RESUMO
Changes in gut microbiota have been associated with several diseases. Here, the International Multiple Sclerosis Microbiome Study (iMSMS) studied the gut microbiome of 576 MS patients (36% untreated) and genetically unrelated household healthy controls (1,152 total subjects). We observed a significantly increased proportion of Akkermansia muciniphila, Ruthenibacterium lactatiformans, Hungatella hathewayi, and Eisenbergiella tayi and decreased Faecalibacterium prausnitzii and Blautia species. The phytate degradation pathway was over-represented in untreated MS, while pyruvate-producing carbohydrate metabolism pathways were significantly reduced. Microbiome composition, function, and derived metabolites also differed in response to disease-modifying treatments. The therapeutic activity of interferon-ß may in part be associated with upregulation of short-chain fatty acid transporters. Distinct microbial networks were observed in untreated MS and healthy controls. These results strongly support specific gut microbiome associations with MS risk, course and progression, and functional changes in response to treatment.
Assuntos
Microbioma Gastrointestinal , Esclerose Múltipla , Ácidos Graxos Voláteis , Humanos , Interferon beta , Ácido Fítico , PiruvatosRESUMO
The tumor suppressor p53 is critical for tumor suppression, but the regulatory role of p53 in alcohol-induced fatty liver remains unclear. Here, we show a role for p53 in regulating ethanol metabolism via acetaldehyde dehydrogenase 2 (ALDH2), a key enzyme responsible for the oxidization of alcohol. By repressing ethanol oxidization, p53 suppresses intracellular levels of acetyl-CoA and histone acetylation, leading to the inhibition of the stearoyl-CoA desaturase-1 (SCD1) gene expression. Mechanistically, p53 directly binds to ALDH2 and prevents the formation of its active tetramer and indirectly limits the production of pyruvate that promotes the activity of ALDH2. Notably, p53-deficient mice exhibit increased lipid accumulation, which can be reversed by ALDH2 depletion. Moreover, liver-specific knockdown of SCD1 alleviates ethanol-induced hepatic steatosis caused by p53 loss. By contrast, overexpression of SCD1 in liver promotes ethanol-induced fatty liver development in wild-type mice, while it has a mild effect on p53-/- or ALDH2-/- mice. Overall, our findings reveal a previously unrecognized function of p53 in alcohol-induced fatty liver and uncover pyruvate as a natural regulator of ALDH2.
Assuntos
Aldeído-Desidrogenase Mitocondrial , Fígado Gorduroso Alcoólico , Fígado Gorduroso , Proteína Supressora de Tumor p53 , Animais , Camundongos , Aldeído-Desidrogenase Mitocondrial/genética , Aldeído-Desidrogenase Mitocondrial/metabolismo , Etanol/toxicidade , Etanol/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso Alcoólico/genética , Fígado Gorduroso Alcoólico/metabolismo , Fígado/metabolismo , Piruvatos/metabolismo , Piruvatos/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In neurons, fast axonal transport (FAT) of vesicles occurs over long distances and requires constant and local energy supply for molecular motors in the form of adenosine triphosphate (ATP). FAT is independent of mitochondrial metabolism. Indeed, the glycolytic machinery is present on vesicles and locally produces ATP, as well as nicotinamide adenine dinucleotide bonded with hydrogen (NADH) and pyruvate, using glucose as a substrate. It remains unclear whether pyruvate is transferred to mitochondria from the vesicles as well as how NADH is recycled into NAD+ on vesicles for continuous glycolysis activity. The optimization of a glycolytic activity test for subcellular compartments allowed the evaluation of the kinetics of vesicular glycolysis in the brain. This revealed that glycolysis is more efficient on vesicles than in the cytosol. We also found that lactate dehydrogenase (LDH) enzymatic activity is required for effective vesicular ATP production. Indeed, inhibition of LDH or the forced degradation of pyruvate inhibited ATP production from axonal vesicles. We found LDHA rather than the B isoform to be enriched on axonal vesicles suggesting a preferential transformation of pyruvate to lactate and a concomitant recycling of NADH into NAD+ on vesicles. Finally, we found that LDHA inhibition dramatically reduces the FAT of both dense-core vesicles and synaptic vesicle precursors in a reconstituted cortico-striatal circuit on-a-chip. Together, this shows that aerobic glycolysis is required to supply energy for vesicular transport in neurons, similar to the Warburg effect.
Assuntos
Glicólise , NAD , NAD/metabolismo , Glicólise/fisiologia , Axônios/metabolismo , Trifosfato de Adenosina/metabolismo , Piruvatos/metabolismoRESUMO
The bacterial determinants that facilitate Mycobacterium tuberculosis (Mtb) adaptation to the human host environment are poorly characterized. We have sought to decipher the pressures facing the bacterium in vivo by assessing Mtb genes that are under positive selection in clinical isolates. One of the strongest targets of selection in the Mtb genome is lldD2, which encodes a quinone-dependent L-lactate dehydrogenase (LldD2) that catalyzes the oxidation of lactate to pyruvate. Lactate accumulation is a salient feature of the intracellular environment during infection and lldD2 is essential for Mtb growth in macrophages. We determined the extent of lldD2 variation across a set of global clinical isolates and defined how prevalent mutations modulate Mtb fitness. We show the stepwise nature of lldD2 evolution that occurs as a result of ongoing lldD2 selection in the background of ancestral lineage-defining mutations and demonstrate that the genetic evolution of lldD2 additively augments Mtb growth in lactate. Using quinone-dependent antibiotic susceptibility as a functional reporter, we also find that the evolved lldD2 mutations functionally increase the quinone-dependent activity of LldD2. Using 13C-lactate metabolic flux tracing, we find that lldD2 is necessary for robust incorporation of lactate into central carbon metabolism. In the absence of lldD2, label preferentially accumulates in dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) and is associated with a discernible growth defect, providing experimental evidence for accrued lactate toxicity via the deleterious buildup of sugar phosphates. The evolved lldD2 variants increase lactate incorporation to pyruvate while altering triose phosphate flux, suggesting both an anaplerotic and detoxification benefit to lldD2 evolution. We further show that the mycobacterial cell is transcriptionally sensitive to the changes associated with altered lldD2 activity which affect the expression of genes involved in cell wall lipid metabolism and the ESX- 1 virulence system. Together, these data illustrate a multifunctional role of LldD2 that provides context for the selective advantage of lldD2 mutations in adapting to host stress.
Assuntos
Mycobacterium tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , L-Lactato Desidrogenase , Ácido Láctico/metabolismo , Piruvatos/metabolismo , Quinonas/metabolismo , Fosfatos/metabolismoRESUMO
Mitochondria provide essential metabolites and adenosine triphosphate (ATP) for the regulation of energy homeostasis. For instance, liver mitochondria are a vital source of gluconeogenic precursors under a fasted state. However, the regulatory mechanisms at the level of mitochondrial membrane transport are not fully understood. Here, we report that a liver-specific mitochondrial inner-membrane carrier SLC25A47 is required for hepatic gluconeogenesis and energy homeostasis. Genome-wide association studies found significant associations between SLC25A47 and fasting glucose, HbA1c, and cholesterol levels in humans. In mice, we demonstrated that liver-specific depletion of SLC25A47 impaired hepatic gluconeogenesis selectively from lactate, while significantly enhancing whole-body energy expenditure and the hepatic expression of FGF21. These metabolic changes were not a consequence of general liver dysfunction because acute SLC25A47 depletion in adult mice was sufficient to enhance hepatic FGF21 production, pyruvate tolerance, and insulin tolerance independent of liver damage and mitochondrial dysfunction. Mechanistically, SLC25A47 depletion leads to impaired hepatic pyruvate flux and malate accumulation in the mitochondria, thereby restricting hepatic gluconeogenesis. Together, the present study identified a crucial node in the liver mitochondria that regulates fasting-induced gluconeogenesis and energy homeostasis.
Assuntos
Estudo de Associação Genômica Ampla , Gluconeogênese , Humanos , Camundongos , Animais , Gluconeogênese/fisiologia , Glucose/metabolismo , Fígado/metabolismo , Metabolismo Energético/fisiologia , Piruvatos/metabolismoRESUMO
Complex I (CI) deficiency in mitochondrial oxidative phosphorylation (OXPHOS) is the most common cause of mitochondrial diseases, and limited evidence-based treatment options exist. Although CI provides the most electrons to OXPHOS, complex II (CII) is another entry point of electrons. Enhancement of this pathway may compensate for a loss of CI; however, the effects of boosting CII activity on CI deficiency are unclear at the animal level. 5-Aminolevulinic acid (5-ALA) is a crucial precursor of heme, which is essential for CII, complex III, complex IV (CIV) and cytochrome c activities. Here, we show that feeding a combination of 5-ALA hydrochloride and sodium ferrous citrate (5-ALA-HCl + SFC) increases ATP production and suppresses defective phenotypes in Drosophila with CI deficiency. Knockdown of sicily, a Drosophila homolog of the critical CI assembly protein NDUFAF6, caused CI deficiency, accumulation of lactate and pyruvate and detrimental phenotypes such as abnormal neuromuscular junction development, locomotor dysfunctions and premature death. 5-ALA-HCl + SFC feeding increased ATP levels without recovery of CI activity. The activities of CII and CIV were upregulated, and accumulation of lactate and pyruvate was suppressed. 5-ALA-HCl + SFC feeding improved neuromuscular junction development and locomotor functions in sicily-knockdown flies. These results suggest that 5-ALA-HCl + SFC shifts metabolic programs to cope with CI deficiency. Bullet outline 5-Aminolevulinic acid (5-ALA-HCl + SFC) increases ATP production in flies with complex I deficiency.5-ALA-HCl + SFC increases the activities of complexes II and IV.5-ALA-HCl + SFC corrects metabolic abnormalities and suppresses the detrimental phenotypes caused by complex I deficiency.
Assuntos
Doenças Mitocondriais , Dermatopatias , Animais , Ácido Aminolevulínico/farmacologia , Drosophila/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Lactatos , Trifosfato de Adenosina , PiruvatosRESUMO
Nephron endowment is defined by fetal kidney growth and crucially dictates renal health in adults. Defects in the molecular regulation of nephron progenitors contribute to only a fraction of reduced nephron mass cases, suggesting alternative causative mechanisms. The importance of MAPK/ERK activation in nephron progenitor maintenance has been previously demonstrated, and here, we characterized the metabolic consequences of MAPK/ERK deficiency. Liquid chromatography/mass spectrometry-based metabolomics profiling identified 42 reduced metabolites, of which 26 were supported by in vivo transcriptional changes in MAPK/ERK-deficient nephron progenitors. Among these, mitochondria, ribosome and amino acid metabolism, together with diminished pyruvate and proline metabolism, were the most affected pathways. In vitro cultures of mouse kidneys demonstrated a dosage-specific function for pyruvate in controlling the shape of the ureteric bud tip, a regulatory niche for nephron progenitors. In vivo disruption of proline metabolism caused premature nephron progenitor exhaustion through their accelerated differentiation in pyrroline-5-carboxylate reductases 1 (Pycr1) and 2 (Pycr2) double-knockout kidneys. Pycr1/Pycr2-deficient progenitors showed normal cell survival, indicating no changes in cellular stress. Our results suggest that MAPK/ERK-dependent metabolism functionally participates in nephron progenitor maintenance by monitoring pyruvate and proline biogenesis in developing kidneys.
Assuntos
Sistema de Sinalização das MAP Quinases , Organogênese , Aminoácidos/metabolismo , Animais , Diferenciação Celular/genética , Rim/metabolismo , Camundongos , Néfrons/metabolismo , Oxirredutases/metabolismo , Prolina/metabolismo , Piruvatos/metabolismo , Células-Tronco/metabolismoRESUMO
Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.
Assuntos
Proteínas Quinases Ativadas por AMP , Vírus da Febre Suína Clássica , Mitofagia , Piruvato Quinase , Serina-Treonina Quinases TOR , Proteínas não Estruturais Virais , Replicação Viral , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Antivirais , Peste Suína Clássica/metabolismo , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/crescimento & desenvolvimento , Vírus da Febre Suína Clássica/fisiologia , Desenho de Fármacos , Glicólise , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Piruvatos/metabolismo , Transdução de Sinais , Suínos/metabolismo , Suínos/virologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Microbial pathogens balance growth against tissue damage to achieve maximum fitness. Central carbon metabolism is connected to growth, but how it influences growth/damage balance is largely unknown. Here we examined how carbon flux through the exclusively fermentative metabolism of the pathogenic lactic acid bacterium Streptococcus pyogenes impacts patterns of growth and tissue damage. Using a murine model of soft tissue infection, we systematically examined single and pair-wise mutants that constrained carbon flux through the three major pathways that S. pyogenes employs for reduction of the glycolytic intermediate pyruvate, revealing distinct disease outcomes. Its canonical lactic acid pathway (via lactate dehydrogenase) made a minimal contribution to virulence. In contrast, its two parallel pathways for mixed-acid fermentation played important, but non-overlapping roles. Anaerobic mixed acid fermentation (via pyruvate formate lyase) was required for growth in tissue, while aerobic mixed-acid pathway (via pyruvate dehydrogenase) was not required for growth, but instead regulated levels of tissue damage. Infection of macrophages in vitro revealed that pyruvate dehydrogenase was required to prevent phagolysosomal acidification, which altered expression of the immunosuppressive cytokine IL-10. Infection of IL-10 deficient mice confirmed that the ability of aerobic metabolism to regulate levels of IL-10 plays a key role in the ability of S. pyogenes to modulate levels of tissue damage. Taken together, these results show critical non-overlapping roles for anaerobic and aerobic metabolism in soft tissue infection and provide a mechanism for how oxygen and carbon flux act coordinately to regulate growth/damage balance. Therapies targeting carbon flux could be developed to mitigate tissue damage during severe S. pyogenes infection.
Assuntos
Infecções dos Tecidos Moles , Streptococcus pyogenes , Animais , Camundongos , Streptococcus pyogenes/metabolismo , Interleucina-10 , Oxirredutases , Ácido Láctico/metabolismo , Piruvatos , CarbonoRESUMO
BACKGROUND: Sepsis-induced cardiomyopathy (SIC) is a cardiac dysfunction caused by sepsis, with mitochondrial dysfunction being a critical contributor. Pyruvate dehydrogenase kinase 4 (PDK4) is a kinase of pyruvate dehydrogenase with multifaceted actions in mitochondrial metabolism. However, its role in SIC remains unknown. METHODS: Serum PDK4 levels were measured and analyzed in 27 children with SIC, 30 children with sepsis, and 29 healthy children. In addition, for mice exhibiting SIC, the effects of PDK4 knockdown or inhibition on the function and structure of the myocardium and mitochondria were assessed. RESULTS: The findings from the analysis of children with SIC revealed that PDK4 was significantly elevated and correlated with disease severity and organ injury. Nonsurvivors displayed higher serum PDK4 levels than survivors. Furthermore, mice with SIC benefited from PDK4 knockdown or inhibition, showing improved myocardial contractile function, reduced myocardial injury, and decreased mitochondrial structural injury and dysfunction. In addition, inhibition of PDK4 decreased the inhibitory phosphorylation of PDHE1α (pyruvate dehydrogenase complex E1 subunit α) and improved abnormal pyruvate metabolism and mitochondrial dysfunction. CONCLUSIONS: PDK4 is a potential biomarker for the diagnosis and prognosis of SIC. In experimental SIC, PDK4 promoted mitochondrial dysfunction with increased phosphorylation of PDHE1α and abnormal pyruvate metabolism.
Assuntos
Cardiomiopatias , Doenças Mitocondriais , Proteínas Quinases , Sepse , Animais , Criança , Humanos , Camundongos , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Miocárdio/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Piruvatos/metabolismo , Sepse/complicações , Sepse/metabolismoRESUMO
High-intensity exercise stimulates glycolysis, subsequently leading to elevated lactate production within skeletal muscle. While lactate produced within the muscle is predominantly released into the circulation via the monocarboxylate transporter 4 (MCT4), recent research underscores lactate's function as an intercellular and intertissue signalling molecule. However, its specific intracellular roles within muscle cells remains less defined. In this study, our objective was to elucidate the effects of increased intramuscular lactate accumulation on skeletal muscle adaptation to training. To achieve this, we developed MCT4 knockout mice and confirmed that a lack of MCT4 indeed results in pronounced lactate accumulation in skeletal muscle during high-intensity exercise. A key finding was the significant enhancement in endurance exercise capacity at high intensities when MCT4 deficiency was paired with high-intensity interval training (HIIT). Furthermore, metabolic adaptations supportive of this enhanced exercise capacity were evident with the combination of MCT4 deficiency and HIIT. Specifically, we observed a substantial uptick in the activity of glycolytic enzymes, notably hexokinase, glycogen phosphorylase and pyruvate kinase. The mitochondria also exhibited heightened pyruvate oxidation capabilities, as evidenced by an increase in oxygen consumption when pyruvate served as the substrate. This mitochondrial adaptation was further substantiated by elevated pyruvate dehydrogenase activity, increased activity of isocitrate dehydrogenase - the rate-limiting enzyme in the TCA cycle - and enhanced function of cytochrome c oxidase, pivotal to the electron transport chain. Our findings provide new insights into the physiological consequences of lactate accumulation in skeletal muscle during high-intensity exercises, deepening our grasp of the molecular intricacies underpinning exercise adaptation. KEY POINTS: We pioneered a unique line of monocarboxylate transporter 4 (MCT4) knockout mice specifically tailored to the ICR strain, an optimal background for high-intensity exercise studies. A deficiency in MCT4 exacerbates the accumulation of lactate in skeletal muscle during high-intensity exercise. Pairing MCT4 deficiency with high-intensity interval training (HIIT) results in a synergistic boost in high-intensity exercise capacity, observable both at the organismal level (via a treadmill running test) and at the muscle tissue level (through an ex vivo muscle contractile function test). Coordinating MCT4 deficiency with HIIT enhances both the glycolytic enzyme activities and mitochondrial capacity to oxidize pyruvate.
Assuntos
Treinamento Intervalado de Alta Intensidade , Transportadores de Ácidos Monocarboxílicos , Músculo Esquelético , Animais , Camundongos , Lactatos , Camundongos Endogâmicos ICR , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Piruvatos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismoRESUMO
A high-salt diet significantly impacts various diseases, ilncluding cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, we determined that the pyruvate dehydrogenase phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmosensing mechanism. Additionally, we demonstrate that Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Collectively, our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of pyruvate dehydrogenase phosphorylation by direct osmosensing within mitochondria.
Assuntos
Mitocôndrias , Pressão Osmótica , Oxirredutases , Piruvatos , Humanos , Células HeLa , Mitocôndrias/metabolismo , Oxirredutases/metabolismo , Fosforilação , Piruvatos/metabolismo , Células RAW 264.7 , Animais , CamundongosRESUMO
Fuel interactions in contracting muscle represent a complex interplay between enzymes regulating carbohydrate and fatty acid catabolism, converging in the mitochondrial matrix. While increasing exercise intensity promotes carbohydrate use at the expense of fatty acid oxidation, the mechanisms underlying this effect remain poorly elucidated. As a potential explanation, we investigated whether exercise-induced reductions in intramuscular pH (acidosis) attenuate carnitine palmitoyltransferase-I (CPT-I)-supported bioenergetics, the rate-limiting step for fatty acid oxidation within mitochondria. Specifically, we assessed the effect of a physiologically relevant reduction in pH (pH 7.2 versus 6.8) on single and mixed substrate respiratory responses in murine skeletal muscle isolated mitochondria and permeabilized fibers. While pH did not influence oxidative phosphorylation stoichiometry (ADP/O ratios), coupling efficiency, oxygen affinity, or ADP respiratory responses, acidosis impaired lipid bioenergetics by attenuating respiration with L-carnitine and palmitoyl-CoA, while enhancing the inhibitory effect of malonyl-CoA on CPT-I. These acidotic effects were largely retained following a single bout of intense exercise. At rest, pyruvate and succinate-supported respiration were also impaired by acidosis. However, providing more pyruvate and ADP at pH 6.8 to model increases in glycolytic flux and ATP turnover with intense exercise overcame the acidotic attenuation of carbohydrate-linked oxidative phosphorylation. Importantly, this situation is fundamentally different from lipids where CPT-I substrate sensitivity and availability is impaired at higher power outputs suggesting lipid metabolism may be more susceptible to the effects of acidosis, possibly contributing to fuel shifts with increasing exercise intensity.
Assuntos
Acidose , Carnitina O-Palmitoiltransferase , Metabolismo Energético , Metabolismo dos Lipídeos , Condicionamento Físico Animal , Animais , Camundongos , Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Oxirredução , Piruvatos/metabolismo , Piruvatos/farmacologia , Acidose/metabolismo , Camundongos Endogâmicos C57BL , Condicionamento Físico Animal/fisiologia , Concentração de Íons de Hidrogênio , Metabolismo dos Carboidratos , Transporte de ElétronsRESUMO
In recent years, lactate has been recognized as an important circulating energy substrate rather than only a dead-end metabolic waste product generated during glucose oxidation at low levels of oxygen. The term "aerobic glycolysis" has been coined to denote increased glucose uptake and lactate production despite normal oxygen levels and functional mitochondria. Hence, in "aerobic glycolysis," lactate production is a metabolic choice, whereas in "anaerobic glycolysis," it is a metabolic necessity based on inadequate levels of oxygen. Interestingly, lactate can be taken up by cells and oxidized to pyruvate and thus constitutes a source of pyruvate that is independent of insulin. Here, we show that the transcription factor Foxp1 regulates glucose uptake and lactate production in adipocytes and myocytes. Overexpression of Foxp1 leads to increased glucose uptake and lactate production. In addition, protein levels of several enzymes in the glycolytic pathway are upregulated, such as hexokinase 2, phosphofructokinase, aldolase, and lactate dehydrogenase. Using chromatin immunoprecipitation and real-time quantitative PCR assays, we demonstrate that Foxp1 directly interacts with promoter consensus cis-elements that regulate expression of several of these target genes. Conversely, knockdown of Foxp1 suppresses these enzyme levels and lowers glucose uptake and lactate production. Moreover, mice with a targeted deletion of Foxp1 in muscle display systemic glucose intolerance with decreased muscle glucose uptake. In primary human adipocytes with induced expression of Foxp1, we find increased glycolysis and glycolytic capacity. Our results indicate Foxp1 may play an important role as a regulator of aerobic glycolysis in adipose tissue and muscle.
Assuntos
Adipócitos , Fatores de Transcrição Forkhead , Glicólise , Células Musculares , Fatores de Transcrição , Animais , Camundongos , Adipócitos/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Glucose/metabolismo , Glicólise/genética , Ácido Láctico/metabolismo , Células Musculares/metabolismo , Piruvatos , Fatores de Transcrição/metabolismo , Ratos , Linhagem Celular , TranscriptomaRESUMO
Rhodopseudomonas palustris TIE-1 grows photoautotrophically with Fe(II) as an electron donor and photoheterotrophically with a variety of organic substrates. However, it is unclear whether R. palustris TIE-1 conducts Fe(II) oxidation in conditions where organic substrates and Fe(II) are available simultaneously. In addition, the effect of organic co-substrates on Fe(II) oxidation rates or the identity of Fe(III) minerals formed is unknown. We incubated R. palustris TIE-1 with 2 mM Fe(II), amended with 0.6 mM organic co-substrate, and in the presence/absence of CO2 . We found that in the absence of CO2 , only the organic co-substrates acetate, lactate and pyruvate, but not Fe(II), were consumed. When CO2 was present, Fe(II) and all organic substrates were consumed. Acetate, butyrate and pyruvate were consumed before Fe(II) oxidation commenced, whereas lactate and glucose were consumed at the same time as Fe(II) oxidation proceeded. Lactate, pyruvate and glucose increased the Fe(II) oxidation rate significantly (by up to threefold in the case of lactate). 57 Fe Mössbauer spectroscopy revealed that short-range ordered Fe(III) oxyhydroxides were formed under all conditions. This study demonstrates phototrophic Fe(II) oxidation proceeds even in the presence of organic compounds, and that the simultaneous oxidation of organic substrates can stimulate Fe(II) oxidation.
Assuntos
Dióxido de Carbono , Compostos Férricos , Rodopseudomonas , Oxirredução , Ácido Láctico , Compostos Ferrosos , Piruvatos , Acetatos , GlucoseRESUMO
The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells.
Assuntos
Glucose , Hepatócitos , Hepatócitos/metabolismo , Glucose/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Ácido Láctico , Trifosfato de Adenosina/metabolismo , PiruvatosRESUMO
MAIN CONCLUSION: Hydroxy(phenyl)pyruvic acid reductase from Actaea racemosa catalyzes dual reactions in reducing 4-hydroxyphenylpyruvic acid as well as ß-hydroxypyruvic acid. It thus qualifies to be part of fukinolic and cimicifugic acid biosynthesis and also photorespiration. The accumulation of fukinolic acid and cimicifugic acids is mainly restricted to Actaea racemosa (Ranunculaceae) and other species of the genus Actaea/Cimicifuga. Cimicifugic and fukinolic acids are composed of a hydroxycinnamic acid part esterified with a benzyltartaric acid moiety. The biosynthesis of the latter is unclear. We isolated cDNA encoding a hydroxy(phenyl)pyruvic acid reductase (GenBank OR393286) from suspension-cultured material of A. racemosa (ArH(P)PR) and expressed it in E. coli for protein production. The heterologously synthesized enzyme had a mass of 36.51 kDa and catalyzed the NAD(P)H-dependent reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid or ß-hydroxypyruvic acid to glyceric acid, respectively. The optimal temperature was at 38 °C and the pH optimum at pH 7.5. NADPH is the preferred cosubstrate (Km 23 ± 4 µM). Several substrates are accepted by ArH(P)PR with ß-hydroxypyruvic acid (Km 0.26 ± 0.12 mM) followed by 4-hydroxyphenylpyruvic acid (Km 1.13 ± 0.12 mM) as the best ones. Thus, ArH(P)PR has properties of ß-hydroxypyruvic acid reductase (involved in photorespiration) as well as hydroxyphenylpyruvic acid reductase (possibly involved in benzyltartaric acid formation).
Assuntos
Ácidos Cafeicos , Cimicifuga , Fenilacetatos , Ácidos Fenilpirúvicos , Piruvatos , Cimicifuga/química , Ácido Pirúvico , Oxirredutases , Escherichia coli/genética , Extratos VegetaisRESUMO
MAIN CONCLUSION: Optimal levels of indole-3-butyric acid (IBA) applied at the stem base promote adventitious root (AR) initiation and primordia formation, thus promoting the rooting of leafy micro-cuttings of tetraploid Robinia pseudoacacia. Tetraploid Robinia pseudoacacia L. is a widely cultivated tree in most regions of China that has a hard-rooting capability, propagated by stem cuttings. This study utilizes histological, physiological, and transcriptomic approaches to explore how root primordia are induced after indole butyric acid (IBA) treatment of micro-cuttings. IBA application promoted cell divisions in some cells within the vasculature, showing subcellular features associated with adventitious root (AR) founder cells. The anatomical structure explicitly showed that AR initiated from the cambium layer and instigate the inducible development of AR primordia. Meanwhile, the hormone data showed that similar to that of indole-3-acetic acid, the contents of trans-zeatin and abscisic acid peaked at early stages of AR formation and increased gradually in primordia formation across the subsequent stages, suggesting their indispensable roles in AR induction. On the contrary, 24-epibrassinolide roughly maintained at extremely high levels during primordium initiation thoroughly, indicating its presence was involved in cell-specific reorganization during AR development. Furthermore, antioxidant activities transiently increased in the basal region of micro-cuttings and may serve as biochemical indicators for distinct rooting phases, potentially aiding in AR formation. Transcriptomic analysis during the early stages of root formation shows significant downregulation of the abscisic acid and jasmonate signaling pathways, while ethylene and cytokinin signaling seems upregulated. Network analysis of genes involved in carbon metabolism and photosynthesis indicates that the basal region of the micro-cuttings undergoes rapid reprogramming, which results in the breakdown of sugars into pyruvate. This pyruvate is then utilized to fuel the tricarboxylic acid cycle, thereby sustaining growth through aerobic respiration. Collectively, our findings provide a time-course morphophysiological dissection and also suggest the regulatory role of a conserved auxin module in AR development in these species.
Assuntos
Ácido Abscísico , Robinia , Ácido Abscísico/farmacologia , Ácido Abscísico/metabolismo , Robinia/genética , Tetraploidia , Ácidos Indolacéticos/metabolismo , Perfilação da Expressão Gênica , Piruvatos/metabolismo , Raízes de Plantas/metabolismoRESUMO
PURPOSE: To use the hepatocyte-specific gadolinium-based contrast agent gadoxetate combined with hyperpolarized (HP) [1-13 C]pyruvate MRI to selectively suppress metabolic signals from normal hepatocytes while preserving the signals arising from tumors. METHODS: Simulations were performed to determine the expected changes in HP 13 C MR signal in liver and tumor under the influence of gadoxetate. CC531 colon cancer cells were implanted into the livers of five Wag/Rij rats. Liver and tumor metabolism were imaged at 3 T using HP [1-13 C] pyruvate chemical shift imaging before and 15 min after injection of gadoxetate. Area under the curve for pyruvate and lactate were measured from voxels containing at least 75% of normal-appearing liver or tumor. RESULTS: Numerical simulations predicted a 36% decrease in lactate-to-pyruvate (L/P) ratio in liver and 16% decrease in tumor. In vivo, baseline L/P ratio was 0.44 ± 0.25 in tumors versus 0.21 ± 0.08 in liver (p = 0.09). Following administration of gadoxetate, mean L/P ratio decreased by an average of 0.11 ± 0.06 (p < 0.01) in normal-appearing liver. In tumors, mean L/P ratio post-gadoxetate did not show a statistically significant change from baseline. Compared to baseline levels, the relative decrease in L/P ratio was significantly greater in liver than in tumors (-0.52 ± 0.16 vs. -0.19 ± 0.25, p < 0.05). CONCLUSIONS: The intracellular hepatobiliary contrast agent showed a greater effect suppressing HP 13 C MRI metabolic signals (through T1 shortening) in normal-appearing liver when compared to tumors. The combined use of HP MRI with selective gadolinium contrast agents may allow more selective imaging in HP 13 C MRI.
Assuntos
Meios de Contraste , Neoplasias Hepáticas , Ratos , Animais , Meios de Contraste/farmacologia , Gadolínio/farmacologia , Hepatócitos/metabolismo , Gadolínio DTPA , Fígado/metabolismo , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/metabolismo , Imageamento por Ressonância Magnética/métodos , Piruvatos/metabolismo , Lactatos/metabolismoRESUMO
Using captured CO2 and C1-feedstocks like formate and methanol derived from electrochemical activation of CO2 are key solutions for transforming industrial processes towards a circular carbon economy. Engineering formate and CO2-based growth in the biotechnologically relevant yeast Saccharomyces cerevisiae could boost the emergence of a formate-mediated circular bio-economy. This study adopts a growth-coupled selection scheme for modular implementation of the Reductive Glycine Pathway (RGP) and subsequent Adaptive Laboratory Evolution (ALE) to enable formate and CO2 assimilation for biomass formation in yeast. We first constructed a serine biosensor strain and then implemented the serine synthesis module of the RGP into yeast, establishing glycine and serine synthesis from formate and CO2. ALE improved the RGP-dependent growth by 8-fold. 13C-labeling experiments reveal glycine, serine, and pyruvate synthesis via the RGP, demonstrating the complete pathway activity. Further, we re-established formate and CO2-dependent growth in non-evolved biosensor strains via reverse-engineering a mutation in GDH1 identified from ALE. This mutation led to significantly more 13C-formate assimilation than in WT without any selection or overexpression of the RGP. Overall, we demonstrated the activity of the complete RGP, showing evidence for carbon transfer from formate to pyruvate coupled with CO2 assimilation.