Search

Regulation of mitochondrial calcium import is less understood in evolutionarily distinct protozoan parasites, such as Leishmania, as some of the mitochondrial calcium uniporter complex proteins are either missing or functionally diverged. Here, we show that Actin-related protein4 (ARP4), localizes exclusively into the Leishmania mitochondrion and depletion of this protein causes cells to accumulate calcium in the mitochondrion. The ARP4 depleted cells show increased activation of pyruvate dehydrogenase and production of ATP. Overall, our results indicate that ARP4 negatively regulates calcium uptake in the Leishmania mitochondrion.

OBJECTIVES To investigate the activity of key rate-limiting enzymes of glucose metabolism after restoration of spontaneous circulation (ROSC), to explore the potential pathophysiological mechanism of impaired myocardial energy metabolism after cardiopulmonary resuscitation (CPR). METHODS Twenty-one male Sprague-Dawley rats were randomized into three experimental groups assigned in accordance with different observation times after ROSC: 1) Sham, instrumented rats without induced cardiac arrest or resuscitation; 2) Post-resuscitation (PR2 h); 3) PR24 h. In these groups, CPR, including precordial compressions and synchronized mechanical ventilation, was initiated 6 min after asphyxia-induced cardiac arrest. Hearts were harvested after ROSC and samples were used to detect high-energy phosphate and glucose metabolic enzyme activity. RESULTS Compared with sham, the contents of phosphocreatine and adenosine triphosphate reduced in the PR2 h group, while remained unchanged in the PR24 h group. Activities of hexokinase and pyruvate kinase did not change after ROSC. Phosphofructokinase activity decreased only in the PR24 h group. Activities of pyruvate dehydrogenase and citrate synthase fell in PR2 h group and recovered in the PR24 h group. However, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase activities fell in the PR2 h group, but did not recover in the PR24 h group. CONCLUSIONS Lowered key rate-limiting enzymes activity in glucose metabolism resulted in impairment of energy production in the early stage of ROSC, but partially recovered in 24 h. This process has a role in the mechanism of impaired myocardial energy metabolism after CPR. This investigation might shed light on new strategies to treat post resuscitation myocardial dysfunction.

Calcium signaling via mitochondrial calcium uniporter (MCU) complex coordinates mitochondrial bioenergetics with cellular energy demands. Emerging studies show that the stability and activity of the pore-forming subunit of the complex, MCU, is dependent on the mitochondrial phospholipid, cardiolipin (CL), but how this impacts calcium-dependent mitochondrial bioenergetics in CL-deficiency disorder like Barth syndrome (BTHS) is not known. Here we utilized multiple models of BTHS including yeast, mouse muscle cell line, as well as BTHS patient cells and cardiac tissue to show that CL is required for the abundance and stability of the MCU-complex regulatory subunit MICU1. Interestingly, the reduction in MICU1 abundance in BTHS mitochondria is independent of MCU. Unlike MCU and MICU1/MICU2, other subunit and associated factor of the uniporter complex, EMRE and MCUR1, respectively, are not affected in BTHS models. Consistent with the decrease in MICU1 levels, we show that the kinetics of MICU1-dependent mitochondrial calcium uptake is perturbed and acute stimulation of mitochondrial calcium signaling in BTHS myoblasts fails to activate pyruvate dehydrogenase, which in turn impairs the generation of reducing equivalents and blunts mitochondrial bioenergetics. Taken together, our findings suggest that defects in mitochondrial calcium signaling could contribute to cardiac and skeletal muscle pathologies observed in BTHS patients.

У статті розглядаються питання впливу тіаміну та його похідних на перебіг ускладнень цукрового діабету (ЦД). Tіамін слугує коферментом для транскетолази, піруватдегідрогенази й комплексів α-кето­глутаратдегідрогенази, ферменти яких відіграють фундаментальну роль у внутрішньоклітинному метаболізмi глюкози. У літературі повідомляється про взаємозв’язки між тіаміном і ЦД. Рівні тіаміну й активність залежних від тіаміну ферментів знижені у хворих на ЦД. Генетичні дослідження дають можливість встановити зв’язуючі ланки між тіаміном і ЦД. Встановлено, що тіамін і його деривати запобігають активації біохімічних процесів (посилене виділення поліоловим шляхом, надмірне утворення кінцевих продуктів глікування, активація протеїнкінази C і посилення гексозамінового шляху біосинтезу), спричинених гіперглікемією за ЦД. Підкреслюється значення тіаміну при ендотеліальних судинних хворобах при ЦД (мікро- і макроангіопатія), порушеннях ліпідного обміну, при ретино-, нефро-, кардіо- і нейропатії.

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site., The pyruvate dehydrogenase complex (PDHc) is a large multienzyme complex that converts pyruvate into acetyl-coenzyme A and in E. coli the core of the PDHc is formed by 24 copies of dihydrolipoyl transacetylase. Here, the authors present the cryo-EM structure of the E. coli dihydrolipoyl transacetylase 24-mer core in a native resting state including lipoyl domains, and discuss the mechanism of substrate shuttling by the lipoyl domains.

Macrophages play an essential role in alcohol-induced inflammation and oxidative stress. We investigated the effects of nicotinamide riboside (NR), a natural nicotinamide adenine dinucleotide (NAD+) precursor, on alcohol-induced inflammation and oxidative stress in macrophages. NR significantly decreased ethanol-induced inflammatory gene expression, with a concomitant decrease in nuclear translocation of nuclear factor κB p65 in RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs). In macrophages incubated with ethanol or acetaldehyde, NR abolished the accumulation of cellular reactive oxygen species. Ethanol decreased sirtuin 1 (SIRT1) expression and activity, and cellular NAD+ level while inducing pro-inflammatory gene expression. However, NR markedly attenuated the changes. SIRT1 inhibition augmented ethanol-induced inflammatory gene expression, but its activation elicited opposing effects. Also, ethanol did not alter glycolysis but increased glycolytic capacity, glycolytic reserve, and non-glycolytic acidification, with concomitant increases in hypoxia-induced factor 1α expression and activity, phosphorylation of pyruvate dehydrogenase, and extracellular lactate levels. Interestingly, ethanol increased mitochondrial respiration and ATP production but decreased maximal respiration and spare respiration capacity. The latter was linked to decreases in mitochondrial copy numbers. NR abolished the ethanol-induced metabolic changes in the glycolytic and oxidative phosphorylation pathways in RAW 264.7 macrophages. In conclusion, NR exerts anti-inflammatory and antioxidant properties by abrogating the inhibitory effects of ethanol on the SIRT1 pathway by increasing Sirt1 expression and its activator, NAD+. Also, SIRT1 activation and normalization of ethanol-induced changes in NAD+/NADH ratios by NR are likely crucial to counteract the changes in energy phenotypes of macrophages exposed to ethanol.

Background We previously reported that glioma stemlike cells (GSCs) exist in the area of the tumor periphery showing no gadolinium enhancement on magnetic resonance imaging. In the present work, we analyzed glucose metabolism to investigate whether lactate could be predictive of tumor invasiveness and of use in detection of the tumor invasion area in glioblastoma multiforme (GBM). Methods The expression of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase (PDH) was investigated in 20 patients. In GSC lines, LDH-A and PDH expression also was examined in parallel to assessments of mitochondrial respiration. We then investigated the relationship between lactate/creatine ratios in the tumor periphery measured by magnetic resonance spectroscopy, using learning-compression-model algorithms and phenotypes of GBMs. Results In 20 GBMs, high-invasive GBM expressed LDH-A at significantly higher expression than did low-invasive GBM, whereas low-invasive GBM showed significantly higher expression of PDH than did high-invasive GBM. The highly invasive GSC line showed higher expression of LDH-A and lower expression of PDH compared with low-invasive GSC lines. The highly invasive GSC line also showed the lowest consumption of oxygen and the lowest production of adenosine triphosphate. Lactate levels, as measured by magnetic resonance spectroscopy, showed a significant positive correlation with LDH-A transcript levels, permitting classification of the GBMs into high-invasive and low-invasive phenotypes based on a cutoff value of 0.66 in the lactate/creatine ratio. Conclusions In the tumor periphery area of the highly invasive GBM, aerobic glycolysis was the predominant pathway for glucose metabolism, resulting in the accumulation of lactate. The level of lactate may facilitate prediction of the tumor-infiltrating area on GBM.

Hydrogen sulphide (H2S) inhibits vascular smooth muscle cell (VSMC) proliferation induced by hyperglycaemia and hyperlipidaemia; however, the mechanisms are unclear. Here, we observed lower H2S levels and higher expression of the proliferation‐related proteins PCNA and cyclin D1 in db/db mouse aortae and vascular smooth muscle cells treated with 40 mmol/L glucose and 500 μmol/L palmitate, whereas exogenous H2S decreased PCNA and cyclin D1 expression. The nuclear translocation of mitochondrial pyruvate dehydrogenase complex‐E1 (PDC‐E1) was significantly increased in VSMCs treated with high glucose and palmitate, and it increased the level of acetyl‐CoA and histone acetylation (H3K9Ac). Exogenous H2S inhibited PDC‐E1 translocation from the mitochondria to the nucleus because PDC‐E1 was modified by S‐sulfhydration. In addition, PDC‐E1 was mutated at Cys101. Overexpression of PDC‐E1 mutated at Cys101 increased histone acetylation (H3K9Ac) and VSMC proliferation. Based on these findings, H2S regulated PDC‐E1 S‐sulfhydration at Cys101 to prevent its translocation from the mitochondria to the nucleus and to inhibit VSMC proliferation under diabetic conditions.

Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with wide-ranging applications in the areas of medicine, light chemical industry, wastewater treatment, and agriculture. However, the production cost of γ-PGA is high for the requirement of adding the expensive precursor L-glutamic acid during fermentation, which hinders its widespread application. In this study, in order to improve γ-PGA yield, central carbon metabolism was engineered to enhance the carbon flux of tricarboxylic acid (TCA) cycle and glutamic acid synthesis in a γ-PGA production strain Bacillus licheniformis WX-02. Firstly, pyruvate dehydrogenase (PdhABCD) and citrate synthase (CitA) were overexpressed to strengthen the flux of pyruvate into TCA cycle, resulting in 34.93% and 11.14% increase of γ-PGA yield in B. licheniformis WX-02, respectively. Secondly, the carbon flux to glyoxylate shunt was rewired via varying the expression of isocitrate lyase (AceA), and a 23.24% increase of γ-PGA yield was obtained in AceA down-regulated strain WXPbacAaceBA. Thirdly, deletion of pyruvate formate-lyase gene pflB led to a 30.70% increase of γ-PGA yield. Finally, combinatorial metabolic engineering was applied, and γ-PGA titer was enhanced to 12.02 g/L via overexpressing pdhABCD and citA, repressing aceA, and deleting pflB, with a 69.30% improvement compared to WX-02. Collectively, metabolic engineering of central carbon metabolism is an effective strategy for enhanced γ-PGA production in B. licheniformis, and this research provided a promising strain for industrial production of γ-PGA.

The accumulation of carbon dioxide in the atmosphere as a result of human activities has caused a number of adverse circumstances in the world. For this reason, the proposed solutions lie within the aim of reducing carbon dioxide emissions have been quite valuable. However, as the human activity continues to increase on this planet, the possibility of reducing carbon dioxide emissions decreases with the use of conventional methods. The emergence of compounds than can be used in different fields by converting the released carbon dioxide into different chemicals will construct a fundamental solution to the problem. Although electro-catalysis or photolithography methods have emerged for this purpose, they have not been able to achieve successful results. Alternatively, another proposed solution are enzyme based systems. Among the enzyme-based systems, pyruvate decarboxylase, carbonic anhydrase and dehydrogenases have been the most studied enzymes. Pyruvate dehydrogenase and carbonic anhydrase have either been an expensive method or were incapable of producing the desired result due to the reaction cascade they catalyze. However, the studies reporting the production of industrial chemicals from carbon dioxide using dehydrogenases and in particular, the formate dehydrogenase enzyme, have been remarkable. Moreover, reported studies have shown the existence of more active and stable enzymes, especially the dehydrogenase family that can be identified from the biome. In addition to this, their redesign through protein engineering can have an immense contribution to the increased use of enzyme-based methods in CO2 reduction, resulting in an enormous expansion of the industrial capacity.

The metabolism in tumors is reprogrammed to meet its energetic and substrate demands. However, this metabolic reprogramming creates metabolic vulnerabilities, providing new opportunities for cancer therapy. Metabolic vulnerability as a therapeutic target in esophageal squamous cell carcinoma (ESCC) has not been adequately clarified. Here, we identified pyruvate dehydrogenase (PDH) component X (PDHX) as a metabolically essential gene for the cell growth of ESCC. PDHX expression was required for the maintenance of PDH activity and the production of ATP, and its knockdown inhibited the proliferation of cancer stem cells (CSCs) and in vivo tumor growth. PDHX was concurrently upregulated with the CD44 gene, a marker of CSCs, by co‐amplification at 11p13 in ESCC tumors and these genes coordinately functioned in cancer stemness. Furthermore, CPI‐613, a PDH inhibitor, inhibited the proliferation of CSCs in vitro and the growth of ESCC xenograft tumors in vivo. Thus, our study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex‐associated metabolic vulnerability., Inoue et al. found that pyruvate dehydrogenase (PDH) component X expression is necessary for PDH activity and is involved in the cancer stemness of esophageal squamous cell carcinoma (ESCC) cells, thereby being metabolically essential for the tumor growth of ESCC. This study provides new insights related to the development of novel therapeutic strategies for ESCC by targeting the PDH complex‐associated metabolic vulnerability.

Evidence has validated the prophylactic effects of exercising on different aspects of health. On the opposite side, immobilization may lead to various destructive effects causing neurodegeneration. Here, we investigated the association between exercising and mitochondrial quality for preventing the destructive effects of restraint stress in different rat brain regions. Twenty-four male Wistar rats, were randomized into four groups (n = 6), exercise, stress, exercise + stress, and control. The exercise procedure consisted of running on a rodent treadmill for 8 weeks, and rats in the stress group were immobilized for 6 h. Rats were then euthanized by decapitation and tricarboxylic acid (TCA) cycle enzyme activity, antioxidant levels, and mitochondrial biogenesis factors were assessed in the frontal, hippocampus, parietal and temporal regions using spectrophotometer and western blot technique. Based on our results, increased activity of TCA cycle enzymes in the exercised and exercise-stressed groups was detected, except for malate dehydrogenase which was decreased in exercise-stressed group, and fumarase that did not change. Furthermore, the level of antioxidant agents (superoxide dismutase and reduced glutathione), mitochondrial biogenesis factors (peroxisome proliferator-activated receptor gamma coactivator 1-alpha and mitochondrial transcription factor A), and dynamics markers (Mitofusin 2, dynamic related protein 1, PTEN induced putative kinase-1, and parkin) increased in both mentioned groups. Interestingly our results also revealed that the majority of the mitochondrial factors increased in the frontal and parietal lobes, which may be in relation with the location of motor and sensory areas. Exercise can be used as a prophylactic approach against bioenergetics and mitochondrial dysfunction.

Graphical abstract, This study evaluated the effect of Crataegus aronia (C. aronia) aqueous extract on cardiac substrate utilization and insulin signaling in adult male healthy Wistar rats. Rats (n = 18/group) were either administered normal saline (vehicle) or treated with C. aronia aqueous extract (200 mg/kg) for 7 days, daily. Fasting plasma glucose and insulin levels were not significantly changed in C. aronia-treated rats but were significantly reduced after both the intraperitoneal glucose or insulin tolerance tests. Besides, C. aronia significantly increased the left ventricular (LV) activities of phosphofructokinase (PFK) and pyruvate dehydrogenase (PDH), two markers of glycolysis and glucose oxidation, respectively, and suppressed the levels of pyruvate dehydrogenase kinase 4 (PDK4), an inhibitor of PDH. Concomitantly, it significantly reduced the LV levels of carnitine palmitoyltransferase 1 (CPT1) and PPARα, two markers of fatty acid (FAs) oxidations. Under basal and insulin stimulation, C. aronia aqueous extract boosted insulin signaling in the LV of rats by increasing the protein levels of p-IRS (Tyr612) and p-Akt (Ser473) and suppressing protein levels of p-mTOR (Ser 2448) and p-IRS (Ser307). In parallel, C. aronia also increased the protein levels of GLUT-4 in the membrane fraction of the treated LVs. All these effects were also associated with a significant increase in AMPK activity (phosphorylation at Thr172), a major energy modulator that stimulates glucose utilization. In conclusion, short-term administration of C. aronia aqueous extract shifts the cardiac metabolism toward glucose utilization, thus making this plant a potential therapeutic medication in cardiac disorders with impaired metabolism.

Metabolic and epigenetic reprogramming are two key hallmarks of cancer. Rapidly proliferating cancer cells often exhibit a higher and differentially reprogrammed transcriptional activity than normal cells of the same tissue. To support transcriptional reprogramming and increased transcriptional load, sustained chromatin acetylation is required to loosen the chromatin and enable RNA transcription. Histone acetylation also requires a constant supply of acetyl-CoA, a labile, membrane impermeable metabolite that is the obligatory donor of the acetyl groups for histone acetylation. However, the pathways responsible for increased synthesis of acetyl-CoA inside the nucleus remain largely elusive. Using estrogen-responsive (MCF7) and their matched endocrine therapy resistant breast cancer cells (LCC9 and MCF7:5C), we show that blocking p300-mediated acetyltransferase activity is crucial for proliferation of endocrine therapy-resistant breast cancer cells. Furthermore, we found nuclear expression of two acetyl-CoA producing enzymes, i.e., pyruvate dehydrogenase complex (PDC), and ATP citrate lyase (ACLY), which implies their functional role inside the nucleus. PDC is a multimeric protein complex enzyme made up of five different subunits. Canonically, PDC is found in mitochondria and is responsible for the conversion of pyruvate to acetyl-CoA, linking glycolysis to the tricarboxylic acid (TCA) cycle. Our results show that multiple PDC enzyme subunits, e.g., pyruvate dehydrogenase (PDH) E1 alpha (E1 alpha), dihydrolipoal dehydrogenase (DLD), and PDHX (also known as E3BP) are expressed in the nucleus of MCF7, LCC9 and MCF7:5C cells. Moreover, expression of nuclear DLD and PDHX subunit is higher in endocrine resistant cells (LCC9 and MCF7:5C) than in their parental MCF7 cells, strongly suggesting a possible role in endocrine resistance. Notably, high expression of PDHX is correlated with poor relapse-free survival in estrogen-receptor positive breast cancer patients in four independent publicly available datasets. ACLY is a tetramer of identical subunits catalyzes conversion of citric acid and co-enzyme A to acetyl-CoA. ACLY is also overexpressed in the nucleus of endocrine-resistant breast cancer cells (LCC9 and MCF7:5C) when compared with their endocrine sensitive MCF7 controls. This study investigates the role of nuclear PDC and ACLY mediated acetyl-CoA synthesis and its impact on histone acetylation enabling reprogrammed transcriptional activity supporting proliferation of endocrine therapy-resistant breast cancer cells. Citation Format: Surojeet Sengupta, Shuait Nair, Lu Jin, Catherine M Sevigny, Brandon Jones, Robert Clarke. Nuclear expression of acetyl-CoA producing enzymes and their roles in epigenetic reprogramming in breast cancer cells [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-50.

Fermentation conditions affect pectinase production. Among these conditions, fermentation period is one of the important parameters to be optimized for saving the process costs. However, information regarding whether metabolic regulation plays a role is not available. Here, a Bacillus licheniformis strain DY2 was incubated in submerged fermentation for pectinase production for 48 h. The pectinase activity was assayed every 4 h from 8th h of fermentation. The pectinase activity was low at 8 h, middle at 28 h, and peak at 44 h. Consistently, metabolomics analysis showed that the metabolic shift is related to the fermentation period. The metabolomes exhibited almost of differential metabolites in the enriched metabolic pathways, which were decreased at 28 h and 44 h compared to 8 h in a time-dependent manner. Furthermore, reduced palmitic acid and stearic acid in a time-dependent manner were identified as crucial biomarkers at 48 h. These findings in our work reveal that the reduced fatty acid biosynthesis is required for pectinase yield, which was further demonstrated by addition of inhibitors furfural and triclosan to inhibit pyruvate dehydrogenase and fatty acid biosynthesis, respectively. Our work reveals a potential possibility to elevate pectinase yield through metabolomics modulation.

Pt(IV) prodrugs have gained tremendous attention due to their indisputable advantages compared to cisplatin. Herein, new Pt(IV) derivatives with cinnamic acid at the first axial position, and inhibitor of matrix metalloproteinases-2 and -9, histone deacetylase, cyclooxygenase or pyruvate dehydrogenase at the second axial position are constructed to develop multi-action prodrugs. We demonstrate that Pt(IV) prodrugs are reducible and have superior antiproliferative activity with IC50 values at submicromolar concentrations. Notably, Pt(IV) prodrugs exhibit highly potent anti-tumour activity in an in vivo breast cancer model. Our results support the view that a triple-action Pt(IV) prodrug acts via a synergistic mechanism, which involves the effects of CDDP and the effects of axial moieties, thus jointly leading to the death of tumour cells. These findings provide a practical strategy for the rational design of more effective Pt(IV) prodrugs to efficiently kill tumour cells by enhancing their cellular accumulation and tuning their canonical mechanism.

Objectives Early-life stress (ELS) increases the risk of metabolic disorders in later life. The present study investigated the ELS effect on pancreatic pyruvate dehydrogenase (PDH) protein level, α-ketoglutarate dehydrogenase (α-KGDH), and aconitase activities as metabolic enzymes in response to young adulthood stress in male rat offspring. Methods Male Wistar rats were divided into six groups: Control, early life stress (Early STR), young adult foot-shock stress (Y. adult F-SH STR), early + young adult foot-shock stress (Early + Y. adult F-SH STR), young adult psychological stress (Y. adult Psy STR) and early + young adult psychological stress (Early + Y. adult Psy STR). Stress was induced by a communication box at 2 weeks of age and young adulthood for five consecutive days. The blood samples were collected in young adult rats, then pancreases were removed to measure its PDH protein level and aconitase and α-KGDH activities. Results In ELS animals, applying foot-shock stress in young adulthood increased PDH protein level, decreased α-KGDH and aconitase activities, and increased plasma glucose, insulin, and corticosterone concentrations. However, exposure to young adulthood psychological stress only decreased α-KGDH and aconitase activities. Conclusions It seems that ELS altered metabolic response to young adulthood stress through changes of Krebs cycle-related enzymes activities, though the type of adulthood stress was determinant.

Made available in DSpace on 2021-06-25T10:46:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The aim of this study is to evaluate whether the alterations in glucose metabolism and insulin resistance are mechanisms presented in cardiac remodelling induced by the toxicity of cigarette smoke. Male Wistar rats were assigned to the control group (C; n = 12) and the cigarette smoke-exposed group (exposed to cigarette smoke over 2 months) (CS; n = 12). Transthoracic echocardiography, blood pressure assessment, serum biochemical analyses for catecholamines and cotinine, energy metabolism enzymes activities assay; HOMA index (homeostatic model assessment); immunohistochemistry; and Western blot for proteins involved in energy metabolism were performed. The CS group presented concentric hypertrophy, systolic and diastolic dysfunction, and higher oxidative stress. It was observed changes in energy metabolism, characterized by a higher HOMA index, lower concentration of GLUT4 (glucose transporter 4) and lower 3-hydroxyl-CoA dehydrogenase activity, suggesting the presence of insulin resistance. Yet, the cardiac glycogen was depleted, phosphofructokinase (PFK) and lactate dehydrogenase (LDH) increased, with normal pyruvate dehydrogenase (PDH) activity. The activity of citrate synthase, mitochondrial complexes and ATP synthase (adenosine triphosphate synthase) decreased and the expression of Sirtuin 1 (SIRT1) increased. In conclusion, exposure to cigarette smoke induces cardiac remodelling and dysfunction. The mitochondrial dysfunction and heart damage induced by cigarette smoke exposure are associated with insulin resistance and glucose metabolism changes. Department of Internal Medicine Botucatu Medical School São Paulo State University-UNESP Faculty of Nutrition UFG – Univ Federal de Goiás Department of Chemical and Biological Sciences São Paulo State University-UNESP Department of Pathology and Legal Medicine Ribeirão Preto Medical School University of São Paulo Experimental Research Unit – UNIPEX Botucatu Medical School São Paulo State University-UNESP Department of Internal Medicine Botucatu Medical School São Paulo State University-UNESP Department of Chemical and Biological Sciences São Paulo State University-UNESP Experimental Research Unit – UNIPEX Botucatu Medical School São Paulo State University-UNESP CAPES: 001 FAPESP: 2014/24197-0 FAPESP: 2016/14547-0 FAPESP: 2016/25676-5

The purpose of this study was to determine whether intramyocellular glucose partitioning was altered in primary human myotubes derived from severely obese women with type 2 diabetes. Human skeletal muscle cells were obtained from lean nondiabetic and severely obese Caucasian females with type 2 diabetes [body mass index (BMI): 23.6 ± 2.6 vs. 48.8 ± 1.9 kg/m2, fasting glucose: 86.9 ± 1.6 vs. 135.6 ± 12.0 mg/dL, n = 9/group]. 1-[14C]-Glucose metabolism (glycogen synthesis, glucose oxidation, and nonoxidized glycolysis) and 1- and 2-[14C]-pyruvate oxidation were examined in fully differentiated myotubes under basal and insulin-stimulated conditions. Tricarboxylic acid cycle intermediates were determined via targeted metabolomics. Myotubes derived from severely obese individuals with type 2 diabetes exhibited impaired insulin-mediated glucose partitioning with reduced rates of glycogen synthesis and glucose oxidation and increased rates of nonoxidized glycolytic products, when compared with myotubes derived from the nondiabetic individuals ( P < 0.05). Both 1- and 2-[14C]-pyruvate oxidation rates were significantly blunted in myotubes from severely obese women with type 2 diabetes compared with myotubes from the nondiabetic controls. Lastly, concentrations of tricarboxylic acid cycle intermediates, namely, citrate ( P < 0.05), cis-aconitic acid ( P = 0.07), and α-ketoglutarate ( P < 0.05), were lower in myotubes from severely obese women with type 2 diabetes. These data suggest that intramyocellular insulin-mediated glucose partitioning is intrinsically altered in the skeletal muscle of severely obese women with type 2 diabetes in a manner that favors the production of glycolytic end products. Defects in pyruvate dehydrogenase and tricarboxylic acid cycle may be responsible for this metabolic derangement associated with type 2 diabetes.

The objective of the present investigation was to understand the impact of exogenously applied melatonin on mitochondrial respiration and sugar metabolism in two contrasting rice cultivars, viz., Khitish (arsenic-susceptible) and Muktashri (arsenic-tolerant) under arsenic-stress. Melatonin effectively restored the level of organic acids like pyruvic acid, malic acid and more particularly citric acid by 33 % in Khitish which were lowered during arsenic-stress, whereas their levels were further elevated in Muktashri to provide energy for defence against arsenic-induced injury. Arsenic-exposure led to a significant inhibition in enzyme activities as well as corresponding transcript level of key respiratory enzymes, viz., pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, intriguingly more prominently in case of Khitish. Conversely, melatonin supplementation, irrespective of cultivars, considerably improved the activity of the above enzymes and corresponding gene expressions during stress, indicating acceleration in the rate of Krebs cycle. Melatonin supplementation also stimulated the accumulation of total soluble sugars by 62 % and 25 %, reducing sugars by 50 % and 44 % and non-reducing sugars by 75 % and 14 % in Khitish and Muktashri respectively, concomitant with higher activities of acid invertase, sucrose synthase and sucrose phosphate synthase enzymes, along with the expression of corresponding genes. Enhanced starch accumulation via regulation of alpha amylase and starch phosphorylase activities and gene expression, by melatonin also contributed towards better stress tolerance. Overall, this work illustrated the efficacy of melatonin in the regulation of representative organic acids and enzymes of respiratory cycle along with starch and sugar metabolism in rice cultivars under arsenic toxicity.

Diabetic peripheral neuropathy (DPN) is a common complication of uncontrolled diabetes. The pathogenesis of DPN is associated with chronic inflammation in dorsal root ganglion (DRG), eventually causing structural and functional changes. Studies on DPN have primarily focused on neuronal component, and there is limited knowledge about the role of satellite glial cells (SGCs), although they completely enclose neuronal soma in DRG. Lipocalin-2 (LCN2) is a pro-inflammatory acute-phase protein found in high levels in diverse neuroinflammatory and metabolic disorders. In diabetic DRG, the expression of LCN2 was increased exclusively in the SGCs. This upregulation of LCN2 in SGCs correlated with increased inflammatory responses in DRG and sciatic nerve. Furthermore, diabetes-induced inflammation and morphological changes in DRG, as well as sciatic nerve, were attenuated in Lcn2 knockout (KO) mice. Lcn2 gene ablation also ameliorated neuropathy phenotype as determined by nerve conduction velocity and intraepidermal nerve fiber density. Mechanistically, studies using specific gene KO mice, adenovirus-mediated gene overexpression strategy, and primary cultures of DRG SGCs and neurons have demonstrated that LCN2 enhances the expression of mitochondrial gate-keeping regulator pyruvate dehydrogenase kinase-2 (PDK2) through PPARβ/δ, thereby inhibiting pyruvate dehydrogenase activity and increasing production of glycolytic end product lactic acid in DRG SGCs and neurons of diabetic mice. Collectively, our findings reveal a crucial role of glial LCN2-PPARβ/δ-PDK2-lactic acid axis in progression of DPN. Our results establish a link between pro-inflammatory LCN2 and glycolytic PDK2 in DRG SGCs and neurons and propose a novel glia-based mechanism and drug target for therapy of DPN. MAIN POINTS: Diabetes upregulates LCN2 in satellite glia, which in turn increases pyruvate dehydrogenase kinase-2 (PDK2) expression and lactic acid production in dorsal root ganglia (DRG). Glial LCN2-PDK2-lactic acid axis in DRG plays a crucial role in the pathogenesis of diabetic neuropathy.

Background The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible decarboxylation of pyruvate into acetyl-CoA. PDC deficiency can be caused by alterations in any of the genes encoding its several subunits. The resulting phenotype, though very heterogeneous, mainly affects the central nervous system. The aim of this study is to describe and discuss the clinical, biochemical and genotypic information from thirteen PDC deficient patients, thus seeking to establish possible genotype–phenotype correlations. Results The mutational spectrum showed that seven patients carry mutations in the PDHA1 gene encoding the E1α subunit, five patients carry mutations in the PDHX gene encoding the E3 binding protein, and the remaining patient carries mutations in the DLD gene encoding the E3 subunit. These data corroborate earlier reports describing PDHA1 mutations as the predominant cause of PDC deficiency but also reveal a notable prevalence of PDHX mutations among Portuguese patients, most of them carrying what seems to be a private mutation (p.R284X). The biochemical analyses revealed high lactate and pyruvate plasma levels whereas the lactate/pyruvate ratio was below 16; enzymatic activities, when compared to control values, indicated to be independent from the genotype and ranged from 8.5% to 30%, the latter being considered a cut-off value for primary PDC deficiency. Concerning the clinical features, all patients displayed psychomotor retardation/developmental delay, the severity of which seems to correlate with the type and localization of the mutation carried by the patient. The therapeutic options essentially include the administration of a ketogenic diet and supplementation with thiamine, although arginine aspartate intake revealed to be beneficial in some patients. Moreover, in silico analysis of the missense mutations present in this PDC deficient population allowed to envisage the molecular mechanism underlying these pathogenic variants. Conclusion The identification of the disease-causing mutations, together with the functional and structural characterization of the mutant protein variants, allow to obtain an insight on the severity of the clinical phenotype and the selection of the most appropriate therapy.

Objective Monoamine oxidase A (MAO-A) is a mitochondrial protein involved in tumourigenesis in different types of cancer. However, the biological function of MAO-A in gastric cancer development remains unknown. Methods We examined MAO-A expression in gastric cancer tissues and in gastric cancer cell lines by immunohistochemistry and Western blot analyses. CCK8, FACS and bromodeoxyuridine incorporation assays were performed to assess the effects of MAO-A on gastric cancer cell proliferation. The role of MAO-A in mitochondrial function was determined through MitoSOX Red staining, ATP generation and glycolysis assays. Results In the present study, we observed that MAO-A was significantly upregulated in gastric cancer tissues and in AGS and MGC803 cells. The observed MAO-A inhibition indicated decreased cell cycle progression and proliferation. Silencing MAO-A expression was associated with suppressed migration and invasion of gastric cancer cells in vitro. Moreover, alleviated mitochondrial damage in these cells was demonstrated by decreased levels of mitochondrial reactive oxygen species and increased ATP generation. MAO-A knockdown also regulated the expression of the glycolysis rate-limiting enzymes hexokinase 2 and pyruvate dehydrogenase. Finally, we observed that the glycolysis-mediated effect was weakened in AGS and MGC803 cells when MAO-A was blocked. Conclusion The findings of the present study indicate that MAO-A is responsible for mitochondrial dysfunction and aerobic glycolysis, which in turn leads to the proliferation and metastasis of human gastric tumour cells.

The objective of the article was to determine the variability of meningococcal house-keeping gene alleles circulating in Belarus. House-keeping genes sequencing was made by Sanger (ABI 3500). The phylogenetic analysis was done in MEGA X. SNPs were analyzed at pubMLST.org. 60 Belarusian meningococci, collected during 8 years, contain 17 alleles of abcZ gene (5.9 % first identified in Belarus – abcZ 1016) encoding 5 variants of the ABC transporter; 16 adk gene alleles – 2 variants of adenylate cyclase; 17 alleles of aroE gene (11.8 % Belarusian – aroE 944 and aroE 972) – 14 variants of shikimat dehydrogenase; 24 alleles of fumC gene (4.2 % Belarusian – fumC 988) – 4 variants of fumarate dehydratase; 18 alleles of gdh gene (16.7 % first identified in Belarus – gdh 560, gdh 985 and gdh 1083) – 4 variants of glucose-6-phosphate dehydrogenase; 18 alleles of pdhC gene – 11 variants of pyruvate dehydrogenase subunit and 20 alleles of pgm gene – 13 variants of phosphoglucomutase (5.6 and 5 % of Belarusian alleles − pdhC 888 and pgm 1099 respectively). Dominant alleles are abcZ 8 – 25 %, adk 5 – 30, aroE 6 – 28.3, fumC 17 – 30, gdh 560 – 20, pdhC 18 – 21.7, pgm 2 – 25 %. The Belarusian meningococcal population is diverse and includes both its own house-keeping gene alleles (7.7 %) and those circulating in other countries (92.3 %). The number of SNPs is varied from 29 (adk) to 125 (aroE). Single nucleotide polymorphisms are mostly synonymous and, on average, lead to amino acid substitutions in the range from 0.6 % in adenylate cyclase and up to 26.4 % in shikimat dehydrogenase.

Termites are generally deficient in pyruvate dehydrogenase (PDH), which links glycolysis to the Krebs cycle; however, one termite species, Coptotermes formosanus, has some PDH activity, though it is not enough to maintain the respiration of the termite by itself. We obtained a high quality annotated draft genome of C. formosanus. We found that all genes constituting the PDH complex and controlling PDH activity are present in the genome of C. formosanus, except for the PDH protein X component that is essential for a functional PDH complex. Additionally, we found that C. formosanus has three endo-s-1,4-glucanases (EGs), for which the amino acid sequences differ from those of previously reported EGs. Despite the ability of termites to convert cellulose to glucose and the resulting glucose to pyruvate, PDH is likely to function poorly due to a missing X component of the PDH complex.

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.

The Warburg effect is a unique metabolic feature of the majority of tumor cells and is closely related to chemotherapeutic resistance. Pyruvate dehydrogenase kinase 1 (PDK1) is considered a 'switch' that controls the fate of pyruvate in glucose metabolism. However, to date, to the best of our knowledge, there are only a few studies to available which had studied the reduction of chemotherapeutic resistance via the metabolic reprogramming of tumor cells with PDK1 as a target. In the present study, it was found dicoumarol (DIC) reduced the phosphorylation of pyruvate dehydrogenase (PDH) by inhibiting the activity of PDK1, which converted the metabolism of human hepatocellular carcinoma (HCC) cells to oxidative phosphorylation, leading to an increase in mitochondrial reactive oxygen species ROS (mtROS) and a decrease in mitochondrial membrane potential (MMP), thereby increasing the apoptosis induced by oxaliplatin (OXA). Furthermore, the present study elucidated that the targeting of PDK1 may be a potential strategy for targeting metabolism in the chemotherapy of HCC. In addition, DIC as an 'old drug' exhibits novel efficacy, bringing new hope for antitumor therapy.

Protein interactions in mitochondria isolated from S. cerevisiae grown on glycerol or glucose medium were analyzed by XL-MS. The non-cleavable cross-linker BS3 proved suitable for elucidating differences in protein-protein interactions under both conditions. XL-MS analysis of interprotein interactions using stable-isotope-labeled BS3 revealed certain limitations in the quantitative application of cross-linking to quite different cellular states. The results from glycerol-grown mitochondria show that Ndi1 interacts directly with CIII in an ETC supercomplex and that Min8 promotes Cox12 assembly into CIV., Graphical Abstract Highlights XL-MS reveals new PPIs in yeast mitochondria under glycerol and glucose condition. Significant but limited results from quantitative XL-MS experiments. Ndi1 participates in a CIII2CIV2 respiratory supercomplex. Min8 promotes assembly of Cox12 into an intermediate complex IV., Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

To determine the role of pyruvate dehydrogenase complex (PDHC) in Klebsiella pneumoniae, the growth and metabolism of PDHC-deficient mutant in glycerol-based medium were analyzed and compared with those of other strains. Under aerobic conditions, the PDHC activity was fourfold higher than that of pyruvate formate lyase (PFL), and blocking of PDHC caused severe growth defect and pyruvate accumulation, indicating that the carbon flux through pyruvate to acetyl coenzyme A mainly depended on PDHC. Under anaerobic conditions, although the PDHC activity was only 50% of that of PFL, blocking of PDHC resulted in more growth defect than blocking of PFL. Subsequently, combined with the requirement of CO2 and intracellular redox status, it was presumed that the critical role of PDHC was to provide NADH for the anaerobic growth of K. pneumoniae. This presumption was confirmed in the PDHC-deficient mutant by further blocking one of the formate dehydrogenases, FdnGHI. Besides, based on our data, it can also be suggested that an improvement in the carbon flux in the PFL-deficient mutant could be an effective strategy to construct highyielding 1,3-propanediol-producing K. pneumoniae strain.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

Cachexia is a life-threatening complication of cancer traditionally characterized by weight loss and muscle dysfunction. Cachexia, however, is a systemic disease that also involves remodeling of nonmuscle organs. The liver exerts major control over systemic metabolism, yet its role in cancer cachexia is not well understood. To advance the understanding of how the liver contributes to cancer cachexia, we used quantitative proteomics and bioinformatics to identify hepatic pathways and cellular processes dysregulated in mice with moderate and severe colon-26 tumor-induced cachexia; ~300 differentially expressed proteins identified during the induction of moderate cachexia were also differentially regulated in the transition to severe cachexia. KEGG pathway enrichment revealed representation by oxidative phosphorylation, indicating altered hepatic mitochondrial function as a common feature across cachexia severity. Glycogen catabolism was also observed in cachexic livers along with decreased pyruvate dehydrogenase protein X component (Pdhx), increased lactate dehydrogenase A chain (Ldha), and increased lactate transporter Mct1. Together this suggests altered lactate metabolism and transport in cachexic livers, which may contribute to energetically inefficient interorgan lactate cycling. Acyl-CoA synthetase-1 (ACSL1), known for activating long-chain fatty acids, was decreased in moderate and severe cachexia based on LC-MS/MS analysis and immunoblotting. ACSL1 showed strong linear relationships with percent body weight change and muscle fiber size (R2 = 0.73–0.76, P < 0.01). Mitochondrial coupling efficiency, which is compromised in cachexic livers to potentially increase energy expenditure and weight loss, also showed a linear relationship with ACSL1. Findings suggest altered mitochondrial and substrate metabolism of the liver in cancer cachexia, and possible hepatic targets for intervention.

Toxoplasma gondii is a common protozoan parasite that infects a wide range of hosts, including livestock and humans. Previous studies have suggested that the type 2 fatty acid synthesis (FAS2) pathway, located in the apicoplast (a nonphotosynthetic plastid relict), is crucial for the parasite's survival. Here we examined the physiological relevance of fatty acid synthesis in T. gondii by focusing on the pyruvate dehydrogenase complex and malonyl-CoA-[acyl carrier protein] transacylase (FabD), which are located in the apicoplast to drive de novo fatty acid biosynthesis. Our results disclosed unexpected metabolic resilience of T. gondii tachyzoites, revealing that they can tolerate CRISPR/Cas9–assisted genetic deletions of three pyruvate dehydrogenase subunits or FabD. All mutants were fully viable in prolonged cultures, albeit with impaired growth and concurrent loss of the apicoplast. Even more surprisingly, these mutants displayed normal virulence in mice, suggesting an expendable role of the FAS2 pathway in vivo. Metabolic labeling of the Δpdh-e1α mutant showed reduced incorporation of glucose-derived carbon into fatty acids with medium chain lengths (C14:0 and C16:0), revealing that FAS2 activity was indeed compromised. Moreover, supplementation of exogenous C14:0 or C16:0 significantly reversed the growth defect in the Δpdh-e1α mutant, indicating salvage of these fatty acids. Together, these results demonstrate that the FAS2 pathway is dispensable during the lytic cycle of Toxoplasma because of its remarkable flexibility in acquiring fatty acids. Our findings question the long-held assumption that targeting this pathway has significant therapeutic potential for managing Toxoplasma infections.

Brominated azo dyes (BADs) have been identified as predominant indoor brominated pollutants in daycare dust; thus, their potential health risk to children is of concern. However, the toxicities of BADs remain elusive. In this study, the toxicokinetics of two predominant BADs, Disperse Blue 373 (DB373) and Disperse Violet 93 (DV93), and their suspect metabolite 2-bromo-4,6-dinitroaniline (BDNA) was investigated in embryos of zebrafish (Danio rerio). The bioconcentration factor of DV93 at 120 hpf is 6.2-fold lower than that of DB373. The nontarget analysis revealed distinct metabolism routes between DB373 and DV93 by reducing nitro groups to nitroso (DB373) or amine (DV93), despite their similar structures. NAD(P)H quinone oxidoreductase 1 (NQO1) and pyruvate dehydrogenase were predicted as the enzymes responsible for the reduction of DB373 and DV93 by correlating time courses of the metabolites and enzyme development. Further in vitro recombinant enzyme and in vivo inhibition results validated NQO1 as the enzyme specifically reducing DB373, but not DV93. Global proteome profiling revealed that the expression levels of proteins from the "apoptosis-induced DNA fragmentation" pathway were significantly upregulated by all three BADs, supporting the bioactivation of BADs to mutagenic aromatic amines. This study discovered the bioactivation of BADs via distinct eukaryotic enzymes, implying their potential health risks.

The pyruvate dehydrogenase complex (PDC) is a multi-enzyme complex of the mitochondria that provides a link between glycolysis and the Krebs cycle. PDC plays an essential role in producing acetyl-CoA from glucose and the regulation of fuel consumption. In general, PDC enzyme is regulated in two different ways, end-product inhibition and posttranslational modifications (more extensive phosphorylation and dephosphorylation subunit E1). Posttranslational modifications of this enzyme are regulated by various factors. Sirtuins are the class III of histone deacylatases that catalyze protein posttranslational modifications, including deacetylation, adenosine diphosphate ribosylation, and deacylation. Sirt3, Sirt4, and Sirt5 are mitochondrial sirtuins that control the posttranslational modifications of mitochondrial protein. Considering the comprehensive role of sirtuins in post-translational modifications and regulation of metabolic processes, the aim of this review is to explore the role of mitochondrial sirtuins in the regulation of the PDC. PDC deficiency is a common metabolic disorder that causes pyruvate to be converted to lactate and alanine rather than to acetyl-CoA. because this enzyme is in the gateway of complete oxidation, glucose products entering the Krebs cycle and resulting in physiological and structural changes in the organs. Metabolic blockage such as ketogenic diet broken up by b -oxidation and producing acetyl-CoA can improve the patients. Sirtuins play a role in the production of acetyl-CoA through oxidation of fatty acids and other pathways. Thus, we hypothesize that the targets and bioactive compounds targeting mitochondrial sirtuins can be involved in the treatment of PDC deficiency. In general, this review discusses the present knowledge on how mitochondrial sirtuins are involved in the regulation of PDC as well as their possible roles in the treatment of PDC deficiency.

Summary Designing synthetic pathways for efficient CO2 fixation and conversion is essential for sustainable chemical production. Here we have designed a synthetic acetate‐acetyl‐CoA/malonyl‐CoA (AAM) bypass to overcome an enzymatic activity of pyruvate dehydrogenase complex. This synthetic pathway utilizes acetate assimilation and carbon rearrangements using a methyl malonyl‐CoA carboxyltransferase. We demonstrated direct conversion of CO2 into acetyl‐CoA‐derived acetone as an example in photosynthetic Synechococcus elongatus PCC 7942 by increasing the acetyl‐CoA pools. The engineered cyanobacterial strain with the AAM‐bypass produced 0.41 g/L of acetone at 0.71 m/day of molar productivity. This work clearly shows that the synthetic pyruvate dehydrogenase bypass (AAM‐bypass) is a key factor for the high‐level production of an acetyl‐CoA‐derived chemical in photosynthetic organisms.

The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC’s implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA. Electronic supplementary material The online version of this article (10.1007/s00429-020-02026-5) contains supplementary material, which is available to authorized users.

Background: Mitochondrial dysfunction may involve in the progression of human non-small cell lung cancers (NSCLCs). We analyzed the mitochondrial DNA (mtDNA) copy number, the expression levels of mitochondrial biogenesis-related proteins including pyruvate dehydrogenase, mitochondrial transcription factor A (TFAM) and mtDNA-encoded peptide NADH dehydrogenase subunit 1 (ND1), and the expression level of hexokinase II (HK-II) in human NSCLCs both ex vivo and in vitro. Materials and Methods: Paired cancerous and non-cancerous pathological specimens from 20 resected NSCLCs and an NSCLC cell line, the H23, were used in this study. H23 was infected by lentiviral particles to knockdown (KD) the expression of TFAM. TFAM-Null and TFAM-KD represent the control and TFAM knocked-down H23 cells, respectively. The mtDNA copy number was measured by quantitative real-time polymerase chain reaction and the protein expression levels were measured by immunohistochemical staining and Western blotting, respectively. Results: Low TFAM expression (P = 0.066) and low mtDNA copy number of NSCLCs (P = 0.009) were poor prognostic variables in NSCLC patients. Advanced T4 NSCLCs had lower TFAM expression (P = 0.021), lower expression of mtDNA-encoded ND1 polypeptide (P = 0.049), and lower mtDNA copy number (P = 0.050) than did T1 or T2/T3 NSCLCs, respectively. TFAM-KD cells expressed lower levels of TFAM protein (P Conclusion: Mitochondrial dysfunction caused by lower levels of TFAM, mtDNA copy number, and mtDNA-encoded ND1 polypeptide may play an important role in the progression of NSCLCs.

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O(2) consumption rate (R(N)) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour R(N) measurements following leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids) identified phosphoenolpyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf R(N). Using metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling R(N). Many amino acids, as well as Glc analogs, were found to potently inhibit the R(N) stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro- and Ala-stimulated R(N) were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway. Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

The role of reactive oxygen species (ROS) in myeloid development is well established. However, its aberrant generation alters hematopoiesis. Thus, a comprehensive understanding of events controlling ROS homeostasis forms the central focus of this study. We show that, in homeostasis, myeloid-like blood progenitor cells of the Drosophila larvae, which reside in a specialized hematopoietic organ termed the lymph gland, use TCA to generate ROS. However, excessive ROS production leads to lymph gland growth retardation. Therefore, to moderate blood progenitor ROS, Drosophila larvae rely on olfaction and its downstream systemic GABA. GABA internalization and its breakdown into succinate by progenitor cells activates pyruvate dehydrogenase kinase (PDK), which controls inhibitory phosphorylation of pyruvate dehydrogenase (PDH). PDH is the rate-limiting enzyme that connects pyruvate to the TCA cycle and to oxidative phosphorylation. Thus, GABA metabolism via PDK activation maintains TCA activity and blood progenitor ROS homeostasis, and supports normal lymph gland growth. Consequently, animals that fail to smell also fail to sustain TCA activity and ROS homeostasis, which leads to lymph gland growth retardation. Overall, this study describes the requirement of animal odor-sensing and GABA in myeloid ROS regulation and hematopoietic growth control., Summary: Ablation of olfactory receptor neurons reveals that odor-sensing and GABA are involved in myeloid reactive oxygen species regulation and hematopoietic growth control.

Rice (Oryza sativa L.) is one of the major crops in the world and significant increase in grain yield is constant demand for breeders to meet the needs of a rapidly growing population. The size of grains is one of major components determining rice yield and a vital trait for domestication and breeding. To increase the grain size in rice, OsSPL16/qGW8 was mutagenized through CRISPR/Cas9, and proteomic analysis was performed to reveal variations triggered by mutations. More specifically, mutants were generated with two separate guide RNAs targeting recognition sites on opposite strands and genomic insertions and deletions were characterized. Mutations followed Mendelian inheritance and homozygous and heterozygous mutants lacking any T-DNA and off-target effects were screened. The mutant lines showed a significant increase in grain yield without any change in other agronomic traits in T0, T1, and T2 generations. Proteomic screening found a total of 44 differentially expressed proteins (DEPs), out of which 33 and 11 were up and downregulated, respectively. Most of the DEPs related to pyruvate kinase, pyruvate dehydrogenase, and cell division and proliferation were upregulated in the mutant plants. Pathway analysis revealed that DEPs were enriched in the biosynthesis of secondary metabolites, pyruvate metabolism, glycolysis/gluconeogenesis, carbon metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and citrate cycle. Gene Ontology (GO) analysis presented that most of the DEPs were involved in the pyruvate metabolic process and pyruvate dehydrogenase complex. Proteins related to pyruvate dehydrogenase E1 component subunit alpha-1 displayed higher interaction in the protein-protein interaction (PPI) network. Thus, the overall results revealed that CRISPR/Cas9-guided OsSPL16 mutations have the potential to boost the grain yield of rice. Additionally, global proteome analysis has broad applications for discovering molecular components and dynamic regulation underlying the targeted gene mutations.

Fusarium oxysporum is one of the most important pathogenic fungi with a broad range of plant and animal hosts. The first key step of its infection cycle is conidial germination, but there is limited information available on the molecular events supporting this process. We show here that germination is accompanied by a sharp decrease in expression of FoSir5, an ortholog of the human lysine deacetylase SIRT5. We observe that FoSir5 decrotonylates a subunit of the fungal pyruvate dehydrogenase complex (FoDLAT) at K148, resulting in inhibition of the activity of the complex in mitochondria. Moreover, FoSir5 decrotonylates histone H3K18, leading to a downregulation of transcripts encoding enzymes of aerobic respiration pathways. Thus, the activity of FoSir5 coordinates regulation in different organelles to steer metabolic flux through respiration. As ATP content is positively related to fungal germination, we propose that FoSir5 negatively modulates conidial germination in F. oxysporum through its metabolic impact. These findings provide insights into the multifaceted roles of decrotonylation, catalysed by FoSir5, that support conidial germination in F. oxysporum.

International audience; α-oxoacid dehydrogenase complexes are large, tripartite enzymatic machineries carrying out key reactions in central metabolism. Extremely conserved across the tree of life, they have been, so far, all considered to be structured around a high–molecular weight hollow core, consisting of up to 60 subunits of the acyltransferase component. We provide here evidence that Actinobacteria break the rule by possessing an acetyltranferase component reduced to its minimally active, trimeric unit, characterized by a unique C-terminal helix bearing an actinobacterial specific insertion that precludes larger protein oligomerization. This particular feature, together with the presence of an odhA gene coding for both the decarboxylase and the acyltransferase domains on the same polypetide, is spread over Actinobacteria and reflects the association of PDH and ODH into a single physical complex. Considering the central role of the pyruvate and 2-oxoglutarate nodes in central metabolism, our findings pave the way to both therapeutic and metabolic engineering applications.

Lipoic acid (LA) is an organic compound that plays a key role in cellular metabolism. It participates in a posttranslational modification (PTM) named lipoylation, an event that is highly conserved and that occurs in multimeric metabolic enzymes of very distinct microorganisms such as Plasmodium sp. and Staphylococcus aureus, including pyruvate dehydrogenase (PDH) and α-ketoglutarate dehydrogenase (KDH). In this mini review, we revisit the recent literature regarding LA metabolism in Plasmodium sp. and Staphylococcus aureus, by covering the lipoate ligase proteins in both microorganisms, the role of lipoate ligase proteins and insights for possible inhibitors of lipoate ligases.

18 Páginas.-- 12 Figuras.-- 2 Tablas, Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon metabolism in most bacteria and eukaryotes. LA is synthetized by the concerted activity of the octanoyltransferase (LIP2, EC 2.3.1.181) and lipoyl synthase (LIP1, EC 2.8.1.8) enzymes. In plants, pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase or glycine decarboxylase are essential complexes that need to be lipoylated. These lipoylated enzymes and complexes are located in the mitochondria, while PDH is also present in plastids where it provides acetyl-CoA for de novo fatty acid biosynthesis. As such, lipoylation of PDH could regulate fatty acid synthesis in both these organelles. In the present work, the sunflower LIP1 and LIP2 genes (HaLIP1m and HaLIP2m) were isolated sequenced, cloned, and characterized, evaluating their putative mitochondrial location. The expression of these genes was studied in different tissues and protein docking was modeled. The genes were also expressed in Escherichia coli and Arabidopsis thaliana, where their impact on fatty acid and glycerolipid composition was assessed. Lipidomic studies in Arabidopsis revealed lipid remodeling in lines overexpressing these enzymes and the involvement of both sunflower proteins in the phenotypes observed is discussed in the light of the results obtained., This work was financed by the PID2020-113134RB-I00/AEI/10.13039/501100011033 project granted by the Spanish State Research Agency within the State Programs for Research and Innovation Oriented to the Challenges of Society.

Oxidative metabolism is fine-tuned machinery that combines two tightly coupled fluxes of glucose and glutamine-derived carbons. Hypoxia interrupts the coordination between the metabolism of these two nutrients and leads to a decrease of the system efficacy and may eventually cause cell death. The subterranean blind mole rat, Spalax, is an underexplored, underground, hypoxia-tolerant mammalian group which spends its life under sharply fluctuating oxygen levels. Primary Spalax cells are an exceptional model to study the metabolic strategies that have evolved in mammals inhabiting low-oxygen niches. In this study we explored the metabolic frame of glutamine (Gln) homeostasis in Spalax skin cells under normoxic and hypoxic conditions and their impacts on the metabolism of rat cells. Targeted metabolomics employing liquid chromatography and mass spectrometry (LC-MS) was used to track the fate of heavy glutamine carbons (13C5 Gln) after 24 h under normoxia or hypoxia (1% O2). Our results indicated that large amounts of glutamine-originated carbons were detected as proline (Pro) and hydroxyproline (HPro) in normoxic Spalax cells with a further increase under hypoxia, suggesting a strategy for reduced Gln carbons storage in proteins. The intensity of the flux and the presence of HPro suggests collagen as a candidate protein that is most abundant in animals, and as the primary source of HPro. An increased conversion of αKG to 2 HG that was indicated in hypoxic Spalax cells prevents the degradation of hypoxia-inducible factor 1α (HIF-1α) and, consequently, maintains cytosolic and mitochondrial carbons fluxes that were uncoupled via inhibition of the pyruvate dehydrogenase complex. A strong antioxidant defense in Spalax cells can be attributed, at least in part, to the massive usage of glutamine-derived glutamate for glutathione (GSH) production. The present study uncovers additional strategies that have evolved in this unique mammal to support its hypoxia tolerance, and probably contribute to its cancer resistance, longevity, and healthy aging.

Proximal tubular epithelial cells (TECs) demand high energy and rely on mitochondrial oxidative phosphorylation as the main energy source. However, this is disturbed in renal fibrosis. Acetylation is an important post-translational modification for mitochondrial metabolism. The mitochondrial protein NAD+-dependent deacetylase sirtuin 3 (SIRT3) regulates mitochondrial metabolic function. Therefore, we aimed to identify the changes in the acetylome in tubules from fibrotic kidneys and determine their association with mitochondria. We found that decreased SIRT3 expression was accompanied by increased acetylation in mitochondria that have separated from TECs during the early phase of renal fibrosis. Sirt3 knockout mice were susceptible to hyper-acetylated mitochondrial proteins and to severe renal fibrosis. The activation of SIRT3 by honokiol ameliorated acetylation and prevented renal fibrosis. Analysis of the acetylome in separated tubules using LC–MS/MS showed that most kidney proteins were hyper-acetylated after unilateral ureteral obstruction. The increased acetylated proteins with 26.76% were mitochondrial proteins which were mapped to a broad range of mitochondrial pathways including fatty acid β-oxidation, the tricarboxylic acid cycle (TCA cycle), and oxidative phosphorylation. Pyruvate dehydrogenase E1α (PDHE1α), which is the primary link between glycolysis and the TCA cycle, was hyper-acetylated at lysine 385 in TECs after TGF-β1 stimulation and was regulated by SIRT3. Our findings showed that mitochondrial proteins involved in regulating energy metabolism were acetylated and targeted by SIRT3 in TECs. The deacetylation of PDHE1α by SIRT3 at lysine 385 plays a key role in metabolic reprogramming associated with renal fibrosis.

Anti-mitochondrial antibodies (AMA) are directed against the E2 subunits of the 2-oxo acid dehydrogenase complexes (PDC-E2) and are the typical biomarkers of primary biliary cholangitis (PBC), being present in 90-95% of patients, with increasing sensitivity at increasing titers. Albeit being highly specific for PBC diagnosis, AMA can be detected in less than 1% of healthy subjects, and thus the management subjects with no sign or symptom of liver disease is still a challenge and data concerning clinical risk of developing PBC in this subgroup of patients are controversial. Moreover, AMA can also be detected in patients affected by overlap syndrome, as well as hepatic diseases (i.e., NASH and viral hepatitis), while the association with autoimmune diseases, in particular Sjogren's syndrome, systemic sclerosis, and systemic lupus erythematosus, is well established. Furthermore, new associations are being identified with inflammatory myositis and heart disease. AMA are directed towards the pyruvate dehydrogenase multi enzyme complex (PDC-E2) subunit, which represents an epithelial specific autoantigen for PBC. This review focuses on the main characteristics of AMA, their association with autoimmune diseases and liver diseases.