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1.
J Biosci Bioeng ; 130(2): 149-158, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32414665

RESUMO

The hyperthermophilic archaeon Thermococcus kodakarensis can grow on pyruvate or maltooligosaccharides through H2 fermentation. H2 production levels of members of the Thermococcales are high, and studies to improve their production potential have been reported. Although H2 production is primary metabolism, here we aimed to partially uncouple cell growth and H2 production of T. kodakarensis. Additional A1-type ATPase genes were introduced into T. kodakarensis KU216 under the control of two promoters; the strong constitutive cell surface glycoprotein promoter, Pcsg, and the sugar-inducible fructose-1,6-bisphosphate aldolase promoter, Pfba. Whereas cells with the A1-type ATPase genes under the control of Pcsg displayed only trace levels of growth, cells with Pfba (strain KUA-PF) displayed growth sufficient for further analysis. Increased levels of A1-type ATPase protein were detected in KUA-PF cells grown on pyruvate or maltodextrin, when compared to the levels in the host strain KU216. The growth and H2 production levels of strain KUA-PF with pyruvate or maltodextrin as a carbon and electron source were analyzed and compared to those of the host strain KU216. Compared to a small decrease in total H2 production, significantly larger decreases in cell growth were observed, resulting in an increase in cell-specific H2 production. Quantification of the substrate also revealed that ATPase overexpression led to increased cell-specific pyruvate and maltodextrin consumptions. The results clearly indicate that ATPase production results in partial uncoupling of cell growth and H2 production in T. kodakarensis.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Regulação da Expressão Gênica em Archaea , Hidrogênio/metabolismo , Thermococcus/enzimologia , Thermococcus/genética , Carbono/metabolismo , Dosagem de Genes/fisiologia , Regulação da Expressão Gênica em Archaea/genética , Organismos Geneticamente Modificados/metabolismo , Polissacarídeos/metabolismo , Ácido Pirúvico/metabolismo
2.
Electron. j. biotechnol ; 44: 19-24, Mar. 2020. ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1087631

RESUMO

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.


Assuntos
Ácido Pirúvico/metabolismo , Yarrowia/genética , Yarrowia/metabolismo , Pressão Osmótica , Leveduras , Carbono/metabolismo , Cloreto de Sódio , Reatores Biológicos , Tolerância ao Sal/genética , Fermentação , Glicerol/metabolismo , Mutação
3.
BMC Genomics ; 21(1): 145, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041545

RESUMO

BACKGROUND: The glyoxalase pathway is evolutionarily conserved and involved in the glutathione-dependent detoxification of methylglyoxal (MG), a cytotoxic by-product of glycolysis. It acts via two metallo-enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII), to convert MG into D-lactate, which is further metabolized to pyruvate by D-lactate dehydrogenases (D-LDH). Since D-lactate formation occurs solely by the action of glyoxalase enzymes, its metabolism may be considered as the ultimate step of MG detoxification. By maintaining steady state levels of MG and other reactive dicarbonyl compounds, the glyoxalase pathway serves as an important line of defence against glycation and oxidative stress in living organisms. Therefore, considering the general role of glyoxalases in stress adaptation and the ability of Sorghum bicolor to withstand prolonged drought, the sorghum glyoxalase pathway warrants an in-depth investigation with regard to the presence, regulation and distribution of glyoxalase and D-LDH genes. RESULT: Through this study, we have identified 15 GLYI and 6 GLYII genes in sorghum. In addition, 4 D-LDH genes were also identified, forming the first ever report on genome-wide identification of any plant D-LDH family. Our in silico analysis indicates homology of putatively active SbGLYI, SbGLYII and SbDLDH proteins to several functionally characterised glyoxalases and D-LDHs from Arabidopsis and rice. Further, these three gene families exhibit development and tissue-specific variations in their expression patterns. Importantly, we could predict the distribution of putatively active SbGLYI, SbGLYII and SbDLDH proteins in at least four different sub-cellular compartments namely, cytoplasm, chloroplast, nucleus and mitochondria. Most of the members of the sorghum glyoxalase and D-LDH gene families are indeed found to be highly stress responsive. CONCLUSION: This study emphasizes the role of glyoxalases as well as that of D-LDH in the complete detoxification of MG in sorghum. In particular, we propose that D-LDH which metabolizes the specific end product of glyoxalases pathway is essential for complete MG detoxification. By proposing a cellular model for detoxification of MG via glyoxalase pathway in sorghum, we suggest that different sub-cellular organelles are actively involved in MG metabolism in plants.


Assuntos
Lactato Desidrogenases/genética , Lactoilglutationa Liase/genética , Proteínas de Plantas/genética , Aldeído Pirúvico/metabolismo , Ácido Pirúvico/metabolismo , Sorghum/enzimologia , Tioléster Hidrolases/genética , Estudo de Associação Genômica Ampla , Lactato Desidrogenases/classificação , Lactoilglutationa Liase/classificação , Filogenia , Proteínas de Plantas/classificação , Sorghum/genética , Estresse Fisiológico/genética , Tioléster Hidrolases/classificação
4.
Invest Ophthalmol Vis Sci ; 61(2): 10, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32049346

RESUMO

Purpose: Aiming to clarify the metabolic interrogation in cell fate decision of cultured human corneal endothelial cells (cHCECs). Methods: To analyze the metabolites in the culture supernatants (CS), 34 metabolome measurements were carried out for mature differentiated and a variety of cHCECs with cell state transition through a facility service. Integrated proteomics research for cell lysates by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed for 3 aliquots of each high-quality or low-quality cHCEC subpopulations (SP). The investigations for the focused genes involved in cHCEC metabolism were performed by using DAVID and its options "KEGG_PATHWAY." Results: The clusters of metabolites coincided well with the distinct content of CD44-/+ SPs. Both secreted pyruvic acid and lactic acid in the CS were negatively correlated with the content of high-quality SPs. Lactic acid and pyruvic acid in the CS exhibited the positive correlation with that of Ile, Leu, and Ser, whereas the negative correlation was with glutamine. Platelet-derived growth factor-ßß in the CS negatively correlated with lactic acid in CS, indicating indirectly the positive correlation with the content of CD44-/+ SPs. Upregulated glycolytic enzymes and influx of glutamine to the tricarboxylic acid cycle may be linked with a metabolic rewiring converting oxidative metabolism in mature differentiated CD44-/+SPs into a glycolytic flux-dependent state in immature SPs with cell state transition. Conclusions: The findings suggest that the cell fate decision of cHCECs may be dictated at least partly through metabolic rewiring.


Assuntos
Células Endoteliais/metabolismo , Epitélio Posterior/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Epitélio Posterior/citologia , Feminino , Humanos , Ácido Láctico/metabolismo , Masculino , Metaboloma/fisiologia , Fosforilação/fisiologia , Proteoma/metabolismo , Ácido Pirúvico/metabolismo
5.
Nat Commun ; 11(1): 698, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019928

RESUMO

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.


Assuntos
Aconitato Hidratase/metabolismo , Macrófagos/enzimologia , Óxido Nítrico/metabolismo , Quinase Piruvato Desidrogenase (Transferência de Acetil)/metabolismo , Aconitato Hidratase/genética , Animais , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Quinase Piruvato Desidrogenase (Transferência de Acetil)/genética , Ácido Pirúvico/metabolismo
6.
Oncol Rep ; 43(2): 711-717, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894283

RESUMO

The aim of the present study was to investigate the metabolic and anticancer effects of troglitazone (TGZ) with a focus on the potential role of mitochondrial pyruvate utilization. 2­Deoxyglucose (2­DG) was more cytotoxic in CT26 cancer cells compared with T47D cells, despite a smaller suppression of glucose uptake. On the other hand, TGZ caused a more prominent shift to glycolytic metabolism and was more cytotoxic in T47D cells. Both effects of TGZ on T47D cells were dose­dependently reversed by addition of methyl pyruvate (mPyr), indicating suppression of mitochondrial pyruvate availability. Furthermore, UK5099, a specific mitochondrial pyruvate carrier inhibitor, closely simulated the metabolic and antitumor effects of TGZ and their reversal by mPyr. This was accompanied by a substantial reduction of activated p70S6K. In CT26 cells, UK5099 did not reduce activated p70S6K and only modestly decreased cell proliferation. In these cells, combining glutamine restriction with UK5099 further increased glucose uptake and completely suppressed cell proliferation. Thus, TGZ­mediated inhibition of mitochondrial pyruvate utilization is an effective treatment for cancer cells that are more dependent on mitochondrial glucose metabolism. By contrast, cancer cells that are more glycolysis­dependent may require suppression of glutamine utilization in addition to blocking mitochondrial pyruvate availability for a full antitumor effect.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Troglitazona/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Feminino , Glicólise/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
7.
Arch Microbiol ; 202(5): 1043-1048, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31974932

RESUMO

Halophilic bacteria are receiving increasing attention for industrial chemical production processes due to their unique properties. Herein, an alkaliphilic and halophilic bacterium was isolated from a commercial Spirulina culture at Nghe An province in Vietnam and found to secrete pyruvate. Pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals, and is employed for production of crop protection agents, polymers, cosmetics, and food additives. Phenotypic and chemotaxonomic characterization, and the 16S rRNA gene sequence homology with Halomonas hydrothermalis strain DSM 15,725 (99.2%) predicted that the strain belongs to the Halomonas genus, thus we named this strain as H. hydrothermalis strain C22. We investigated the biocharacteristics and capacity of strain C22 and determined the draft genome sequence comprising 3,934,166 bp with a G + C content of 60.2% encoding 3,668 proteins, 58 tRNAs, 9 rRNAs, and 1 tmRNA. Maximal pyruvate secretion reached 51.1 g/l after 84 h of cultivation. The results will facilitate future studies on the genetic and metabolic diversity of halophilic bacteria and expand our understanding of important bioprocesses in this microorganism.


Assuntos
DNA Bacteriano/genética , Genoma Bacteriano/genética , Halomonas/genética , Halomonas/isolamento & purificação , Ácido Pirúvico/metabolismo , Composição de Bases/genética , DNA Ribossômico/genética , Ácidos Graxos/química , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Spirulina/fisiologia , Vietnã
8.
Metabolomics ; 16(1): 10, 2020 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-31902059

RESUMO

INTRODUCTION: Myasthenia gravis (MG) and rheumatoid arthritis (RA) are examples of antibody-mediated chronic, progressive autoimmune diseases. Phenotypically dissimilar, MG and RA share common immunological features. However, the immunometabolomic features common to humoral autoimmune diseases remain largely unexplored. OBJECTIVES: The aim of this study was to reveal and illustrate the metabolomic profile overlap found between these two diseases and describe the immunometabolomic significance. METHODS: Metabolic analyses using acid- and dansyl-labelled was performed on serum from adult patients with seropositive MG (n = 46), RA (n = 23) and healthy controls (n = 49) presenting to the University of Alberta Hospital specialty clinics. Chemical isotope labelling liquid chromatography mass spectrometry (CIL LC-MS) methods were utilized to assess the serum metabolome in patients; 12C/13C-dansyl chloride (DnsCl) was used to label amine/phenol metabolites and 12C/13C-p-dimethylaminophenacyl bromide (DmPA) was used for carboxylic acids. Metabolites matching our criteria for significance were selected if they were present in both groups. Multivariate statistical analysis [including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA)] and biochemical pathway analysis was then conducted to gain understanding of the principal pathways involved in antibody-mediated pathogenesis. RESULTS: We found 20 metabolites dysregulated in both MG and RA when compared to healthy controls. Most prominently, observed changes were related to pathways associated with phenylalanine metabolism, tyrosine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and pyruvate metabolism. CONCLUSION: From these results it is evident that many metabolites are common to humoral disease and exhibit significant immunometabolomic properties. This observation may lead to an enhanced understanding of the metabolic underpinnings common to antibody-mediated autoimmune disease. Further, contextualizing these findings within a larger clinical and systems biology context could provide new insights into the pathogenesis and management of these diseases.


Assuntos
Artrite Reumatoide/metabolismo , Metaboloma , Miastenia Gravis/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/sangue , Feminino , Humanos , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Miastenia Gravis/sangue , Fenilalanina/metabolismo , Ácido Pirúvico/metabolismo , Tirosina/metabolismo , Ubiquinona/metabolismo
9.
Microb Cell Fact ; 19(1): 9, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931839

RESUMO

BACKGROUND: Aerobic growth provides benefits in biomass yield and stress tolerance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Catabolite control protein A (CcpA) is a master regulator involved in the aerobic and anaerobic growth, metabolic production and stress response in L. bulgaricus, but its potential molecular mechanisms remains unclear. The aim of this study is to elucidate the role of CcpA in L. bulgaricus in aerobic growth at the proteomic perspective. RESULTS: The differential proteomic analysis was performed on the L. bulgaricus ATCC11842 and its ccpA inactivated mutant strain using iTRAQ technology. A total of 132 differentially expressed proteins were obtained, among which 58 were up-regulated and 74 were down-regulated. These proteins were mainly involved in the cellular stress response, carbohydrate and energy metabolism, amino acid transport and protein synthesis, genetic information processing. Moreover, inactivation of ccpA negatively affected the expression of key enzymes involved in glycolysis pathway, while it enhanced the expression of proteins related to the pyruvate pathway, supporting the conclusion that CcpA mediated the shift from homolactic fermentation to mixed acid fermentation in L. bulgaricus. CONCLUSIONS: Overall, these results showed that the role of CcpA in L. bulgaricus as a pleiotropic regulator in aerobic metabolism and stress response. This proteomic analysis also provide new insights into the CcpA-mediated regulatory network of L. bulgaricus and potential strategies to improve the production of starter and probiotic cultures based on the metabolic engineering of global regulators.


Assuntos
Proteínas de Bactérias/metabolismo , Lactobacillus delbrueckii/genética , Proteoma , Aerobiose , Proteínas de Bactérias/genética , Deleção de Genes , Genes Bacterianos , Engenharia Metabólica/métodos , Probióticos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteômica , Ácido Pirúvico/metabolismo
10.
Nat Biotechnol ; 38(3): 309-313, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932725

RESUMO

An elevated intracellular NADH:NAD+ ratio, or 'reductive stress', has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain. As the intracellular NADH:NAD+ ratio can be in near equilibrium with the circulating lactate:pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate to pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of purified LOXCAT to the medium of cultured human cells with a defective electron transport chain decreased the extracellular lactate:pyruvate ratio, normalized the intracellular NADH:NAD+ ratio, upregulated glycolytic ATP production and restored cellular proliferation. In mice, tail-vein-injected LOXCAT lowered the circulating lactate:pyruvate ratio, blunted a metformin-induced rise in blood lactate:pyruvate ratio and improved NADH:NAD+ balance in the heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviates intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.


Assuntos
Bactérias/enzimologia , Catalase/metabolismo , Ácido Láctico/sangue , Oxigenases de Função Mista/metabolismo , Engenharia de Proteínas/métodos , Proteínas de Bactérias/metabolismo , Células HeLa , Humanos , Células K562 , NAD/metabolismo , Ácido Pirúvico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(4): 2092-2098, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31964840

RESUMO

Our purpose is to investigate the feasibility of imaging tumor metabolism in breast cancer patients using 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13C label exchange between injected [1-13C]pyruvate and the endogenous tumor lactate pool. Treatment-naïve breast cancer patients were recruited: four triple-negative grade 3 cancers; two invasive ductal carcinomas that were estrogen and progesterone receptor-positive (ER/PR+) and HER2/neu-negative (HER2-), one grade 2 and one grade 3; and one grade 2 ER/PR+ HER2- invasive lobular carcinoma (ILC). Dynamic 13C MRSI was performed following injection of hyperpolarized [1-13C]pyruvate. Expression of lactate dehydrogenase A (LDHA), which catalyzes 13C label exchange between pyruvate and lactate, hypoxia-inducible factor-1 (HIF1α), and the monocarboxylate transporters MCT1 and MCT4 were quantified using immunohistochemistry and RNA sequencing. We have demonstrated the feasibility and safety of hyperpolarized 13C MRI in early breast cancer. Both intertumoral and intratumoral heterogeneity of the hyperpolarized pyruvate and lactate signals were observed. The lactate-to-pyruvate signal ratio (LAC/PYR) ranged from 0.021 to 0.473 across the tumor subtypes (mean ± SD: 0.145 ± 0.164), and a lactate signal was observed in all of the grade 3 tumors. The LAC/PYR was significantly correlated with tumor volume (R = 0.903, P = 0.005) and MCT 1 (R = 0.85, P = 0.032) and HIF1α expression (R = 0.83, P = 0.043). Imaging of hyperpolarized [1-13C]pyruvate metabolism in breast cancer is feasible and demonstrated significant intertumoral and intratumoral metabolic heterogeneity, where lactate labeling correlated with MCT1 expression and hypoxia.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Imagem por Ressonância Magnética/instrumentação , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Simportadores/genética , Simportadores/metabolismo
12.
BMC Bioinformatics ; 21(1): 13, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924164

RESUMO

BACKGROUND: The rapid growth of available knowledge on metabolic processes across thousands of species continues to expand the possibilities of producing chemicals by combining pathways found in different species. Several computational search algorithms have been developed for automating the identification of possible heterologous pathways; however, these searches may return thousands of pathway results. Although the large number of results are in part due to the large number of possible compounds and reactions, a subset of core reaction modules is repeatedly observed in pathway results across multiple searches, suggesting that some subpaths between common compounds were more consistently explored than others.To reduce the resources spent on searching the same metabolic space, a new meta-algorithm for metabolic pathfinding, Hub Pathway search with Atom Tracking (HPAT), was developed to take advantage of a precomputed network of subpath modules. To investigate the efficacy of this method, we created a table describing a network of common hub metabolites and how they are biochemically connected and only offloaded searches to and from this hub network onto an interactive webserver capable of visualizing the resulting pathways. RESULTS: A test set of nineteen known pathways taken from literature and metabolic databases were used to evaluate if HPAT was capable of identifying known pathways. HPAT found the exact pathway for eleven of the nineteen test cases using a diverse set of precomputed subpaths, whereas a comparable pathfinding search algorithm that does not use precomputed subpaths found only seven of the nineteen test cases. The capability of HPAT to find novel pathways was demonstrated by its ability to identify novel 3-hydroxypropanoate (3-HP) synthesis pathways. As for pathway visualization, the new interactive pathway filters enable a reduction of the number of displayed pathways from hundreds down to less than ten pathways in several test cases, illustrating their utility in reducing the amount of presented information while retaining pathways of interest. CONCLUSIONS: This work presents the first step in incorporating a precomputed subpath network into metabolic pathfinding and demonstrates how this leads to a concise, interactive visualization of pathway results. The modular nature of metabolic pathways is exploited to facilitate efficient discovery of alternate pathways.


Assuntos
Algoritmos , Redes e Vias Metabólicas , Ácido Láctico/análogos & derivados , Ácido Láctico/química , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo
13.
Am J Physiol Endocrinol Metab ; 318(1): E72-E86, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31743040

RESUMO

The possibility to use leptin therapeutically for lowering glucose levels in patients with type 1 diabetes has attracted interest. However, earlier animal models of type 1 diabetes are severely catabolic with very low endogenous leptin levels, unlike most patients with diabetes. Here, we aim to test glucose-lowering effects of leptin in novel, more human-like murine models. We examined the glucose-lowering potential of leptin in diabetic models of two types: streptozotocin-treated mice and mice treated with the insulin receptor antagonist S961. To prevent hypoleptinemia, we used combinations of thermoneutral temperature and high-fat feeding. Leptin fully normalized hyperglycemia in standard chow-fed streptozotocin-treated diabetic mice. However, more humanized physiological conditions (high-fat diets or thermoneutral temperatures) that increased adiposity - and thus also leptin levels - in the diabetic mice abrogated the effects of leptin, i.e., the mice developed leptin resistance also in this respect. The glucose-lowering effect of leptin was not dependent on the presence of the uncoupling protein-1 and was not associated with alterations in plasma insulin, insulin-like growth factor 1, food intake or corticosterone but fully correlated with decreased plasma glucagon levels and gluconeogenesis. An important implication of these observations is that the therapeutic potential of leptin as an additional treatment in patients with type 1 diabetes is probably limited. This is because such patients are treated with insulin and do not display low leptin levels. Thus, the potential for a glucose-lowering effect of leptin would already have been attained with standard insulin therapy, and further effects on blood glucose level through additional leptin cannot be anticipated.


Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Leptina/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Glicemia/metabolismo , Corticosterona/metabolismo , Modelos Animais de Doenças , Ingestão de Alimentos , Glucagon/metabolismo , Gluconeogênese , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Leptina/farmacologia , Camundongos , Camundongos Knockout , Consumo de Oxigênio , Peptídeos/farmacologia , Ácido Pirúvico/metabolismo , Receptor de Insulina/antagonistas & inibidores , Transcriptoma , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
14.
J Appl Microbiol ; 128(2): 355-365, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31618501

RESUMO

AIM: This study was carried out to investigate the effects of dietary calcium pyruvate supplementation on growth performance and intestinal health of weaned piglets fed low-protein diets. METHODS AND RESULTS: After a 7-day adaptation period, 60 individually housed piglets (Duroc × Yorkshire-Landrace) weaned at 28 days of age were randomly assigned to receive one of three treatments (20 pigs/treatment) for 28 days: control diet (20·0% crude protein [CP]), low-protein diet (15·5% CP), and experimental (15·5% CP + 1·8% calcium pyruvate). At the end of the experiment, six piglets from each diet group were slaughtered and blood and tissue samples were collected. Compared with the control group, feeding piglets with 15·5% CP decreased the daily body weight gain; lengths of the duodenum, jejunum and ileum; and weights of the stomach, duodenum, jejunum and ileum (P < 0·05), while 15·5% CP + 1·8% calcium pyruvate supplementation removed those differences (P > 0·05). Compared with the control group, the diarrhoea incidence and relative richness of Firmicutes in the colon contents of piglets in both the 15·5% CP and 15·5% CP + 1·8% calcium pyruvate groups was decreased. The relative richness of Bacteriodetes in the colon contents of piglets was higher in the 15·5% CP + 1·8% calcium pyruvate group than in the control and 15·5% CP groups (P < 0·05). CONCLUSION: Calcium pyruvate supplementation for four weeks removed the negative effects of a low-protein diet on the gastrointestinal tract development and daily body weight gain of weaned piglets. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that supplementing a low-protein diet with calcium pyruvate, an effective alternative metabolic fuel to amino acids, was beneficial in improving the intestinal health and maximizing the growth of newly weaned piglets.


Assuntos
Cálcio na Dieta/metabolismo , Dieta com Restrição de Proteínas/veterinária , Trato Gastrointestinal/crescimento & desenvolvimento , Ácido Pirúvico/metabolismo , Suínos/crescimento & desenvolvimento , Aminoácidos/metabolismo , Ração Animal/análise , Animais , Suplementos Nutricionais , Feminino , Trato Gastrointestinal/metabolismo , Intestinos/crescimento & desenvolvimento , Masculino , Suínos/metabolismo , Desmame , Ganho de Peso
15.
Food Chem ; 305: 125438, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31494498

RESUMO

Fifteen vitisin A-type pyranoanthocyanins (vAPs) were determined in bilberry wines fermented with Saccharomyces cerevisiae and Schizosaccharomyces pombe by HPLC-DAD and UPLC-DAD-ESI-MS/MS. The fermentation involving S. pombe enhanced the production of vAPs compared to the fermentation with pure S. cerevisiae. The formation of vAPs correlated significantly with the decrease in the content of monomeric anthocyanins and pyruvic acid during 12 months of aging. vAPs were more stable than their corresponding monomeric anthocyanins. Methylation in the B-ring and glycosylation with galactose and arabinose further improved the stability of vAPs. Aging for 12 months led to depletion of pyruvic acid and reduction of over 50% of monomeric anthocyanins. The content of vAPs increased by 26-54% during the first six months of aging, followed by a 2.2-10.2% reduction over the following six months. More residual pyruvic acid in S. pombe wines after fermentation consequently enhanced the generation of vAPs during aging.


Assuntos
Antocianinas/análise , Sucos de Frutas e Vegetais/análise , Schizosaccharomyces/fisiologia , Vaccinium myrtillus/química , Benzofuranos/química , Reatores Biológicos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Fenóis/química , Ácido Pirúvico/metabolismo , Fatores de Tempo , Vaccinium myrtillus/metabolismo
16.
J Biotechnol ; 307: 15-28, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31639341

RESUMO

E. coli strain NT1259 /pF112aroFBLkan was able to produce 14.3 g L-1 L-tryptophan within 68 h in a fed-batch process from glycerol on a 15 L scale. To gain detailed insight into metabolism of this E. coli strain in the fed-batch process, a sample of L-tryptophan producing cells was withdrawn after 47 h, was separated rapidly and then resuspended in four parallel stirred-tank bioreactors with fresh media. Four different carbon sources (glucose, glycerol, succinate, pyruvate) were supplied individually with varying feeding rates within 19 min and the metabolic reactions of the cells in the four parallel reactors were analyzed by quantification of extracellular and intracellular substrate, product and metabolite concentrations. Data analysis allowed the estimation of intracellular carbon fluxes and of thermodynamic limitations concerning intracellular concentrations and reaction energies. Carbon fluxes and intracellular metabolite concentrations enabled the estimation of elasticities and flux control coefficients by applying metabolic control analysis making use of a metabolic model considering 48 enzymatic reactions and 56 metabolites. As the flux control coefficients describe connections between enzyme activities and metabolic fluxes, they reveal genetic targets for strain improvement. Metabolic control analysis of the recombinant E. coli cells withdrawn from the fed-batch production process clearly indicated that (i) the supply of two precursors for L-tryptophan biosynthesis, L-serine and phosphoribosyl-pyrophosphate, as well as (ii) the formation of aromatic byproducts and (iii) the enzymatic steps of igps and trps2 within the L-tryptophan biosynthesis pathway have major impact on fed-batch production of L-tryptophan from glycerol and should be the targets for further strain improvements.


Assuntos
Carbono/metabolismo , Escherichia coli/metabolismo , Triptofano/metabolismo , Reatores Biológicos , Escherichia coli/genética , Glucose/metabolismo , Glicerol/metabolismo , Ácido Pirúvico/metabolismo , Ácido Succínico/metabolismo
17.
Oxid Med Cell Longev ; 2019: 6840540, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827694

RESUMO

Introduction: Mitochondria supply cellular energy and are key regulators of intrinsic cell death and consequently affect longevity. The nematode Caenorhabditis elegans is frequently used for lifespan assays. Using paraquat (PQ) as a generator of reactive oxygen species, we here describe its effects on the acceleration of aging and the associated dysfunctions at the level of mitochondria. Methods: Nematodes were incubated with various concentrations of paraquat in a heat-stress resistance assay (37°C) using nucleic staining. The most effective concentration was validated under physiological conditions, and chemotaxis was assayed. Mitochondrial membrane potential (ΔΨm) was measured using rhodamine 123, and activity of respiratory chain complexes determined using a Clark-type electrode in isolated mitochondria. Energetic metabolites in the form of pyruvate, lactate, and ATP were determined using commercial kits. Mitochondrial integrity and structure was investigated using transmission electron microscopy. Live imaging after staining with fluorescent dyes was used to measure mitochondrial and cytosolic ROS. Expression of longevity- and mitogenesis-related genes were evaluated using qRT-PCR. Results: PQ (5 mM) significantly increased ROS formation in nematodes and reduced the chemotaxis, the physiological lifespan, and the survival in assays for heat-stress resistance. The number of fragmented mitochondria significantly increased. The ∆Ψm, the activities of complexes I-IV of the mitochondrial respiratory chain, and the levels of pyruvate and lactate were significantly reduced, whereas ATP production was not affected. Transcript levels of genetic marker genes, atfs-1, atp-2, skn-1, and sir-2.1, were significantly upregulated after PQ incubation, which implicates a close connection between mitochondrial dysfunction and oxidative stress response. Expression levels of aak-2 and daf-16 were unchanged. Conclusion: Using paraquat as a stressor, we here describe the association of oxidative stress, restricted energy metabolism, and reduced stress resistance and longevity in the nematode Caenorhabditis elegans making it a readily accessible in vivo model for mitochondrial dysfunction.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crescimento & desenvolvimento , Metabolismo Energético , Resposta ao Choque Térmico , Longevidade , Mitocôndrias/patologia , Estresse Oxidativo , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Herbicidas/farmacologia , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Paraquat/farmacologia , Ácido Pirúvico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
18.
Nat Commun ; 10(1): 5566, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31804482

RESUMO

Overexpressed Aurora-A kinase promotes tumor growth through various pathways, but whether Aurora-A is also involved in metabolic reprogramming-mediated cancer progression remains unknown. Here, we report that Aurora-A directly interacts with and phosphorylates lactate dehydrogenase B (LDHB), a subunit of the tetrameric enzyme LDH that catalyzes the interconversion between pyruvate and lactate. Aurora-A-mediated phosphorylation of LDHB serine 162 significantly increases its activity in reducing pyruvate to lactate, which efficiently promotes NAD+ regeneration, glycolytic flux, lactate production and bio-synthesis with glycolytic intermediates. Mechanistically, LDHB serine 162 phosphorylation relieves its substrate inhibition effect by pyruvate, resulting in remarkable elevation in the conversions of pyruvate and NADH to lactate and NAD+. Blocking S162 phosphorylation by expression of a LDHB-S162A mutant inhibited glycolysis and tumor growth in cancer cells and xenograft models. This study uncovers a function of Aurora-A in glycolytic modulation and a mechanism through which LDHB directly contributes to the Warburg effect.


Assuntos
Aurora Quinase A/metabolismo , Glicólise , L-Lactato Desidrogenase/metabolismo , Animais , Aurora Quinase A/antagonistas & inibidores , Azepinas/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Pirimidinas/farmacologia , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologia , Serina/genética , Serina/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Oxid Med Cell Longev ; 2019: 1652609, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31871539

RESUMO

The Mediterranean plant Silybum marianum L., commonly known as milk thistle, has been used for centuries to treat liver disorders. The flavonolignan silibinin represents a natural antioxidant and the main bioactive ingredient of silymarin (silybin), a standard extract of its seeds. Mitochondrial dysfunction and the associated generation of reactive oxygen/nitrogen species (ROS/RNS) are involved in the development of chronic liver and age-related neurodegenerative diseases. Silibinin A (SIL A) is one of two diastereomers found in silymarin and was used to evaluate the effects of silymarin on mitochondrial parameters including mitochondrial membrane potential and ATP production with and without sodium nitroprusside- (SNP-) induced nitrosative stress, oxidative phosphorylation, and citrate synthase activity in HepG2 and PC12 cells. Both cell lines were influenced by SIL A, but at different concentrations. SIL A significantly weakened nitrosative stress in both cell lines. Low concentrations not only maintained protective properties but also increased basal mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels. However, these effects could not be associated with oxidative phosphorylation. On the other side, high concentrations of SIL A significantly decreased MMP and ATP levels. Although SIL A did not provide a general improvement of the mitochondrial function, our findings show that SIL A protects against SNP-induced nitrosative stress at the level of mitochondria making it potentially beneficial against neurological disorders.


Assuntos
Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Silibina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Citrato (si)-Sintase/metabolismo , Células Hep G2 , Humanos , Ácido Láctico/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Cardo-Mariano/química , Células PC12 , Ácido Pirúvico/metabolismo , Ratos
20.
Microb Cell Fact ; 18(1): 217, 2019 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-31884954

RESUMO

BACKGROUND: Diacetyl provides the buttery aroma in products such as butter and margarine. It can be made via a harsh set of chemical reactions from sugarcane bagasse, however, in dairy products it is normally formed spontaneously from α-acetolactate, a compound generated by selected lactic acid bacteria in the starter culture used. Due to its bacteriostatic properties, it is difficult to achieve high levels of diacetyl by fermentation. Here we present a novel strategy for producing diacetyl based on whole-cell catalysis, which bypasses the toxic effects of diacetyl. RESULTS: By expressing a robust α-acetolactate synthase (ALS) in a metabolically optimized Lactococcus lactis strain we obtained a whole-cell biocatalyst that efficiently converted pyruvate into α-acetolactate. After process optimization, we achieved a titer for α-acetolactate of 172 ± 2 mM. Subsequently we used a two-stage production setup, where pyruvate was produced by an engineered L. lactis strain and subsequently used as the substrate for the biocatalyst. Using this approach, 122 ± 5 mM and 113 ± 3 mM α-acetolactate could be made from glucose or lactose in dairy waste, respectively. The whole-cell biocatalyst was robust and fully active in crude fermentation broth containing pyruvate. CONCLUSIONS: An efficient approach for converting sugar into α-acetolactate, via pyruvate, was developed and tested successfully. Due to the anaerobic conditions used for the biotransformation, little diacetyl was generated, and this allowed for efficient biotransformation of pyruvate into α-acetolactate, with the highest titers reported to date. The use of a two-step procedure for producing α-acetolactate, where non-toxic pyruvate first is formed, and subsequently converted into α-acetolactate, also simplified the process optimization. We conclude that whole cell catalysis is suitable for converting lactose in dairy waste into α-acetolactate, which favors resource utilization.


Assuntos
Lactatos/metabolismo , Lactococcus lactis/metabolismo , Ácido Pirúvico/metabolismo , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Laticínios/análise , Fermentação , Glucose/metabolismo , Lactococcus lactis/genética , Lactose/metabolismo , Resíduos
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