The beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs) on preventing obesity are well known; however, the underlying mechanism by which n-3 PUFAs influence tricarboxylic acid (TCA) cycle under obesity remains unclear. We randomly divided male C57BL/6 mice into 5 groups (n=10) and fed for 12 weeks as follows: mice fed a normal diet (Con, 10% kcal); mice fed a high-fat diet (HFD, lard, 60% kcal); and mice fed a high-fat diet (60% kcal) substituting half the lard with safflower oil (SO), safflower oil and fish oil (SF) and fish oil (FO), respectively. Then we treated HepG2 cells with palmitic acid and DHA for 24 h. We found that body weight in FO group was significantly lower than it in HFD and SO groups. N-3 PUFAs reduced the transcription and translation of TCA cycle enzymes, including IDH1, IDH2, SDHA, FH and MDH2, to enhance mitochondrial function in vivo and vitro. DHA significantly inhibited protein expression of the mTORC1 signaling pathway, increased p-AKT protein expression to alleviate insulin resistance and improved mitochondrial oxygen consumption rate and glycolysis ability in HepG2 cells. In addition, the expressions of IDH2 and SDHB were reduced by rapamycin. N-3 PUFAs could prevent obesity by improving TCA cycle homeostasis and mTORC1 signaling pathway may be upstream.

Acute liver failure (ALF) is a serious clinical disorder with high fatality rates. Mahuang decoction (MHD), a well-known traditional Chinese medicine, has multiple pharmacological effects, such as anti-inflammation, anti-allergy, anti-asthma, and anti-hyperglycemia. In this study, we investigated the protective effect of MHD against ALF. In the lipopolysaccharide and D-galactosamine (LPS/D-GalN)-induced ALF mouse model, the elevated activities of the serum alanine and aspartate transaminases as well as the liver pathological damage were markedly alleviated by MHD. Subsequently, a metabolomics study based on the ultrahigh performance liquid chromatograph coupled with Q Exactive Orbitrap mass spectrometry was carried to clarify the therapeutic mechanisms of MHD against ALF. A total of 36 metabolites contributing to LPS/D-GalN-induced ALF were identified in the serum samples, among which the abnormalities of 27 metabolites were ameliorated by MHD. The analysis of metabolic pathways revealed that the therapeutic effects of MHD are likely due to the modulation of the metabolic disorders of tricarboxylic acid (TCA) cycle, retinol metabolism, tryptophan metabolism, arginine and proline metabolism, nicotinate and nicotinamide metabolism, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan synthesis, as well as cysteine and methionine metabolism. This study demonstrated for the first time that MHD exerted an obvious protective effect against ALF mainly through the regulation of TCA cycle and amino acid metabolism, highlighting the importance of metabolomics to investigate the drug-targeted metabolic pathways.

In multiple sclerosis (MS) a process of white matter degradation leading to demyelination is observed. Oxidative stress, inflammation, apoptosis, necrosis and/or autophagy result together into a progressive loss of oligodendrocytes. 7-ketocholesterol (7KC), found increased in the cerebrospinal fluid of MS patients, triggers a rupture of RedOx homeostasis associated with mitochondrial dysfunctions, aptoptosis and autophagy (oxiapoptophagy) in cultured murine oligodendrocytes (158N). α-tocopherol is able to mild the alterations induced by 7KC partially restoring the cellular homeostasis. In presence of 7KC, the amount of adherent 158N cells was decreased and oxidative stress was enhanced. An increase of caspase-3 and PARP degradation (evidences of apoptosis), and an increased LC3-II/LC3-I ratio (criterion of autophagy), were detected. These events were associated with a decrease of the mitochondrial membrane potential (ΔΨm) and by a decrease of oxidative phosphorylation revealed by reduced NAD+ and ATP. The cellular lactate was higher while pyruvate, citrate, fumarate, succinate (tricarboxylic acid (TCA) cycle intermediates) were significantly reduced in exposed cells, suggesting that an impairment of mitochondrial respiratory functions could lead to an increase of lactate production and to a reduced amount of ATP and acetyl-CoA available for the anabolic pathways. The concentration of sterol precursors lathosterol, lanosterol and desmosterol were significantly reduced together with satured and unsatured long chain fatty acids (C16:0 - C18:0, structural elements of membrane phospholipids). Such reductions were milder with α-tocopherol. It is likely that the cell death induced by 7KC is associated with mitochondrial dysfunctions, including alterations of oxidative phosphorylation, which could result from lipid anabolism dysfunctions, especially on TCA cycle intermediates. A better knowledge of mitochondrial associated dysfunctions triggered by 7KC will contribute to bring new information on the demyelination processes which are linked with oxidative stress and lipid peroxidation, especially in MS.

The optimal substrate for hypothermic machine perfusion preservation of donor hearts is unknown. Fatty acids, acetate, and ketones are preferred substrates of the heart during normothermic perfusion, but cannot replete the tricarboxylic acid (TCA) cycle directly. Propionate, an anaplerotic substrate, can replenish TCA cycle intermediates and may affect cardiac metabolism. The purpose of this study was to determine myocardial substrate preferences during hypothermic machine perfusion and to assess if an anaplerotic substrate was required to maintain the TCA cycle intermediate pool in perfused hearts.

To infer molecular effectors of therapeutic effects and adverse events for dimethyl fumarate (DMF) in patients with relapsing-remitting MS (RRMS) using untargeted plasma metabolomics.

Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable alpha-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO(2). Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of beta-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with beta-oxidation (KOR-dependent), and one that functions in the absence of beta-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host.

The tricarboxylic acid (TCA) cycle, a crucial component of respiratory metabolism, is composed of a set of eight enzymes present in the mitochondrial matrix. However, most of the TCA cycle enzymes are encoded in the nucleus in higher eukaryotes. In addition, evidence has accumulated demonstrating that nuclear genes were acquired from the mitochondrial genome during the course of evolution. For this reason, we here analyzed the evolutionary history of all TCA cycle enzymes in attempt to better understand the origin of these nuclear-encoded proteins. Our results indicate that prior to endosymbiotic events the TCA cycle seemed to operate only as isolated steps in both the host (eubacterial cell) and mitochondria (alphaproteobacteria). The origin of isoforms present in different cell compartments might be associated either with gene-transfer events which did not result in proper targeting of the protein to mitochondrion or with duplication events. Further in silico analyses allow us to suggest new insights into the possible roles of TCA cycle enzymes in different tissues. Finally, we performed coexpression analysis using mitochondrial TCA cycle genes revealing close connections among these genes most likely related to the higher efficiency of oxidative phosphorylation in this specialized organelle. Moreover, these analyses allowed us to identify further candidate genes which might be used for metabolic engineering purposes given the importance of the TCA cycle during development and/or stress situations.

Huanglongbing in citrus is caused by a phloem-limited, uncultivable, gram-negative α-proteobacterium, Candidatus Liberibacter asiaticus (CLas). CLas is transmitted by the phloem-sucking insect, Diaphorina citri (Hemiptera: Liviidae), in a persistent, circulative, and propagative manner. In this study, we investigated the metabolomic and respiration rates changes in D. citri upon infection with CLas using gas chromatography-mass spectrometry (GC-MS) and gas exchange analysis. The level of glycine, L-serine, L-threonine, and gamma-amino butyric acid were higher in CLas-infected D. citri, while L-proline, L-aspartic acid, and L-pyroglutamic acid were lower in CLas-infected D. citri compared with the control. Citric acid was increased in CLas-infected D. citri, whereas malic and succinic acids were reduced. Interestingly, most of the reduced metabolites such as malate, succinate, aspartate, and L-proline are required for the growth of CLas. The increase in citric acid, serine, and glycine indicated that CLas induced glycolysis and the tricarboxylic acid cycle (TCA) in its vector. In agreement with the GC-MS results, the gene expression results also indicated that glycolysis and TCA were induced in CLas-infected D. citri and this was accompanied with an increases in respiration rate. Phosphoric acid and most of the sugar alcohols were higher in CLas-infected D. citri, indicating a response to the biotic stress or cell damage. Only slight increases in the levels of few sugars were observed in CLas-infected D. citri, which indicated that sugars are tightly regulated by D. citri. Our results indicated that CLas induces nutrient and energetic stress in its host insect. This study may provide some insights into the mechanism of colonization of CLas in its vector.

Cachexia is a life-threatening disease characterized by chronic, inflammatory muscle wasting and systemic metabolic impairment. Despite its high prevalence, there are no efficacious therapies for cachexia. Mice chronically infected with the protozoan parasite Toxoplasma gondii represent a novel animal model recapitulating the chronic kinetics of cachexia. To understand how perturbations to metabolic tissue homeostasis influence circulating metabolite availability we used mass spectrometry analysis. Despite the significant reduction in circulating triacylglycerides, non-esterified fatty acids, and glycerol, sphingolipid long-chain bases and a subset of phosphatidylcholines (PCs) were significantly increased in the sera of mice with T. gondii infection-induced cachexia. In addition, the TCA cycle intermediates α-ketoglutarate, 2-hydroxyglutarate, succinate, fumarate, and malate were highly depleted in cachectic mouse sera. Sphingolipids and their de novo synthesis precursors PCs are the major components of the mitochondrial membrane and regulate mitochondrial function consistent with a causal relationship in the energy imbalance driving T. gondii-induced chronic cachexia.

Although the severity of influenza virus infections has been associated with host energy metabolism, the related mechanisms have not yet been clarified. Here we examined the effects of influenza virus infection on host energy metabolism in mice. After infecting mice with intranasal applications of 500 plaque-forming units of A/Puerto Rico/8/34 (H1N1; PR8) virus, the serum levels of most intermediates in the tricarboxylic acid (TCA) cycle and related metabolic pathways were significantly reduced. These data suggest that substrate supply to the TCA cycle is reduced under these conditions, rather than specific metabolic reactions being inhibited. Then, we focused on glucose and fatty acid metabolism that supply substrates to the TCA cycle. Akt phosphorylation following insulin injections was attenuated in the livers of PR8 virus-infected mice. Furthermore, glucose tolerance tests revealed that the PR8 virus-infected mice showed higher blood glucose levels than the vehicle-inoculated control mice. These results suggest that influenza virus infection impairs insulin signaling, which regulates glucose uptake. However, increases in the hepatic expressions of fatty acid-metabolizing enzymes suggest that fatty acids accumulate in liver cells of infected mice. Collectively, our data indicate that influenza virus infection dysregulates host energy metabolism. This line of investigation provides novel insights into the pathogenesis of influenza.

There is an urgent need to elucidate the pathogenesis of myocardial ischemia (MI) and potential drug treatments. Here, the anti-MI mechanism and material basis of Ginkgo biloba L. extract (GBE) were studied from the perspective of energy metabolism flux regulation. Metabolic flux analysis (MFA) was performed to investigate energy metabolism flux disorder and the regulatory nodes of GBE components in isoproterenol (ISO)-induced ischemia-like cardiomyocytes. It showed that [U-13C] glucose derived m+2 isotopologues from the upstream tricarboxylic acid (TCA) cycle metabolites were markedly accumulated in ISO-injured cardiomyocytes, but the opposite was seen for the downstream metabolites, while their total cellular concentrations were increased. This indicates a blockage of carbon flow from glycolysis and enhanced anaplerosis from other carbon sources. A Seahorse test was used to screen for GBE components with regulatory effects on mitochondrial aerobic respiratory dysfunction. It showed that bilobalide protected against impaired mitochondrial aerobic respiration. MFA also showed that bilobalide significantly modulated the TCA cycle flux, reduced abnormal metabolite accumulation, and balanced the demand of different carbon sources. Western blotting and PCR analysis showed that bilobalide decreased the enhanced expression of key metabolic enzymes in injured cells. Bilobalide's efficacy was verified by in vivo experiments in rats. This is the first report to show that bilobalide, the active ingredient of GBE, protects against MI by rescuing impaired TCA cycle flux. This provides a new mechanism and potential drug treatment for MI. It also shows the potential of MFA/Seahorse combination as a powerful strategy for pharmacological research on herbal medicine.

Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial tricarboxylic acid (TCA) cycle metabolism in astrocytes are unknown. We investigated this by mapping 13 C labeling in TCA cycle intermediates and corresponding amino acids after incubation of primary rat astrocytes with [U-13 C]glucose. The presence of metformin did not compromise the viability of cultured astrocytes during 4 hr of incubation, but almost doubled cellular glucose consumption and lactate release. Compared with control cells, the presence of metformin dramatically lowered the molecular 13 C carbon labeling (MCL) of the cellular TCA cycle intermediates citrate, α-ketoglutarate, succinate, fumarate, and malate, as well as the MCL of the TCA cycle intermediate-derived amino acids glutamate, glutamine, and aspartate. In addition to the total molecular 13 C labeling, analysis of the individual isotopomers of TCA cycle intermediates confirmed a severe decline in labeling and a significant lowering in TCA cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs complex I-mediated mitochondrial respiration in astrocytes, which is likely to cause the observed decrease in labeling of mitochondrial TCA cycle intermediates and the stimulation of glycolytic lactate production. © 2017 Wiley Periodicals, Inc.

The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function.

The Warburg effect is a metabolic hallmark of cancer cells; cancer cells, unlike normal cells, exclusively activate glycolysis, even in the presence of enough oxygen. On the other hand, intratumoral heterogeneity is currently of interest in cancer research, including that involving cancer stem cells (CSCs). In the present study, we attempted to gain an understanding of metabolism in CSCs that is distinct from that in non-CSCs. After forming spheroids from the OVTOKO (ovarian clear cell adenocarcinoma) and SiHa (cervical squamous cell carcinoma) cell lines, the metabolites of these cells were compared with the metabolites of cancer cells that were cultured in adherent plates. A principle components analysis clearly divided their metabolic features. Amino acids that participate in tricarboxylic acid (TCA) cycle reactions, such as serine and glutamine, were significantly increased in the spheroids. Indeed, spheroids from each cell line contained more total adenylates than did their corresponding cells in adherent cultures. This study demonstrated that cancer metabolism is not limited to aerobic glycolysis (i.e. the Warburg effect), but is flexible and context-dependent. In addition, activation of TCA cycles was suggested to be a metabolic feature of CSCs that was distinct from non-CSCs. The amino acid metabolic pathways discussed here are already considered as targets for cancer therapy, and they are additionally proposed as potential targets for CSC treatment.

The tricarboxylic acid (TCA) cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (S Typhimurium) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in the TCA cycle, a strain deficient in all fumarase isoforms (ΔfumABC) elicited a unique metabolic profile. Alongside fumarate, S Typhimurium ΔfumABC accumulates intermediates of the glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes toward glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of S Typhimurium by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis and partially restored levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential- to stationary-growth-phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of S Typhimurium and cellular functions and virulence.IMPORTANCE We performed perturbation analyses of the tricarboxylic acid cycle of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium. The defect of fumarase activity led to accumulation of fumarate but also resulted in a global alteration of carbon fluxes, leading to increased storage of glycogen. Gross alterations were observed in proteome and metabolome compositions of fumarase-deficient Salmonella In turn, these changes were linked to aberrant motility patterns of the mutant strain and resulted in highly increased phagocytic uptake by macrophages. Our findings indicate that basic cellular functions and specific virulence functions in Salmonella critically depend on the proper function of the primary metabolism.

It has been claimed that citrate synthase, aconitase and isocitrate dehydrogenase activities are non-functional in Bordetella pertussis and that this might explain why this bacterium's growth is sometimes associated with accumulation of polyhydroxybutyrate (PHB) and/or free fatty acids. However, the sequenced genome includes the entire citric acid pathway genes. Furthermore, these genes were expressed and the corresponding enzyme activities detected at high levels for the pathway when grown on a defined medium imitating the amino acid content of complex media often used for growth of this pathogenic microorganism. In addition, no significant PHB or fatty acids could be detected. Analysis of the carbon balance and stoichiometric flux analysis based on specific rates of amino acid consumption, and estimated biomass requirements coherent with the observed growth rate, clearly indicate that a fully functional tricarboxylic acid cycle operates in contrast to previous reports.

Background: In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Methods: Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations in vivo. Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δψ) and cell ATP levels were assayed using fluorometric and luminescent test. Results: PDK-1 in KRAS mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δψ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. Conclusion: We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in KRAS mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.

Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechanistic link between mitochondrial function and epigenetic regulation of gene expression that involves polyamine and methionine metabolism responding to changes in the tricarboxylic acid (TCA) cycle. Given the implications of these findings, future studies across different physiological contexts and in vivo are warranted.

It is now well-established that perturbations in the tricarboxylic acid (TCA) cycle play an important role in the metabolic transformation occurring in cancer including that of the prostate. A method for simultaneous qualitative and quantitative analysis of TCA cycle intermediates in body fluids, tissues, and cultured cell lines of human origin was developed using a common C18 reversed-phase column by LC-MS/MS technique. This LC-MS/MS method for profiling TCA cycle intermediates offers significant advantages including simple and fast preparation of a wide range of human biological samples. The analytical method was validated according to the guideline of the Royal Society of Chemistry Analytical Methods Committee. The limits of detection were below 60 nM for most of the TCA intermediates with the exception of lactic and fumaric acids. The calibration curves of all TCA analytes showed linearity with correlation coefficients r2 > 0.9998. Recoveries were >95% for all TCA analytes. This method was established taking into consideration problems and limitations of existing techniques. We envisage that its application to different biological matrices will facilitate deeper understanding of the metabolic changes in the TCA cycle from in vitro, ex vivo, and in vivo studies.

This study aimed to elucidate the variations in primary and secondary metabolites during Lycorisradiata flower development using high performance liquid chromatography (HPLC) and gas chromatography time-of-flight mass spectrometry (GC-TOFMS). The result showed that seven carotenoids, seven phenolic acids, three anthocyanins, and galantamine were identified in the L. radiata flowers. Most secondary metabolite levels gradually decreased according to the flower developmental stages. A total of 51 metabolites, including amines, sugars, sugar intermediates, sugar alcohols, amino acids, organic acids, phenolic acids, and tricarboxylic acid (TCA) cycle intermediates, were identified and quantified using GC-TOFMS. Among the hydrophilic compounds, most amino acids increased during flower development; in contrast, TCA cycle intermediates and sugars decreased. In particular, glutamine, asparagine, glutamic acid, and aspartic acid, which represent the main inter- and intracellular nitrogen carriers, were positively correlated with the other amino acids and were negatively correlated with the TCA cycle intermediates. Furthermore, quantitation data of the 51 hydrophilic compounds were subjected to partial least-squares discriminant analyses (PLS-DA) to assess significant differences in the metabolites of L. radiata flowers from stages 1 to 4. Therefore, this study will serve as the foundation for a biochemical approach to understand both primary and secondary metabolism in L. radiata flower development.