Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues.

Aberrantly accumulated metabolites elicit intra- and inter-cellular pro-oncogenic cascades, yet current measurement methods require sample perturbation/disruption and lack spatio-temporal resolution, limiting our ability to fully characterize their function and distribution. Here, we show that Raman spectroscopy (RS) can directly detect fumarate in living cells in vivo and animal tissues ex vivo, and that RS can distinguish between Fumarate hydratase (Fh1)-deficient and Fh1-proficient cells based on fumarate concentration. Moreover, RS reveals the spatial compartmentalization of fumarate within cellular organelles in Fh1-deficient cells: consistent with disruptive methods, we observe the highest fumarate concentration (37 ± 19 mM) in mitochondria, where the TCA cycle operates, followed by the cytoplasm (24 ± 13 mM) and then the nucleus (9 ± 6 mM). Finally, we apply RS to tissues from an inducible mouse model of FH loss in the kidney, demonstrating RS can classify FH status. These results suggest RS could be adopted as a valuable tool for small molecule metabolic imaging, enabling in situ non-destructive evaluation of fumarate compartmentalization.

Influence of fumarate on interspecies electron transfer and the metabolic shift induced in Clostridium pasteurianum by Geobacter sulfurreducens.

AIMS: In previous studies, it was demonstrated that co-culturing Clostridium pasteurianum and Geobacter sulfurreducens triggers a metabolic shift in the former during glycerol fermentation. This shift, attributed to interspecies electron transfer and the exchange of other molecules, enhances the production of 1,3-propanediol at the expense of the butanol pathway. The aim of this investigation is to examine the impact of fumarate, a soluble compound usually used as an electron acceptor for G. sulfurreducens, in the metabolic shift previously described in C. pasteurianum. METHODS AND RESULTS: Experiments were conducted by adding along with glycerol, acetate, and different quantities of fumarate in co-cultures of G. sulfurreducens and C. pasteurianum. A metabolic shift was exhibited in all the co-culture conditions. This shift was more pronounced at higher fumarate concentrations. Additionally, we observed G. sulfurreducens growing even in the absence of fumarate and utilizing small amounts of this compound as an electron donor rather than an electron acceptor in the co-cultures with high fumarate addition. CONCLUSIONS: This study provided evidence that interspecies electron transfer continues to occur in the presence of a soluble electron acceptor, and the metabolic shift can be enhanced by promoting the growth of G. sulfurreducens.

Fumaric acid production by Rhizopus species from acid hydrolysate of oil palm empty fruit bunches.

The hemicellulosic fraction of lignocellulosic biomass is a very important material, due to the significant concentration of pentoses present in its composition and that can be used sustainably in biotechnological processes such as the production of fumaric acid. Research efforts are currently being promoted for the proper disposal and valorization of empty fruit bunches (EFB) from oil palm. In this work, seventeen Rhizopus species were evaluated in a fermentation medium with EFB hydrolyzate, without detoxification, as a carbon source for fumaric acid production. Rhizopus circicans 1475 and Rhizopus 3271 achieved productions of 5.65 g.L-1 and 5.25 g.L-1 of fumaric acid at 30 °C, 120 rpm for 96 h, respectively. The percentage of consumed sugars, mainly pentoses, was 24.88% and 34.02% for R. circicans 1475 and R 3271, respectively. Soy peptone and ammonium sulfate were evaluated as nitrogen sources, where soy peptone stimulated the formation of biomass pellets while ammonium sulfate produced mycelia and clamps.

Fumaric acid production from fermented oil palm empty fruit bunches using fungal isolate K20: a comparison between free and immobilized cells.

This study investigated the potential of using steam-exploded oil palm empty fruit bunches (EFB) as a renewable feedstock for producing fumaric acid (FA), a food additive widely used for flavor and preservation, through a separate hydrolysis and fermentation process using the fungal isolate K20. The efficiency of FA production by free and immobilized cells was compared. The maximum FA concentration (3.25 g/L), with 0.034 g/L/h productivity, was observed after incubation with the free cells for 96 h. Furthermore, the production was scaled up in a 3-L air-lift fermenter using oil palm EFB-derived glucose as the substrate. The FA concentration, yield, and productivity from 100 g/L initial oil palm EFB-derived glucose were 44 g/L, 0.39 g/g, and 0.41 g/L/h, respectively. The potential for scaling up the fermentation process indicates favorable results, which could have significant implications for industrial applications.

Fumarate induces LncRNA-MIR4435-2HG to regulate glutamine metabolism remodeling and promote the development of FH-deficient renal cell carcinoma.

Fumarate hydratase (FH) deficient renal cell carcinoma (RCC) is a type of tumor with definite metabolic disorder, but the mechanism of metabolic remodeling is still unclear. LncRNA was reported to closely correlate with cancer metabolism, however the biological role of LncRNA in the development of progression of FH-deficent RCC was not well studied either. FH-deficient RCC samples were collected in my hospital and used for RNA-sequencing and Mass spectrometry analysis. FH-deficient RCC cell line UOK262 and control pFH cells were used for in vitro experiments, including proliferation assay, transwell assay, western-blot, mass spectrometry and so on. PDX mouse model was used for further drug inhibition experiments in vivo. In this study, we analyzed the profiles of LncRNA and mRNA in FH-deficienct RCC samples, and we found that the LncRNA-MIR4435-2GH was specifically highly expressed in FH-deficient RCC compared with ccRCC. In vitro experiments demonstrated that MIR4435-2HG was regulated by Fumarate through histone demethylation, and the deletion of this gene could inhibit glutamine metabolism. RNA-pulldown experiments showed that MIR4435-2HG specifically binds to STAT1, which can transcriptionally activate GLS1. GLS1 inhibitor CB-839 could significantly suppress tumor growth in PDX tumor models. This study analyzed the molecular mechanism of MIR4435-2HG in regulating metabolic remodeling of FH-deficient RCC in clinical samples, cells and animal models by combining transcriptional and metabolic methods. We found that that GLS1 was a therapeutic target for this tumor, and MIR4435-2HG can be used as a drug sensitivity marker.

Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized.NEW & NOTEWORTHY Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.

Nitrate-driven anaerobic oxidation of ethane and butane by bacteria.

The short-chain gaseous alkanes (ethane, propane, and butane; SCGAs) are important components of natural gas, yet their fate in environmental systems is poorly understood. Microbially mediated anaerobic oxidation of SCGAs coupled to nitrate reduction has been demonstrated for propane, but is yet to be shown for ethane or butane-despite being energetically feasible. Here we report two independent bacterial enrichments performing anaerobic ethane and butane oxidation, respectively, coupled to nitrate reduction to dinitrogen gas and ammonium. Isotopic 13C- and 15N-labelling experiments, mass and electron balance tests, and metabolite and meta-omics analyses collectively reveal that the recently described propane-oxidizing "Candidatus Alkanivorans nitratireducens" was also responsible for nitrate-dependent anaerobic oxidation of the SCGAs in both these enrichments. The complete genome of this species encodes alkylsuccinate synthase genes for the activation of ethane/butane via fumarate addition. Further substrate range tests confirm that "Ca. A. nitratireducens" is metabolically versatile, being able to degrade ethane, propane, and butane under anoxic conditions. Moreover, our study proves nitrate as an additional electron sink for ethane and butane in anaerobic environments, and for the first time demonstrates the use of the fumarate addition pathway in anaerobic ethane oxidation. These findings contribute to our understanding of microbial metabolism of SCGAs in anaerobic environments.

Role of fumarate reductase on the fermentation properties of Lactobacillus delbrueckii ssp. bulgaricus.

Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus are symbiotic starters widely used in yogurt fermentation. They exchange metabolites to meet their nutritional demands during fermentation, promoting mutual growth. Although S. thermophilus produces fumaric acid, and the addition of fumaric acid has been shown to promote the growth of L. bulgaricus monoculture, whether fumaric acid produced by S. thermophilus is used by L. bulgaricus during coculture remains unclear. Furthermore, the importance of fumaric acid metabolism in the growth of L. bulgaricus is yet to be elucidated. Therefore, in this study, we investigated the importance of fumaric acid metabolism in L. bulgaricus monocultures and coculture with S. thermophilus. We deleted the fumarate reductase gene (frd), which is responsible for the metabolism of fumaric acid to succinic acid, in L. bulgaricus strains 2038 and NCIMB 701373. Both Δfrd strains exhibited longer fermentation times than their parent strains, and fumaric acid was metabolized to malic acid rather than succinic acid. Coculture of Δfrd strains with S. thermophilus 1131 also resulted in a longer fermentation time, and the accumulation of malic acid was observed. These results indicated that fumaric acid produced by S. thermophilus is used by L. bulgaricus as a symbiotic substance during yogurt fermentation and that the metabolism of fumaric acid to succinic acid by fumarate reductase is a key factor determining the fermentation ability of L. bulgaricus.

Organic-inorganic composite of polypropylene fumarate and nanohydroxyapatite as carrier of antibiotics for the treatment of bone infections.

Current treatment approaches in clinics to treat the infectious lesions have partial success thus demanding the need for development of advanced treatment modalities. In this study we fabricated an organic-inorganic composite of polypropylene fumarate (PPF) and nanohydroxyapatite (nHAP) by photo-crosslinking as a carrier of two clinically used antibiotics, ciprofloxacin (CIP) and rifampicin (RFP) for the treatment of bone infections. Carboxy terminal-PPF was first synthesized by cis-trans isomerization of maleic anhydride which was then photo-crosslinked using diethylfumarate (DEF) as crosslinker and bis-acylphosphine oxide (BAPO) as photo-initiator under UV lights (P). A composite of PPF and nHAP was fabricated by incorporating 40 % of nHAP in the polymeric matrix of PPF (PH) which was then characterized for different physicochemical parameters. CIP was added along with nHAP to fabricated CIPloaded composite scaffolds (PHC) which was then coated with RFP to synthesize RFP coated CIP-loaded scaffolds (PHCR). It was observed that there was a temporal separation in the in vitro release of two antibiotics after coating PHC with RFP with 80.48 ± 0.40 % release of CIP from PHC and 62.43 ± 0.21 % release of CIP from PHCR for a period of 60 days. Moreover, in vitro protein adsorption was also found to be maximum in PHCR (154.95 ± 0.07 µg/mL) as observed in PHC (75.42 ± 0.06 µg/mL), PH (24.47 ± 0.08 µg/mL) and P alone (4.47 ± 0.02 µg/mL). The scaffolds were also evaluated using in vivo infection model to assess their capacity in reducing the bacterial burden at the infection site. The outcome of this study suggests that RFP coated CIP-loaded PPF composite scaffolds could reduce bacterial burden and simultaneously augment bone healing during infection related fractures.

Enterohaemorrhagic E. coli utilizes host- and microbiota-derived L-malate as a signaling molecule for intestinal colonization.

The mammalian gastrointestinal tract is a complex environment that hosts a diverse microbial community. To establish infection, bacterial pathogens must be able to compete with the indigenous microbiota for nutrients, as well as sense the host environment and modulate the expression of genes essential for colonization and virulence. Here, we found that enterohemorrhagic Escherichia coli (EHEC) O157:H7 imports host- and microbiota-derived L-malate using the DcuABC transporters and converts these substrates into fumarate to fuel anaerobic fumarate respiration during infection, thereby promoting its colonization of the host intestine. Moreover, L-malate is important not only for nutrient metabolism but also as a signaling molecule that activates virulence gene expression in EHEC O157:H7. The complete virulence-regulating pathway was elucidated; the DcuS/DcuR two-component system senses high L-malate levels and transduces the signal to the master virulence regulator Ler, which in turn activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence to epithelial cells of the large intestine. Disruption of this virulence-regulating pathway by deleting either dcuS or dcuR significantly reduced colonization by EHEC O157:H7 in the infant rabbit intestinal tract; therefore, targeting these genes and altering physiological aspects of the intestinal environment may offer alternatives for EHEC infection treatment.

The connection between tricarboxylic acid cycle enzyme mutations and pseudohypoxic signaling in pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors originating from chromaffin cells, holding significant clinical importance due to their capacity for excessive catecholamine secretion and associated cardiovascular complications. Roughly 80% of cases are associated with genetic mutations. Based on the functionality of these mutated genes, PPGLs can be categorized into distinct molecular clusters: the pseudohypoxia signaling cluster (Cluster-1), the kinase signaling cluster (Cluster-2), and the WNT signaling cluster (Cluster-3). A pivotal factor in the pathogenesis of PPGLs is hypoxia-inducible factor-2α (HIF2α), which becomes upregulated even under normoxic conditions, activating downstream transcriptional processes associated with pseudohypoxia. This adaptation provides tumor cells with a growth advantage and enhances their ability to thrive in adverse microenvironments. Moreover, pseudohypoxia disrupts immune cell communication, leading to the development of an immunosuppressive tumor microenvironment. Within Cluster-1a, metabolic perturbations are particularly pronounced. Mutations in enzymes associated with the tricarboxylic acid (TCA) cycle, such as succinate dehydrogenase (SDHx), fumarate hydratase (FH), isocitrate dehydrogenase (IDH), and malate dehydrogenase type 2 (MDH2), result in the accumulation of critical oncogenic metabolic intermediates. Notable among these intermediates are succinate, fumarate, and 2-hydroxyglutarate (2-HG), which promote activation of the HIFs signaling pathway through various mechanisms, thus inducing pseudohypoxia and facilitating tumorigenesis. SDHx mutations are prevalent in PPGLs, disrupting mitochondrial function and causing succinate accumulation, which competitively inhibits α-ketoglutarate-dependent dioxygenases. Consequently, this leads to global hypermethylation, epigenetic changes, and activation of HIFs. In FH-deficient cells, fumarate accumulation leads to protein succination, impacting cell function. FH mutations also trigger metabolic reprogramming towards glycolysis and lactate synthesis. IDH1/2 mutations generate D-2HG, inhibiting α-ketoglutarate-dependent dioxygenases and stabilizing HIFs. Similarly, MDH2 mutations are associated with HIF stability and pseudohypoxic response. Understanding the intricate relationship between metabolic enzyme mutations in the TCA cycle and pseudohypoxic signaling is crucial for unraveling the pathogenesis of PPGLs and developing targeted therapies. This knowledge enhances our comprehension of the pivotal role of cellular metabolism in PPGLs and holds implications for potential therapeutic advancements.

Asuc_0142 of Actinobacillus succinogenes 130Z is the l-aspartate/C4-dicarboxylate exchanger DcuA.

Anaerobic bacteria often use antiporters DcuB (malate/succinate antiport) or DcuA (l-aspartate/succinate antiport) for the excretion of succinate during fumarate respiration. The rumen bacterium Actinobacillus succinogenes is able to produce large amounts of succinate by fumarate respiration, using the DcuB-type transporter DcuE for l-malate/succinate antiport. Asuc_0142 was annotated as a second DcuB-type transporter. Deletion of Asuc_0142 decreased the uptake rate for l-[14C]aspartate into A. succinogenes cells. Properties of transport by heterologously expressed Asuc_0142 were investigated in an Escherichia coli mutant deficient of anaerobic C4DC transporters. Expression of Asuc_0142 resulted in high uptake activity for l-[14C]fumarate or l-[14C]aspartate, but the former showed a strong competitive inhibition by l-aspartate. In E. coli loaded with l-[14C]aspartate, [14C]succinate or [14C]fumarate, extracellular C4DCs initiated excretion of the intracellular substrates, with a preference for l-aspartateex/succinatein or l-aspartateex/fumaratein antiport. These findings indicate that Asuc_0142 represents a DcuA-type transporter for l-aspartate uptake and l-aspartateex/C4DCin antiport, differentiating it from the DcuB-type transporter DcuE for l-malateex/succinatein antiport. Sequence analysis and predicted structural characteristics confirm structural similarity of Asuc_0142 to DcuA, and Asuc_0142 was thus re-named as DcuAAs. The bovine rumen fluid contains l-aspartate (99.6 µM), whereas fumarate and l-malate are absent. Therefore, bovine rumen colonisers depend on l-aspartate as an exogenous substrate for fumarate respiration. A. succinogenes encodes HemG (protoporphyrinogen oxidase) and PyrD (dihydroorotate dehydrogenase) for haem and pyrimidine biosynthesis. The enzymes require fumarate as an electron acceptor, suggesting an essential role for l-aspartate, DcuAAs, and fumarate respiration for A. succinogenes growing in the bovine rumen.

An oviduct glycan increases sperm lifespan by diminishing the production of ubiquinone and reactive oxygen species†.

Sperm storage by females after mating for species-dependent periods is used widely among animals with internal fertilization to allow asynchrony between mating and ovulation. Many mammals store sperm in the lower oviduct where specific glycans on oviduct epithelial cells retain sperm to form a reservoir. Binding to oviduct cells suppresses sperm intracellular Ca2+ and increases sperm longevity. We investigated the mechanisms by which a specific oviduct glycan, 3-O-sulfated Lewis X trisaccharide (suLeX), prolongs the lifespan of porcine sperm. Using targeted metabolomics, we found that binding to suLeX diminishes the abundance of 4-hydroxybenzoic acid, the precursor to ubiquinone (also known as Coenzyme Q), 30 min after addition. Ubiquinone functions as an electron acceptor in the electron transport chain (ETC). 3-O-sulfated Lewis X trisaccharide also suppressed the formation of fumarate. A component of the citric acid cycle, fumarate is synthesized by succinate-coenzyme Q reductase, which employs ubiquinone and is also known as Complex II in the ETC. Consistent with the reduced activity of the ETC, the production of harmful reactive oxygen species (ROS) was diminished. The enhanced sperm lifespan in the oviduct may be because of suppressed ROS production because high ROS concentrations have toxic effects on sperm.

Supplementation with Amino Acid Sources Facilitates Fermentative Growth of Shewanella oneidensis MR-1 in Defined Media.

Shewanella oneidensis MR-1 is a facultative anaerobe that grows by respiration using a variety of electron acceptors. This organism serves as a model to study how bacteria thrive in redox-stratified environments. A glucose-utilizing engineered derivative of MR-1 has been reported to be unable to grow in glucose minimal medium (GMM) in the absence of electron acceptors, despite this strain having a complete set of genes for reconstructing glucose to lactate fermentative pathways. To gain insights into why MR-1 is incapable of fermentative growth, this study examined a hypothesis that this strain is programmed to repress the expression of some carbon metabolic genes in the absence of electron acceptors. Comparative transcriptomic analyses of the MR-1 derivative were conducted in the presence and absence of fumarate as an electron acceptor, and these found that the expression of many genes involved in carbon metabolism required for cell growth, including several tricarboxylic acid (TCA) cycle genes, was significantly downregulated in the absence of fumarate. This finding suggests a possibility that MR-1 is unable to grow fermentatively on glucose in minimal media owing to the shortage of nutrients essential for cell growth, such as amino acids. This idea was demonstrated in subsequent experiments that showed that the MR-1 derivative fermentatively grows in GMM containing tryptone or a defined mixture of amino acids. We suggest that gene regulatory circuits in MR-1 are tuned to minimize energy consumption under electron acceptor-depleted conditions, and that this results in defective fermentative growth in minimal media. IMPORTANCE It is an enigma why S. oneidensis MR-1 is incapable of fermentative growth despite having complete sets of genes for reconstructing fermentative pathways. Understanding the molecular mechanisms behind this defect will facilitate the development of novel fermentation technologies for the production of value-added chemicals from biomass feedstocks, such as electro-fermentation. The information provided in this study will also improve our understanding of the ecological strategies of bacteria living in redox-stratified environments.

Computational Modeling Analysis of Kinetics of Fumarate Reductase Activity and ROS Production during Reverse Electron Transfer in Mitochondrial Respiratory Complex II.

Reverse electron transfer in mitochondrial complex II (CII) plays an important role in hypoxia/anoxia, in particular, in ischemia, when the blood supply to an organ is disrupted and oxygen is not available. A computational model of CII was developed in this work to facilitate the quantitative analysis of the kinetics of quinol-fumarate reduction as well as ROS production during reverse electron transfer in CII. The model consists of 20 ordinary differential equations and 7 moiety conservation equations. The parameter values were determined at which the kinetics of electron transfer in CII in both forward and reverse directions would be explained simultaneously. The possibility of the existence of the "tunnel diode" behavior in the reverse electron transfer in CII, where the driving force is QH2, was tested. It was found that any high concentrations of QH2 and fumarate are insufficient for the appearance of a tunnel effect. The results of computer modeling show that the maximum rate of succinate production cannot provide a high concentration of succinate in ischemia. Furthermore, computational modeling results predict a very low rate of ROS production, about 50 pmol/min/mg mitochondrial protein, which is considerably less than 1000 pmol/min/mg protein observed in CII in forward direction.

Dissecting Reaction Mechanisms and Catalytic Contributions in Flavoprotein Fumarate Reductases.

The interconversion between fumarate and succinate is fundamental to the energy metabolism of nearly all organisms. This redox reaction is catalyzed by a large family of enzymes, fumarate reductases and succinate dehydrogenases, using hydride and proton transfers from a flavin cofactor and a conserved Arg side-chain. These flavoenzymes also have substantial biomedical and biotechnological importance. Therefore, a detailed understanding of their catalytic mechanisms is valuable. Here, calibrated electronic structure calculations in a cluster model of the active site of the Fcc3 fumarate reductase were employed to investigate various reaction pathways and possible intermediates in the enzymatic environment and to dissect interactions that contribute to catalysis of fumarate reduction. Carbanion, covalent adduct, carbocation, and radical intermediates were examined. Significantly lower barriers were obtained for mechanisms via carbanion intermediates, with similar activation energies for hydride and proton transfers. Interestingly, the carbanion bound to the active site is best described as an enolate. Hydride transfer is stabilized by a preorganized charge dipole in the active site and by the restriction of the C1-C2 bond in a twisted conformation of the otherwise planar fumarate dianion. But, protonation of a fumarate carboxylate and quantum tunneling effects are not critical for catalysis of the hydride transfer. Calculations also suggest that the driving force for enzyme turnover is provided by regeneration of the catalytic Arg, either coupled with flavin reduction and decomposition of a proposed transient state or directly from the solvent. The detailed mechanistic description of enzymatic reduction of fumarate provided here clarifies previous contradictory views and provides new insights into catalysis by essential flavoenzyme reductases and dehydrogenases.

Cancer-cell-derived fumarate suppresses the anti-tumor capacity of CD8+ T cells in the tumor microenvironment.

Metabolic alterations in the microenvironment significantly modulate tumor immunosensitivity, but the underlying mechanisms remain obscure. Here, we report that tumors depleted of fumarate hydratase (FH) exhibit inhibition of functional CD8+ T cell activation, expansion, and efficacy, with enhanced malignant proliferative capacity. Mechanistically, FH depletion in tumor cells accumulates fumarate in the tumor interstitial fluid, and increased fumarate can directly succinate ZAP70 at C96 and C102 and abrogate its activity in infiltrating CD8+ T cells, resulting in suppressed CD8+ T cell activation and anti-tumor immune responses in vitro and in vivo. Additionally, fumarate depletion by increasing FH expression strongly enhances the anti-tumor efficacy of anti-CD19 CAR T cells. Thus, these findings demonstrate a role for fumarate in controlling TCR signaling and suggest that fumarate accumulation in the tumor microenvironment (TME) is a metabolic barrier to CD8+ T cell anti-tumor function. And potentially, fumarate depletion could be an important strategy for tumor immunotherapy.

Monomethyl fumarate prevents alloimmune rejection in mouse heart transplantation by inducing tolerogenic dendritic cells.

Dendritic cells (DCs) are important targets for eliciting allograft rejection after transplantation. Previous studies have demonstrated that metabolic reprogramming of DCs can transform their immune functions and induce their differentiation into tolerogenic DCs. In this study, we aim to investigate the protective effects and mechanisms of monomethyl fumarate (MMF), a bioactive metabolite of fumaric acid esters, in a mouse model of allogeneic heart transplantation. Bone marrow-derived DCs are harvested and treated with MMF to determine the impact of MMF on the phenotype and immunosuppressive function of DCs by flow cytometry and T-cell proliferation assays. RNA sequencing and Seahorse analyses are performed for mature DCs and MMF-treated DCs (MMF-DCs) to investigate the underlying mechanism. Our results show that MMF prolongs the survival time of heart grafts and inhibits the activation of DCs in vivo. MMF-DCs exhibit a tolerogenic phenotype and function in vitro. RNA sequencing and Seahorse analyses reveal that MMF activates the Nrf2 pathway and mediates metabolic reprogramming. Additionally, MMF-DC infusion prolongs cardiac allograft survival, induces regulatory T cells, and inhibits T-cell activation. MMF prevents allograft rejection in mouse heart transplantation by inducing tolerogenic DCs.

Assessment of the sensitivity of 2 H MR spectroscopy measurements of [2,3-2 H2 ]fumarate metabolism for detecting tumor cell death.

Imaging the metabolism of [2,3-2 H2 ]fumarate to produce malate can be used to detect tumor cell death post-treatment. Here, we assess the sensitivity of the technique for detecting cell death by lowering the concentration of injected [2,3-2 H2 ]fumarate and by varying the extent of tumor cell death through changes in drug concentration. Mice were implanted subcutaneously with human triple negative breast cancer cells (MDA-MB-231) and injected with 0.1, 0.3, and 0.5 g/kg [2,3-2 H2 ]fumarate before and after treatment with a multivalent TRAlL-R2 agonist (MEDI3039) at 0.1, 0.4, and 0.8 mg/kg. Tumor conversion of [2,3-2 H2 ]fumarate to [2,3-2 H2 ]malate was assessed from a series of 13 spatially localized 2 H MR spectra acquired over 65 min using a pulse-acquire sequence with a 2-ms BIR4 adiabatic excitation pulse. Tumors were then excised and stained for histopathological markers of cell death: cleaved caspase 3 (CC3) and DNA damage (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]). The rate of malate production and the malate/fumarate ratio plateaued at tumor fumarate concentrations of 2 mM, which were obtained with injected [2,3-2 H2 ]fumarate concentrations of 0.3 g/kg and above. Tumor malate concentration and the malate/fumarate ratio increased linearly with the extent of cell death determined histologically. At an injected [2,3-2 H2 ]fumarate concentration of 0.3 g/kg, 20% CC3 staining corresponded to a malate concentration of 0.62 mM and a malate/fumarate ratio of 0.21. Extrapolation indicated that there would be no detectable malate at 0% CC3 staining. The use of low and nontoxic fumarate concentrations and the production of [2,3-2 H2 ]malate at concentrations that are within the range that can be detected clinically suggest this technique could translate to the clinic.

Effects of dietary disodium fumarate supplementation on muscle quality, chemical composition, oxidative stress and lipid metabolism of Hu sheep induced by high concentrate diet.

Long-term feeding of high-concentrate (HC) diet causes the decrease of rumen pH, and induces subacute rumen acidosis (SARA), which results in metabolic disorders in sheep. This not only reduces animal performance, but also increases the risk of oxidative stress and inflammatory reaction. Disodium fumarate can improve the rumen buffering capacity and increase rumen pH. This experiment was conducted to investigate the effects of high concentrate diet on muscle quality, chemical composition, oxidative damage and lipid metabolism of Hu sheep, and the regulating effect of disodium fumarate. The results showed that HC diet induced SARA by reducing rumen pH value, thus causing oxidative stress and lipid metabolism disorder in longissimus lumborum (LL) muscle of Hu sheep, which also reduced meat quality by increasing shear force, drip loss, cooking loss, chewiness and hardness, and reducing the contents of crude fat and crude protein in LL muscle. However, disodium fumarate can improve meat quality of SARA Hu sheep by regulating rumen pH, inhibiting muscle oxidative stress and promoting lipid metabolism.