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Acetate is a major nutrient that supports acetyl-coenzyme A (Ac-CoA) metabolism and thus lipogenesis and protein acetylation. However, its source is unclear. Here, we report that pyruvate, the end product of glycolysis and key node in central carbon metabolism, quantitatively generates acetate in mammals. This phenomenon becomes more pronounced in the context of nutritional excess, such as during hyperactive glucose metabolism. Conversion of pyruvate to acetate occurs through two mechanisms: (1) coupling to reactive oxygen species (ROS) and (2) neomorphic enzyme activity from keto acid dehydrogenases that enable function as pyruvate decarboxylases. Further, we demonstrate that de novo acetate production sustains Ac-CoA pools and cell proliferation in limited metabolic environments, such as during mitochondrial dysfunction or ATP citrate lyase (ACLY) deficiency. By virtue of de novo acetate production being coupled to mitochondrial metabolism, there are numerous possible regulatory mechanisms and links to pathophysiology.
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Acetatos/metabolismo , Glucose/metabolismo , Ácido Pirúvico/metabolismo , ATP Citrato (pro-S)-Liase/fisiologia , Acetilcoenzima A/biossíntese , Acetilcoenzima A/metabolismo , Acetilação , Animais , Feminino , Glicólise/fisiologia , Lipogênese/fisiologia , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Oxirredutases , Piruvato Descarboxilase/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ácido Láctico/metabolismo , Neoplasias Pulmonares/metabolismo , Animais , Análise Química do Sangue , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Feminino , Ácidos Glicéricos/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transplante de Neoplasias , Simportadores/genética , Simportadores/metabolismoRESUMO
Lactate accumulates to a significant amount in glioblastomas (GBMs), the most common primary malignant brain tumor with an unfavorable prognosis. However, it remains unclear whether lactate is metabolized by GBMs. Here, we demonstrated that lactate rescued patient-derived xenograft (PDX) GBM cells from nutrient-deprivation-mediated cell death. Transcriptome analysis, ATAC-seq, and ChIP-seq showed that lactate entertained a signature of oxidative energy metabolism. LC/MS analysis demonstrated that U-13C-lactate elicited substantial labeling of TCA-cycle metabolites, acetyl-CoA, and histone protein acetyl-residues in GBM cells. Lactate enhanced chromatin accessibility and histone acetylation in a manner dependent on oxidative energy metabolism and the ATP-citrate lyase (ACLY). Utilizing orthotopic PDX models of GBM, a combined tracer experiment unraveled that lactate carbons were substantially labeling the TCA-cycle metabolites. Finally, pharmacological blockage of oxidative energy metabolism extended overall survival in two orthotopic PDX models in mice. These results establish lactate metabolism as a novel druggable pathway for GBM.
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Glioblastoma , Acetilação , Animais , Linhagem Celular Tumoral , Epigênese Genética , Glioblastoma/genética , Glioblastoma/patologia , Histonas/metabolismo , Humanos , Ácido Láctico/metabolismo , CamundongosRESUMO
The metabolic pathways fueling tumor growth have been well characterized, but the specific impact of transforming events on network topology and enzyme essentiality remains poorly understood. To this end, we performed combinatorial CRISPR-Cas9 screens on a set of 51 carbohydrate metabolism genes that represent glycolysis and the pentose phosphate pathway (PPP). This high-throughput methodology enabled systems-level interrogation of metabolic gene dispensability, interactions, and compensation across multiple cell types. The metabolic impact of specific combinatorial knockouts was validated using 13C and 2H isotope tracing, and these assays together revealed key nodes controlling redox homeostasis along the KEAP-NRF2 signaling axis. Specifically, targeting KEAP1 in combination with oxidative PPP genes mitigated the deleterious effects of these knockouts on growth rates. These results demonstrate how our integrated framework, combining genetic, transcriptomic, and flux measurements, can improve elucidation of metabolic network alterations and guide precision targeting of metabolic vulnerabilities based on tumor genetics.
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Sistemas CRISPR-Cas , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Redes e Vias Metabólicas , Fator 2 Relacionado a NF-E2/metabolismo , Transcriptoma , Glicólise , Células HeLa , Homeostase , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/antagonistas & inibidores , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Via de Pentose Fosfato , Transdução de SinaisRESUMO
Sphingolipids (SPs) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by the serine palmitoyltransferase (SPT). In budding yeast (Saccharomyces cerevisiae), SPT is negatively regulated by the two proteins, Orm1 and Orm2. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane or the lysosome (or vacuole in yeast). Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, measuring lipid metabolic enzyme activity in different cellular sub-compartments has been challenging. Combining a nanobody recruitment approach with SP flux analysis, we show that the nuclear endoplasmic reticulum (ER)-localized SPT and the peripheral ER localized SPT pools are differentially active. Thus, our data add another layer to the complex network of SPT regulation. Moreover, combining lipid metabolic enzyme re-localization with flux analysis serves as versatile tool to measure lipid metabolism with subcellular resolution.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Proteínas de Membrana/metabolismo , Esfingolipídeos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.
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Metabolismo Energético , Túbulos Renais , Mitocôndrias , Animais , Camundongos , Mitocôndrias/metabolismo , Túbulos Renais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Consumo de Oxigênio , Biogênese de Organelas , Transporte Biológico , Glicólise/fisiologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Protein turnover (PT) has been formally defined only in equilibrium conditions, which is ill-suited to quantify PT during dynamic processes that occur during embryogenesis or (extra) cellular signaling. In this Hypothesis, we propose a definition of PT in an out-of-equilibrium regime that allows the quantification of PT in virtually any biological context. We propose a simple mathematical and conceptual framework applicable to a broad range of available data, such as RNA sequencing coupled with pulsed-SILAC datasets. We apply our framework to a published dataset and show that stimulation of mouse dendritic cells with LPS leads to a proteome-wide change in PT. This is the first quantification of PT out-of-equilibrium, paving the way for the analysis of biological systems in other contexts.
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Proteoma , Animais , Camundongos , ProteóliseRESUMO
SignificancePhotosynthesis metabolites are quickly labeled when 13CO2 is fed to leaves, but the time course of labeling reveals additional contributing processes involved in the metabolic dynamics of photosynthesis. The existence of three such processes is demonstrated, and a metabolic flux model is developed to explore and characterize them. The model is consistent with a slow return of carbon from cytosolic and vacuolar sugars into the Calvin-Benson cycle through the oxidative pentose phosphate pathway. Our results provide insight into how carbon assimilation is integrated into the metabolic network of photosynthetic cells with implications for global carbon fluxes.
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Carbono/metabolismo , Redes e Vias Metabólicas , Fotossíntese , Açúcares/metabolismo , Ciclo do Carbono , Dióxido de Carbono/metabolismo , Citosol/metabolismo , Modelos Biológicos , Folhas de Planta/metabolismo , Fenômenos Fisiológicos VegetaisRESUMO
Metabolic dysfunction-associated steatotic liver disease is the most common form of liver disease and poses significant health risks to patients who progress to metabolic dysfunction-associated steatohepatitis. Fatty acid overload alters endoplasmic reticulum (ER) calcium stores and induces mitochondrial oxidative stress in hepatocytes, leading to hepatocellular inflammation and apoptosis. Obese mice have impaired liver sarco/ER Ca2+-ATPase (SERCA) function, which normally maintains intracellular calcium homeostasis by transporting Ca2+ ions from the cytoplasm to the ER. We hypothesized that restoration of SERCA activity would improve diet-induced steatohepatitis in mice by limiting ER stress and mitochondrial dysfunction. WT and melanocortin-4 receptor KO (Mc4r-/-) mice were placed on either chow or Western diet (WD) for 8 weeks. Half of the WD-fed mice were administered CDN1163 to activate SERCA, which reduced liver fibrosis and inflammation. SERCA activation also restored glucose tolerance and insulin sensitivity, improved histological markers of metabolic dysfunction-associated steatohepatitis, increased expression of antioxidant enzymes, and decreased expression of oxidative stress and ER stress genes. CDN1163 decreased hepatic citric acid cycle flux and liver pyruvate cycling, enhanced expression of mitochondrial respiratory genes, and shifted hepatocellular [NADH]/[NAD+] and [NADPH]/[NADP+] ratios to a less oxidized state, which was associated with elevated PUFA content of liver lipids. In sum, the data demonstrate that pharmacological SERCA activation limits metabolic dysfunction-associated steatotic liver disease progression and prevents metabolic dysfunction induced by WD feeding in mice.
Assuntos
Fígado , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Camundongos , Fígado/metabolismo , Fígado/patologia , Masculino , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Estresse do Retículo Endoplasmático , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Dieta Ocidental/efeitos adversos , Camundongos KnockoutRESUMO
Synechococcus elongatus PCC 11801 and 11802 are closely related cyanobacterial strains that are fast-growing and tolerant to high light and temperature. These strains hold significant promise as chassis for photosynthetic production of chemicals from carbon dioxide. A detailed quantitative understanding of the central carbon pathways would be a reference for future metabolic engineering studies with these strains. We conducted isotopic non-stationary 13 C metabolic flux analysis to quantitively assess the metabolic potential of these two strains. This study highlights key similarities and differences in the central carbon flux distribution between these and other model/non-model strains. The two strains demonstrated a higher Calvin-Benson-Bassham (CBB) cycle flux coupled with negligible flux through the oxidative pentose phosphate pathway and the photorespiratory pathway and lower anaplerosis fluxes under photoautotrophic conditions. Interestingly, PCC 11802 shows the highest CBB cycle and pyruvate kinase flux values among those reported in cyanobacteria. The unique tricarboxylic acid (TCA) cycle diversion in PCC 11801 makes it ideal for the large-scale production of TCA cycle-derived chemicals. Additionally, dynamic labeling transients were measured for intermediates of amino acid, nucleotide, and nucleotide sugar metabolism. Overall, this study provides the first detailed metabolic flux maps of S. elongatus PCC 11801 and 11802, which may aid metabolic engineering efforts in these strains.
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Plasma nucleosides-pseudouridine (PU) and N2N2-dimethyl guanosine (DMG) predict the progression of type 2 diabetic kidney disease (DKD) to end-stage renal disease, but the mechanisms underlying this relationship are not well understood. We used a well-characterized model of type 2 diabetes (db/db mice) and control nondiabetic mice (db/m mice) to characterize the production and excretion of PU and DMG levels using liquid chromatography-mass spectrometry. The fractional excretion of PU and DMG was decreased in db/db mice compared with control mice at 24 wk before any changes to renal function. We then examined the dynamic changes in nucleoside metabolism using in vivo metabolic flux analysis with the injection of labeled nucleoside precursors. Metabolic flux analysis revealed significant decreases in the ratio of urine-to-plasma labeling of PU and DMG in db/db mice compared with db/m mice, indicating significant tubular dysfunction in diabetic kidney disease. We observed that the gene and protein expression of the renal tubular transporters involved with nucleoside transport in diabetic kidneys in mice and humans was reduced. In conclusion, this study strongly suggests that tubular handling of nucleosides is altered in early DKD, in part explaining the association of PU and DMG with human DKD progression observed in previous studies.NEW & NOTEWORTHY Tubular dysfunction explains the association between the nucleosides pseudouridine and N2N2-dimethyl guanosine and diabetic kidney disease.
Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Humanos , Camundongos , Animais , Nefropatias Diabéticas/metabolismo , Pseudouridina/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Nucleosídeos/metabolismo , Eliminação Renal , Rim/metabolismo , Guanosina/metabolismoRESUMO
Regular exercise elicits adaptations in glucose and lipid metabolism that allow the body to meet energy demands of subsequent exercise bouts more effectively and mitigate metabolic diseases including fatty liver. Energy discharged during the acute exercise bouts that comprise exercise training may be a catalyst for liver adaptations. During acute exercise, liver glycogenolysis and gluconeogenesis are accelerated to supply glucose to working muscle. Lower liver energy state imposed by gluconeogenesis and related pathways activates AMP-activated protein kinase (AMPK), which conserves ATP partly by promoting lipid oxidation. This study tested the hypothesis that AMPK is necessary for liver glucose and lipid adaptations to training. Liver-specific AMPKα1α2 knockout (AMPKα1α2fl/fl+AlbCre) mice and littermate controls (AMPKα1α2fl/fl) completed sedentary and exercise training protocols. Liver nutrient fluxes were quantified at rest or during acute exercise following training. Liver metabolites and molecular regulators of metabolism were assessed. Training increased liver glycogen in AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. The inability to increase glycogen led to lower glycogenolysis, glucose production, and circulating glucose during acute exercise in trained AMPKα1α2fl/fl+AlbCre mice. Deletion of AMPKα1α2 attenuated training-induced declines in liver diacylglycerides. In particular, training lowered the concentration of unsaturated and elongated fatty acids comprising diacylglycerides in AMPKα1α2fl/fl mice, but not in AMPKα1α2fl/fl+AlbCre mice. Training increased liver triacylglycerides and the desaturation and elongation of fatty acids in triacylglycerides of AMPKα1α2fl/fl+AlbCre mice. These lipid responses were independent of differences in tricarboxylic acid cycle fluxes. In conclusion, AMPK is required for liver training adaptations that are critical to glucose and lipid metabolism.NEW & NOTEWORTHY This study shows that the energy sensor and transducer, AMP-activated protein kinase (AMPK), is necessary for an exercise training-induced: 1) increase in liver glycogen that is necessary for accelerated glycogenolysis during exercise, 2) decrease in liver glycerolipids independent of tricarboxylic acid (TCA) cycle flux, and 3) decline in the desaturation and elongation of fatty acids comprising liver diacylglycerides. The mechanisms defined in these studies have implications for use of regular exercise or AMPK-activators in patients with fatty liver.
Assuntos
Proteínas Quinases Ativadas por AMP , Fígado Gorduroso , Humanos , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Glicogênio Hepático , Fígado/metabolismo , Glucose/metabolismo , Fígado Gorduroso/metabolismo , Ácidos Graxos/metabolismoRESUMO
Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione. A total of 4.3 g/L glutathione was produced by overexpressing gshA and gshB, which encode cysteine glutamate ligase and glutathione synthetase, respectively, and most of the glutathione was excreted into the culture medium. Further improvements were achieved by inhibiting degradation (Δggt and ΔpepT); deleting gor (Δgor), which encodes glutathione oxide reductase; attenuating glutathione uptake (ΔyliABCD); and enhancing cysteine production (PompF-cysE). The engineered strain KG06 produced 19.6 g/L glutathione after 48 h of fed-batch fermentation with continuous addition of ammonium sulfate as the sulfur source. We also found that continuous feeding of glycine had a crucial role for effective glutathione production. The results of metabolic flux and metabolomic analyses suggested that the conversion of O-acetylserine to cysteine is the rate-limiting step in glutathione production by KG06. The use of sodium thiosulfate largely overcame this limitation, increasing the glutathione titer to 22.0 g/L, which is, to our knowledge, the highest titer reported to date in the literature. This study is the first report of glutathione fermentation without adding cysteine in E. coli. Our findings provide a great potential of E. coli fermentation process for the industrial production of glutathione.
Assuntos
Escherichia coli , Glutationa , Engenharia Metabólica , Escherichia coli/genética , Escherichia coli/metabolismo , Glutationa/metabolismo , Glutationa/biossíntese , Glutationa/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , FermentaçãoRESUMO
Yarrowia lipolytica is an industrial yeast that can convert waste oil to value-added products. However, it is unclear how this yeast metabolizes lipid feedstocks, specifically triacylglycerol (TAG) substrates. This study used 13C-metabolic flux analysis (13C-MFA), genome-scale modeling, and transcriptomics analyses to investigate Y. lipolytica W29 growth with oleic acid, glycerol, and glucose. Transcriptomics data were used to guide 13C-MFA model construction and to validate the 13C-MFA results. The 13C-MFA data were then used to constrain a genome-scale model (GSM), which predicted Y. lipolytica fluxes, cofactor balance, and theoretical yields of terpene products. The three data sources provided new insights into cellular regulation during catabolism of glycerol and fatty acid components of TAG substrates, and how their consumption routes differ from glucose catabolism. We found that (1) over 80% of acetyl-CoA from oleic acid is processed through the glyoxylate shunt, a pathway that generates less CO2 compared to the TCA cycle, (2) the carnitine shuttle is a key regulator of the cytosolic acetyl-CoA pool in oleic acid and glycerol cultures, (3) the oxidative pentose phosphate pathway and mannitol cycle are key routes for NADPH generation, (4) the mannitol cycle and alternative oxidase activity help balance excess NADH generated from ß-oxidation of oleic acid, and (5) asymmetrical gene expressions and GSM simulations of enzyme usage suggest an increased metabolic burden for oleic acid catabolism.
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Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO-K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO-K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.
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Metabolic reaction rates (fluxes) play a crucial role in comprehending cellular phenotypes and are essential in areas such as metabolic engineering, biotechnology, and biomedical research. The state-of-the-art technique for estimating fluxes is metabolic flux analysis using isotopic labelling (13C-MFA), which uses a dataset-model combination to determine the fluxes. Bayesian statistical methods are gaining popularity in the field of life sciences, but the use of 13C-MFA is still dominated by conventional best-fit approaches. The slow take-up of Bayesian approaches is, at least partly, due to the unfamiliarity of Bayesian methods to metabolic engineering researchers. To address this unfamiliarity, we here outline similarities and differences between the two approaches and highlight particular advantages of the Bayesian way of flux analysis. With a real-life example, re-analysing a moderately informative labelling dataset of E. coli, we identify situations in which Bayesian methods are advantageous and more informative, pointing to potential pitfalls of current 13C-MFA evaluation approaches. We propose the use of Bayesian model averaging (BMA) for flux inference as a means of overcoming the problem of model uncertainty through its tendency to assign low probabilities to both, models that are unsupported by data, and models that are overly complex. In this capacity, BMA resembles a tempered Ockham's razor. With the tempered razor as a guide, BMA-based 13C-MFA alleviates the problem of model selection uncertainty and is thereby capable of becoming a game changer for metabolic engineering by uncovering new insights and inspiring novel approaches.
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Teorema de Bayes , Isótopos de Carbono , Escherichia coli , Isótopos de Carbono/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Análise do Fluxo Metabólico/métodos , Modelos Biológicos , Engenharia Metabólica/métodos , Marcação por IsótopoRESUMO
Metabolic flux is the final output of cellular regulation and has been extensively studied for carbon but much less is known about nitrogen, which is another important building block for living organisms. For the tuberculosis pathogen, this is particularly important in informing the development of effective drugs targeting the pathogen's metabolism. Here we performed 13 C15 N dual isotopic labeling of Mycobacterium bovis BCG steady state cultures, quantified intracellular carbon and nitrogen fluxes and inferred reaction bidirectionalities. This was achieved by model scope extension and refinement, implemented in a multi-atom transition model, within the statistical framework of Bayesian model averaging (BMA). Using BMA-based 13 C15 N-metabolic flux analysis, we jointly resolve carbon and nitrogen fluxes quantitatively. We provide the first nitrogen flux distributions for amino acid and nucleotide biosynthesis in mycobacteria and establish glutamate as the central node for nitrogen metabolism. We improved resolution of the notoriously elusive anaplerotic node in central carbon metabolism and revealed possible operation modes. Our study provides a powerful and statistically rigorous platform to simultaneously infer carbon and nitrogen metabolism in any biological system.
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Carbono , Nitrogênio , Carbono/metabolismo , Isótopos de Carbono/metabolismo , Nitrogênio/metabolismo , Análise do Fluxo Metabólico , Teorema de Bayes , Modelos BiológicosRESUMO
Cross-feeding is fundamental to the diversity and function of microbial communities. However, identification of cross-fed metabolites is often challenging due to the universality of metabolic and biosynthetic intermediates. Here, we use 13 C isotope tracing in peptides to elucidate cross-fed metabolites in co-cultures of Saccharomyces cerevisiae and Lactococcus lactis. The community was grown on lactose as the main carbon source with either glucose or galactose fraction of the molecule labelled with 13 C. Data analysis allowing for the possible mass-shifts yielded hundreds of peptides for which we could assign both species identity and labelling degree. The labelling pattern showed that the yeast utilized galactose and, to a lesser extent, lactic acid shared by L. lactis as carbon sources. While the yeast provided essential amino acids to the bacterium as expected, the data also uncovered a complex pattern of amino acid exchange. The identity of the cross-fed metabolites was further supported by metabolite labelling in the co-culture supernatant, and by diminished fitness of a galactose-negative yeast mutant in the community. Together, our results demonstrate the utility of 13 C-based proteomics for uncovering microbial interactions.
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Galactose , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteômica , Carbono/metabolismo , Bactérias/metabolismoRESUMO
Metabolic flux analysis (MFA) is a valuable tool for quantifying cellular phenotypes and to guide plant metabolic engineering. By introducing stable isotopic tracers and employing mathematical models, MFA can quantify the rates of metabolic reactions through biochemical pathways. Recent applications of isotopically nonstationary MFA (INST-MFA) to plants have elucidated nonintuitive metabolism in leaves under optimal and stress conditions, described coupled fluxes for fast-growing algae, and produced a synergistic multi-organ flux map that is a first in MFA for any biological system. These insights could not be elucidated through other approaches and show the potential of INST-MFA to correct an oversimplified understanding of plant metabolism.
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Análise do Fluxo Metabólico , Plantas , Análise do Fluxo Metabólico/métodos , Plantas/metabolismo , Modelos Biológicos , Folhas de Planta/metabolismoRESUMO
OBJECTIVE: Chondrocytes, which typically rely on anaerobic metabolism, exhibit upregulated biosynthetic activity when subjected to conditions that elicit mixed aerobic-anaerobic metabolism. Previously, we observed that increasing media volume resulted in the transition from anaerobic to mixed aerobic-anaerobic metabolism. Maximal extracellular matrix (ECM) accumulation occurred at this transition as a result of changes in hypoxia-inducible factor 1α signaling and associated hypoxic gene expression. This study aimed to explore the effect of further increases in media availability on ECM synthesis and chondrocyte metabolism. METHODS: Primary bovine chondrocytes were grown in 3D high-density tissue culture under varying levels of media availability (4-16 mL/106 cells). Changes in ECM accumulation and metabolism were determined through biochemical assays and 13C-metabolic flux analysis (13C-MFA). RESULTS: Increasing media volumes resulted in higher accumulation of cartilaginous ECM (collagen and proteoglycans) and cellularity. Extracellular metabolite measurements revealed that elevated media availability led to increased glucose and glutamine metabolism, along with increased anaerobic activity. 13C-MFA utilizing [U-13C] glucose demonstrated that increased media availability significantly impacted central carbon metabolism, upregulating all glucose-related metabolic pathways (glycolysis, lactate fermentation, the tricarboxylic acid (TCA) cycle, hexosamine biosynthetic pathway, and the malate-aspartate shuttle). Furthermore, 13C-MFA indicated that glutamine was donating carbons to the TCA cycle, and additional studies involving [U-13C] glutamine tracing supported this notion. CONCLUSIONS: Elevated media availability upregulates ECM synthesis and leads to significant changes in metabolic phenotype. Glutamine plays an important role in chondrocyte metabolism and increases in glutamine metabolism correlate with increases in ECM accumulation.