Ethyl pyruvate ameliorates acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) became a focus of intensive research due to its death toll during the Covid-19 pandemic. An uncontrolled and excessive inflammatory response mediated by proinflammatory molecules such as high mobility group box protein 1 (HMGB1), IL-6, and TNF mounts as a response to infection. In this study, ethyl pyruvate (EP), a known inhibitor of HMGB1, was tested in the model of murine ARDS induced in C57BL/6 mice by intranasal administration of polyinosinic:polycytidylic acid (poly(I:C)). Intraperitoneal administration of EP ameliorated the ARDS-related histopathological changes in the lungs of poly(I:C)-induced ARDS and decreased numbers of immune cells in the lungs, broncho-alveolar lavage fluid and draining lymph nodes (DLN). Specifically, fewer CD8+ T cells and less activated CD4+ T cells were observed in DLN. Consequently, the lungs of EP-treated animals had fewer damage-inflicting CD8+ cells and macrophages. Additionally, the expression and production of proinflammatory cytokines, IL-17, IFN-γ and IL-6 were downregulated in the lungs. The expression of chemokine CCL5 which recruits immune cells into the lungs was also reduced. Finally, EP downregulated the expression of HMGB1 in the lungs. Our results imply that EP should be further evaluated as a potential candidate for ARDS therapy.

Hydroxy(phenyl)pyruvic acid reductase in Actaea racemosa L.: a putative enzyme in cimicifugic and fukinolic acid biosynthesis.

MAIN CONCLUSION: Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as ß-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration. The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or ß-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (Km 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with ß-hydroxypyruvic acid (Km 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (Km 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of ß-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).

Re-investigation of in vitro activity of acetohydroxyacid synthase I holoenzyme from Escherichia coli.

Acetohydroxyacid synthase (AHAS) is one of the key enzymes of the biosynthesis of branched-chain amino acids, it is also an effective target for the screening of herbicides and antibiotics. In this study we present a method for preparing Escherichia coli AHAS I holoenzyme (EcAHAS I) with exceptional stability, which provides a solid ground for us to re-investigate the in vitro catalytic properties of the protein. The results show EcAHAS I synthesized in this way exhibits similar function to Bacillus subtilis acetolactate synthase in its catalysis with pyruvate and 2-ketobutyrate (2-KB) as dual-substrate, producing four 2-hydroxy-3-ketoacids including (S)-2-acetolactate, (S)-2-aceto-2-hydroxybutyrate, (S)-2-propionyllactate, and (S)-2-propionyl-2-hydroxybutyrate. Quantification of the reaction indicates that the two substrates almost totally consume, and compound (S)-2-aceto-2- hydroxybutyrate forms in the highest yield among the four major products. Moreover, the protein also condenses two molecules of 2-KB to furnish (S)-2-propionyl-2-hydroxybutyrate. Further exploration manifests that EcAHAS I ligates pyruvate/2-KB and nitrosobenzene to generate two arylhydroxamic acids N-hydroxy-N-phenylacetamide and N-hydroxy-N-phenyl- propionamide. These findings enhance our comprehension of the catalytic characteristics of EcAHAS I. Furthermore, the application of this enzyme as a catalyst in construction of C-N bonds displays promising potential.

Pseudodesulfovibrio pelocollis sp. nov. a Sulfate-Reducing Bacterium Isolated from a Terrestrial Mud Volcano.

Terrestrial mud volcanoes (TMVs), surface expressions of a deep-subterranean sedimentary volcanism, are widespread throughout the world. The methane and sulfur cycles are recognized as the most important biogeochemical cycles in these environments. Only few anaerobic bacterial strains were recovered from TMVs. We have isolated a novel sulfate-reducing bacterium (strain SB368T) from TMV located at Taman Peninsula, Russia. Optimum growth of strain SB368T was observed at 30 °C, pH 8.0 and 1% NaCl. Strain SB368T utilized lactate, pyruvate and fumarate in the presence of sulfate, sulfite or thiosulfate. Growth with molecular hydrogen was observed only in the presence of acetate. Fermentative growth occurred on pyruvate. Phylogenetic analysis revealed that strain SB368T belongs to the genus Pseudodesulfovibrio but is distinct from all described species. Based on its genomic and phenotypic properties, a new species, Pseudodesulfovibrio pelocollis sp. nov. is proposed with strain SB368T (= DSM 111087 T = VKM B-3585 T) as a type strain.

Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog.

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)-based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure-activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

Phototrophic Fe(II) oxidation by Rhodopseudomonas palustris TIE-1 in organic and Fe(II)-rich conditions.

Rhodopseudomonas palustris TIE-1 grows photoautotrophically with Fe(II) as an electron donor and photoheterotrophically with a variety of organic substrates. However, it is unclear whether R. palustris TIE-1 conducts Fe(II) oxidation in conditions where organic substrates and Fe(II) are available simultaneously. In addition, the effect of organic co-substrates on Fe(II) oxidation rates or the identity of Fe(III) minerals formed is unknown. We incubated R. palustris TIE-1 with 2 mM Fe(II), amended with 0.6 mM organic co-substrate, and in the presence/absence of CO2 . We found that in the absence of CO2 , only the organic co-substrates acetate, lactate and pyruvate, but not Fe(II), were consumed. When CO2 was present, Fe(II) and all organic substrates were consumed. Acetate, butyrate and pyruvate were consumed before Fe(II) oxidation commenced, whereas lactate and glucose were consumed at the same time as Fe(II) oxidation proceeded. Lactate, pyruvate and glucose increased the Fe(II) oxidation rate significantly (by up to threefold in the case of lactate). 57 Fe Mössbauer spectroscopy revealed that short-range ordered Fe(III) oxyhydroxides were formed under all conditions. This study demonstrates phototrophic Fe(II) oxidation proceeds even in the presence of organic compounds, and that the simultaneous oxidation of organic substrates can stimulate Fe(II) oxidation.

3-bromopyruvate induces morphological alteration and may initiate programmed cell death in Cryptococcus neoformans cells.

3-Bromopyruvate (3BP), known for its potent anticancer properties, also exhibits remarkable efficacy against the pathogenic fungus Cryptococcus neoformans. So far it has been proven that the main fungicidal activity of 3BP is based on ATP depletion and a reduction of intracellular level of glutathione. The presented study includes a broad range of methods to further investigate the mechanistic effects of 3BP on C. neoformans cells. The use of flow cytometry allowed a thorough examination of their survival during 3BP treatment, while observations using electron microscopy made it possible to note the changes in cellular morphology. Utilizing ruthenium red, the study suggests a mitochondrial pathway may initiate programmed cell death in response to 3BP. Analysis of free radical generation and gene expression changes supports this hypothesis. These findings enhance comprehension of 3BP's mechanisms in fungal cells, paving the way for its potential application as a therapeutic agent against cryptococcosis.

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.

Pyrimidines maintain mitochondrial pyruvate oxidation to support de novo lipogenesis.

Cellular purines, particularly adenosine 5'-triphosphate (ATP), fuel many metabolic reactions, but less is known about the direct effects of pyrimidines on cellular metabolism. We found that pyrimidines, but not purines, maintain pyruvate oxidation and the tricarboxylic citric acid (TCA) cycle by regulating pyruvate dehydrogenase (PDH) activity. PDH activity requires sufficient substrates and cofactors, including thiamine pyrophosphate (TPP). Depletion of cellular pyrimidines decreased TPP synthesis, a reaction carried out by TPP kinase 1 (TPK1), which reportedly uses ATP to phosphorylate thiamine (vitamin B1). We found that uridine 5'-triphosphate (UTP) acts as the preferred substrate for TPK1, enabling cellular TPP synthesis, PDH activity, TCA-cycle activity, lipogenesis, and adipocyte differentiation. Thus, UTP is required for vitamin B1 utilization to maintain pyruvate oxidation and lipogenesis.

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.

Allosteric-Regulation-Based DNA Circuits in Saccharomyces cerevisiae to Detect Organic Acids and Monitor Hydrocarbon Metabolism In Vitro.

We show the engineering of prokaryotic-transcription-factor-based biosensing devices in Saccharomyces cerevisiae cells for an in vitro detection of common hydrocarbon intermediates/metabolites and potentially, for monitoring of the metabolism of carbon compounds. We employed the bacterial receptor proteins MarR (multiple antibiotic-resistant receptor) and PdhR (pyruvate dehydrogenase-complex regulator) to detect benzoate/salicylate and pyruvate, respectively. The yeast-enhanced green fluorescence protein (yEGFP) was adopted as an output signal. Indeed, the engineered yeast strains showed a strong and dynamic fluorescent output signal in the presence of the input chemicals ranging from 2 fM up to 5 mM. In addition, we describe how to make use of these strains to assess over time the metabolism of complex hydrocarbon compounds due to the hydrocarbon-degrading fungus Trichoderma harzianum (KY488463).

Brain Regional Energy Metabolism in Patients with Traumatic Brain Injury: A Cerebral Microdialysis Guided Study.

BACKGROUND: In traumatic brain injuries (TBI), cerebral microdialysis (CMD)-derived parameters, especially the lactate to pyruvate ratio (LP ratio), have been utilized for cerebral perfusion optimization. The objectives were to identify cerebral ischemia as measured by CMD in TBI patients requiring decompressive craniectomy and to observe the correlation between cerebral perfusion pressure (CPP), intracranial pressure (ICP), and CMD variables in these patients. Our secondary aim was to observe the effect of CPP augmentation on ischemia biomarkers. METHODS: After the Institute Ethics Committee approvals, seven adult patients requiring decompressive craniectomy following TBI were enrolled and CMD data were obtained prospectively for 72 h. CPP was augmented by 20% with noradrenaline infusion if LP ratio >40. Correlations were done with bootstrapping (n = 500) to obtain the confidence intervals (CI) due to the small sample size. RESULTS: One patient had cerebral ischemia (median LP ratio of 265.5 and median pyruvate of 38 µmol/L), while another patient had non-ischemic mitochondrial dysfunction (median LP ratio 40.7 and median pyruvate 278.5). The coefficients of correlation between the LP ratio with CPP and ICP were r = -0.05 (CI = -0.14-0.03) and r = 0.09 (CI = -0.03-0.24), respectively. The coefficient of correlation between cerebral and blood glucose was r = 0.38, (CI - 0.35-0.14). Only two patients needed CPP augmentation, however, postaugmentation cerebral biochemistry did not change appreciably. CONCLUSION: CMD can identify cerebral ischemia, however, no correlations were observed between the LP ratio and CPP or ICP. CPP augmentation did not improve cerebral biochemistry. More studies are required to understand and treat cerebral metabolism in TBI.

Pan-cancer analysis reveals PDK family as potential indicators related to prognosis and immune infiltration.

Pyruvate dehydrogenase kinases (PDKs) play a key role in glucose metabolism by exerting negative regulation over pyruvate dehyrogenase complex (PDC) activity through phosphorylation. Inhibition of PDKs holds the potential to enhance PDC activity, prompting cells to adopt a more aerobic metabolic profile. Consequently, PDKs emerge as promising targets for condition rooted in metabolic dysregulation, including malignance and diabetes. However, a comprehensive exploration of the distinct contribution of various PDK family members, particularly PDK3, across diverse tumor types remain incomplete. This study undertakes a systematic investigation of PDK family expression patterns, forging association with clinical parameters, using data from the TCGA and GTEx datasets. Survival analysis of PDKs is executed through both Kaplan-Meier analysis and COX regression analysis. Furthermore, the extent of immune infiltration is assessed by leveraging the CIBERSORT algorithm. Our study uncovers pronounced genetic heterogeneity among PDK family members, coupled with discernible clinical characteristic. Significantly, the study establishes the potential utility of PDK family genes as prognostic indicators and as predictors of therapeutic response. Additionally, our study sheds light on the immune infiltration profile of PDK family. The results showed the intimate involvement of these genes in immune-related metrics, including immune scoring, immune subtypes, tumor-infiltrating lymphocytes, and immune checkpoints expression. In sum, the findings of this study offer insightful strategies to guide the therapeutic direction, aiming at leveraging the impact of PDK family genes in cancer treatment.

Real-time resolution studies of the regulation of lactate production by hexokinases binding to mitochondria in single cells.

During hypoxia accumulation of lactate may be a key factor in acidosis-induced tissue damage. Binding of hexokinase (HK) to the outer membrane of mitochondria may have a protective effect under these conditions. We have investigated the regulation of lactate metabolism by hexokinases (HKs), using HEK293 cells in which the endogenous hexokinases have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were also transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown HEK cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. Upon inhibition of the mitochondrial electron transfer chain by NaCN this effect was reversed as a rapid increase in lactate developed which was followed by a slow and sustained increase in the continued presence of the inhibitor. Incubation of the HKI/HKII double knockdown HEK cells with the inhibitor of the malic enzyme, ME1*, blocked the delayed accumulation of lactate evoked by NaCN. With replacement by overexpression of HKI or HKII the accumulation of intracellular lactate evoked by NaCN was prevented. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN) abolished the protective effect of HK expression, with NaCN causing again a sustained increase in lactate. The effect of HK was dependent on HK's catalytic activity and interaction with the mitochondrial outer membrane (MOM). Based on these data we propose that transformation of glucose into G6P by HK activates the pentose phosphate pathway which increases the production of NADPH, which then blocks the activity of the malic enzyme to transform malate into pyruvate and lactate.

Monocarboxylate transporter 4 deficiency enhances high-intensity interval training-induced metabolic adaptations in skeletal muscle.

High-intensity exercise stimulates glycolysis, subsequently leading to elevated lactate production within skeletal muscle. While lactate produced within the muscle is predominantly released into the circulation via the monocarboxylate transporter 4 (MCT4), recent research underscores lactate's function as an intercellular and intertissue signalling molecule. However, its specific intracellular roles within muscle cells remains less defined. In this study, our objective was to elucidate the effects of increased intramuscular lactate accumulation on skeletal muscle adaptation to training. To achieve this, we developed MCT4 knockout mice and confirmed that a lack of MCT4 indeed results in pronounced lactate accumulation in skeletal muscle during high-intensity exercise. A key finding was the significant enhancement in endurance exercise capacity at high intensities when MCT4 deficiency was paired with high-intensity interval training (HIIT). Furthermore, metabolic adaptations supportive of this enhanced exercise capacity were evident with the combination of MCT4 deficiency and HIIT. Specifically, we observed a substantial uptick in the activity of glycolytic enzymes, notably hexokinase, glycogen phosphorylase and pyruvate kinase. The mitochondria also exhibited heightened pyruvate oxidation capabilities, as evidenced by an increase in oxygen consumption when pyruvate served as the substrate. This mitochondrial adaptation was further substantiated by elevated pyruvate dehydrogenase activity, increased activity of isocitrate dehydrogenase - the rate-limiting enzyme in the TCA cycle - and enhanced function of cytochrome c oxidase, pivotal to the electron transport chain. Our findings provide new insights into the physiological consequences of lactate accumulation in skeletal muscle during high-intensity exercises, deepening our grasp of the molecular intricacies underpinning exercise adaptation. KEY POINTS: We pioneered a unique line of monocarboxylate transporter 4 (MCT4) knockout mice specifically tailored to the ICR strain, an optimal background for high-intensity exercise studies. A deficiency in MCT4 exacerbates the accumulation of lactate in skeletal muscle during high-intensity exercise. Pairing MCT4 deficiency with high-intensity interval training (HIIT) results in a synergistic boost in high-intensity exercise capacity, observable both at the organismal level (via a treadmill running test) and at the muscle tissue level (through an ex vivo muscle contractile function test). Coordinating MCT4 deficiency with HIIT enhances both the glycolytic enzyme activities and mitochondrial capacity to oxidize pyruvate.

Loss of Cardiac PFKFB2 Drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

BACKGROUND: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. METHODS AND RESULTS: To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control mice, we characterized the impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. cKO mice have a shortened life span of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to control animals. Metabolomic, proteomic, and Western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular dilation, represented by reduced fractional shortening and increased left ventricular internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. CONCLUSIONS: Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart.

Model-guided metabolic rewiring to bypass pyruvate oxidation for pyruvate derivative synthesis by minimizing carbon loss.

Engineering microbial hosts to synthesize pyruvate derivatives depends on blocking pyruvate oxidation, thereby causing severe growth defects in aerobic glucose-based bioprocesses. To decouple pyruvate metabolism from cell growth to improve pyruvate availability, a genome-scale metabolic model combined with constraint-based flux balance analysis, geometric flux balance analysis, and flux variable analysis was used to identify genetic targets for strain design. Using translation elements from a ~3,000 cistronic library to modulate fxpK expression in a bicistronic cassette, a bifido shunt pathway was introduced to generate three molecules of non-pyruvate-derived acetyl-CoA from one molecule of glucose, bypassing pyruvate oxidation and carbon dioxide generation. The dynamic control of flux distribution by T7 RNAP-mediated synthetic small RNA decoupled pyruvate catabolism from cell growth. Adaptive laboratory evolution and multi-omics analysis revealed that a mutated isocitrate dehydrogenase functioned as a metabolic switch to activate the glyoxylate shunt as the only C4 anaplerotic pathway to generate malate from two molecules of acetyl-CoA input and bypass two decarboxylation reactions in the tricarboxylic acid cycle. A chassis strain for pyruvate derivative synthesis was constructed to reduce carbon loss by using the glyoxylate shunt as the only C4 anaplerotic pathway and the bifido shunt as a non-pyruvate-derived acetyl-CoA synthetic pathway and produced 22.46, 27.62, and 6.28 g/L of l-leucine, l-alanine, and l-valine by a controlled small RNA switch, respectively. Our study establishes a novel metabolic pattern of glucose-grown bacteria to minimize carbon loss under aerobic conditions and provides valuable insights into cell design for manufacturing pyruvate-derived products.IMPORTANCEBio-manufacturing from biomass-derived carbon sources using microbes as a cell factory provides an eco-friendly alternative to petrochemical-based processes. Pyruvate serves as a crucial building block for the biosynthesis of industrial chemicals; however, it is different to improve pyruvate availability in vivo due to the coupling of pyruvate-derived acetyl-CoA with microbial growth and energy metabolism via the oxidative tricarboxylic acid cycle. A genome-scale metabolic model combined with three algorithm analyses was used for strain design. Carbon metabolism was reprogrammed using two genetic control tools to fine-tune gene expression. Adaptive laboratory evolution and multi-omics analysis screened the growth-related regulatory targets beyond rational design. A novel metabolic pattern of glucose-grown bacteria is established to maintain growth fitness and minimize carbon loss under aerobic conditions for the synthesis of pyruvate-derived products. This study provides valuable insights into the design of a microbial cell factory for synthetic biology to produce industrial bio-products of interest.

All Three AKT Isoforms Can Upregulate Oxygen Metabolism and Lactate Production in Human Hepatocellular Carcinoma Cell Lines.

Hepatocellular carcinoma (HCC), the main pathological type of liver cancer, is related to risk factors such as viral hepatitis, alcohol intake, and non-alcoholic fatty liver disease (NAFLD). The constitutive activation of the PI3K/AKT signaling pathway is common in HCC and has essential involvement in tumor progression. The serine/threonine kinase AKT has several downstream substrates, which have been implicated in the regulation of cellular metabolism. However, the contribution of each of the three AKT isoforms, i.e., AKT1, AKT2 and AKT3, to HCC metabolism has not been comprehensively investigated. In this study, we analyzed the functional role of AKT1, AKT2 and AKT3 in HCC metabolism. The overexpression of activated AKT1, AKT2 and AKT3 isoforms in the human HCC cell lines Hep3B and Huh7 resulted in higher oxygen consumption rate (OCR), ATP production, maximal respiration and spare respiratory capacity in comparison to vector-transduced cells. Vice versa, lentiviral vector-mediated knockdowns of each AKT isoform reduced OCR in both cell lines. Reduced OCR rates observed in the three AKT isoform knockdowns were associated with reduced extracellular acidification rates (ECAR) and reduced lactate production in both analyzed cell lines. Mechanistically, the downregulation of OCR by AKT isoform knockdowns correlated with an increased phosphorylation of the pyruvate dehydrogenase on Ser232, which negatively regulates the activity of this crucial gatekeeper of mitochondrial respiration. In summary, our data indicate that each of the three AKT isoforms is able to upregulate OCR, ECAR and lactate production independently of each other in human HCC cells through the regulation of the pyruvate dehydrogenase.

PARP-1 inhibitor alleviates cerebral ischemia/reperfusion injury by reducing PARylation of HK-1 and LDH in mice.

Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.

Inhibition of PKM2 suppresses osteoclastogenesis and alleviates bone loss in mouse periodontitis.

BACKGROUND: Chronic periodontitis triggers an increase in osteoclastogenesis, with glycolysis playing a crucial role in this process. Pyruvate kinase M2 (PKM2) is a critical enzyme involved in glycolysis and pyruvate metabolism. Yet, the precise function of PKM2 in osteoclasts and their formation remains unclear and requires further investigation. METHODS: Bioinformatics was used to investigate critical biological processes in osteoclastogenesis. In vitro, osteoclastogenesis was analyzed using tartrate-resistant acid phosphatase (TRAP) staining, phalloidin staining, quantitative real­time PCR (RT-qPCR), and Western blotting. Small interfering RNA (siRNA) of PKM2 and Shikonin, a specific inhibitor of PKM2, were used to verify the role of PKM2 in osteoclastogenesis. The mouse model of periodontitis was used to assess the effect of shikonin on bone loss. Analyses included micro computed tomography, immunohistochemistry, flow cytometry, TRAP staining and HE staining. RESULTS: Bioinformatic analysis revealed a significant impact of glycolysis and pyruvate metabolism on osteoclastogenesis. Inhibition of PKM2 leads to a significant reduction in osteoclastogenesis. In vitro, co-culture of the heat-killed Porphyromonas gingivalis significantly promoted osteoclastogenesis, concomitant with an increased PKM2 expression in osteoclasts. Shikonin weakened the promoting effect of porphyromonas gingivalis on osteoclastogenesis. In vivo experiments demonstrated that inhibition of PKM2 by shikonin alleviated bone loss induced by periodontitis, suppressed excessive osteoclastogenesis in alveolar bone, and reduced tissue inflammation to some extent. CONCLUSION: PKM2 inhibition by shikonin, a specific inhibitor of this enzyme, attenuated osteoclastogenesis and bone resorption in periodontitis. Shikonin appears to be a promising therapeutic agent for treating periodontitis.