution, restores the motoneuron NTR ability, giving place to a hexoses role in SV recruitment. Moreover these results suggest exogenously supplied C3 carbon sources could have an additional role beyond providing energetic support for neural activity.

Metronidazole Induces the Metazoan Death of Giardia Trophozoites with the Aid of Pyruvate

Background: Metronidazole is the most common drug for the treatment of infectious agent Giardia. The trophozoites need to fight against the oxidative stress generated by metronidazole for their survival. It has been reported that trophozoites possesses several enzymes involved in response to oxidative stress like pyruvate-ferredoxin oxidoreductase, NADH oxidase, peroxiredoxin to combat the harsh condition. These enzyme systems generally act on the amitochondriate trophozoites to attenuate the reactive oxygen species generation which causes cytotoxicity but the actual mechanism of trophozoites death due to metronidazole treatment was still not clear. Methods: The present study aims to establish the effects of pyruvate in Giardia trophozoites exposed to metronidazole treatment. Intracellular reactive oxygen species (ROS) production by Giardia trophozoite suspension was monitored in the presence and absence of pyruvate with the help of a dichlorodihydrofluoresceine diacetate (H2DCFDA) based assay. In the present study, we have investigated the effects of pyruvate on DNA damage in the trophozoites during metronidazole stress. We have also looked into the expression levels of some genes to show their relevance to metronidazole stress. Results: The exogenously addition of physiologically relevant concentration of pyruvate was shown to elevate the rate of ROS generation in Giardia suspension under metronidazole stress. Our results provide evidence that exogenously added pyruvate have induced lipid peroxidation of stressed Giardia. Several known genes are modulated due to the exposure of metronidazole in trophozoites. Conclusion: These results suggest that pyruvate is the key regulatory metabolite that helps generation of different radicals to initiate apoptotic like death in Giardia trophozoites during metronidazole exposure.