Tricarboxylic Acid Cycle Enzymes Research Articles

Protective effects of N-acetylcysteine against hyperoxaluria induced mitochondrial dysfunction in male wistar rats.

The purpose of the present study was to evaluate the nephro-protective potential of N-acetylcysteine against hyperoxaluria-induced renal mitochondrial dysfunction in rats. Nine days dosing of 0.4 % ethylene glycol +1 % ammonium chloride, developed hyperoxaluria in male wistar rats which resulted in renal injury and dysfunction as supported by increased level of urinary lactate dehydrogenase, calcium, and decreased creatinine clearance. Mitochondrial oxidative strain in hyperoxaluric animals was evident by decreased levels of superoxide dismutase, glutathione peroxidase, glutathione reductase, reduced glutathione, and an increased lipid peroxidation. Declined activities of respiratory chain enzymes and tricarboxylic acid cycle enzymes showed mitochondrial dysfunction in hyperoxaluric animals. N-acetylcysteine (50 mg/kg, i.p.), by virtue of its -SH reviving power, was able to increase the glutathione levels and thus decrease the oxidative stress in renal mitochondria. Hence, mitochondrial damage is, evidently, an essential event in ethylene glycol-induced hyperoxaluria and N-acetylcysteine presented itself as a safe and effective remedy in combating nephrolithiasis.

Genetic investigation of tricarboxylic acid metabolism during the Plasmodium falciparum life cycle.

New antimalarial drugs are urgently needed to control drug-resistant forms of the malaria parasite Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted life-cycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate-dehydrogenase-deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of (13)C-isotope-labeled TCA mutant parasites showed that P.falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development.

The Variations of Glycolysis and TCA Cycle Intermediate Levels Grown in Iron and Copper Mediums of Trichoderma harzianum.

The efficiency of optimal metabolic function by microorganism depends on various parameters, especially essential metal supplementation. In the present study, the effects of iron and copper metals on metabolism were investigated by determination of glycolysis and tricarboxylic acid (TCA) cycle metabolites' levels with respect to the metal concentrations and incubation period in Trichoderma harzianum. The pyruvate and citrate levels of T. harzianum increased up to 15 mg/L of copper via redirection of carbon flux though glycolysis by suppression of pentose phosphate pathway (PPP). However, the α-ketoglutarate levels decreased at concentration higher than 5 mg/L of copper to overcome damage of oxidative stress. The fumarate levels correlated with the α-ketoglutarate levels because of substrate limitation. Besides, in T. harzianum cells grown in various concentrations of iron-containing medium, the intracellular pyruvate, citrate, and α-ketoglutarate levels showed positive correlation with iron concentration due to modifying of expression of glycolysis and TCA cycle enzymes via a mechanism involving cofactor or allosteric regulation. However, as a result of consuming of prior substrates required for fumarate production, its levels rose up to 10 mg/L.

Brain glucose metabolism in an animal model of depression

An increasing number of data support the involvement of disturbances in glucose metabolism in the pathogenesis of depression. We previously reported that glucose and glycogen concentrations in brain structures important for depression are higher in a prenatal stress model of depression when compared with control animals. A marked rise in the concentrations of these carbohydrates and glucose transporters were evident in prenatally stressed animals subjected to acute stress and glucose loading in adulthood. To determine whether elevated levels of brain glucose are associated with a change in its metabolism in this model, we assessed key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase), products of glycolysis, i.e., pyruvate and lactate, and two selected enzymes of the tricarboxylic acid cycle (pyruvate dehydrogenase and α-ketoglutarate dehydrogenase) in the hippocampus and frontal cortex. Additionally, we assessed glucose-6-phosphate dehydrogenase activity, a key enzyme in the pentose phosphate pathway (PPP). Prenatal stress increased the levels of phosphofructokinase, an important glycolytic enzyme, in the hippocampus and frontal cortex. However, prenatal stress had no effect on hexokinase or pyruvate kinase levels. The lactate concentration was elevated in prenatally stressed rats in the frontal cortex, and pyruvate levels remained unchanged. Among the tricarboxylic acid cycle enzymes, prenatal stress decreased the level of pyruvate dehydrogenase in the hippocampus, but it had no effect on α-ketoglutarate dehydrogenase. Like in the case of glucose and its transporters, also in the present study, differences in markers of glucose metabolism between control animals and those subjected to prenatal stress were not observed under basal conditions but in rats subjected to acute stress and glucose load in adulthood. Glucose-6-phosphate dehydrogenase activity was not reduced by prenatal stress but was found to be even higher in animals exposed to all experimental conditions, i.e., prenatal stress, acute stress, and glucose administration. Our data indicate that glycolysis is increased and the Krebs cycle is decreased in the brain of a prenatal stress animal model of depression.

Mitochondria: key players in the neurotoxic effects of amphetamines.

Amphetamines are a class of psychotropic drugs with high abuse potential, as a result of their stimulant, euphoric, emphathogenic, entactogenic, and hallucinogenic properties. Although most amphetamines are synthetic drugs, of which methamphetamine, amphetamine, and 3,4-methylenedioxymethamphetamine ("ecstasy") represent well-recognized examples, the use of natural related compounds, namely cathinone and ephedrine, has been part of the history of humankind for thousands of years. Resulting from their amphiphilic nature, these drugs can easily cross the blood-brain barrier and elicit their well-known psychotropic effects. In the field of amphetamines' research, there is a general consensus that mitochondrial-dependent pathways can provide a major understanding concerning pathological processes underlying the neurotoxicity of these drugs. These events include alterations on tricarboxylic acid cycle's enzymes functioning, inhibition of mitochondrial electron transport chain's complexes, perturbations of mitochondrial clearance mechanisms, interference with mitochondrial dynamics, as well as oxidative modifications in mitochondrial macromolecules. Additionally, other studies indicate that amphetamines-induced neuronal toxicity is closely regulated by B cell lymphoma 2 superfamily of proteins with consequent activation of caspase-mediated downstream cell death pathway. Understanding the molecular mechanisms at mitochondrial level involved in amphetamines' neurotoxicity can help in defining target pathways or molecules mediating these effects, as well as in developing putative therapeutic approaches to prevent or treat the acute- or long-lasting neuropsychiatric complications seen in human abusers.

Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease.

Cellular senescence can modulate various pathologies and is associated with irreparable DNA double-strand breaks (IrrDSBs). Extracellular senescence metabolomes (ESMs) were generated from fibroblasts rendered senescent by proliferative exhaustion (PEsen) or 20 Gy of γ rays (IrrDSBsen) and compared with those of young proliferating cells, confluent cells, quiescent cells, and cells exposed to repairable levels of DNA damage to identify novel noninvasive markers of senescent cells. ESMs of PEsen and IrrDSBsen overlapped and showed increased levels of citrate, molecules involved in oxidative stress, a sterol, monohydroxylipids, tryptophan metabolism, phospholipid, and nucleotide catabolism, as well as reduced levels of dipeptides containing branched chain amino acids. The ESM overlaps with the aging and disease body fluid metabolomes, supporting their utility in the noninvasive detection of human senescent cells in vivo and by implication the detection of a variety of human pathologies. Intracellular metabolites of senescent cells showed a relative increase in glycolysis, gluconeogenesis, the pentose-phosphate pathway, and, consistent with this, pyruvate dehydrogenase kinase transcripts. In contrast, tricarboxylic acid cycle enzyme transcript levels were unchanged and their metabolites were depleted. These results are surprising because glycolysis antagonizes senescence entry but are consistent with established senescent cells entering a state of low oxidative stress.

CceR and AkgR regulate central carbon and energy metabolism in alphaproteobacteria.

ABSTRACT Many pathways of carbon and energy metabolism are conserved across the phylogeny, but the networks that regulate their expression or activity often vary considerably among organisms. In this work, we show that two previously uncharacterized transcription factors (TFs) are direct regulators of genes encoding enzymes of central carbon and energy metabolism in the alphaproteobacterium Rhodobacter sphaeroides. The LacI family member CceR (RSP_1663) directly represses genes encoding enzymes in the Entner-Doudoroff pathway, while activating those encoding the F1F0 ATPase and enzymes of the tricarboxylic acid (TCA) cycle and gluconeogenesis, providing a direct transcriptional network connection between carbon and energy metabolism. We identified bases that are important for CceR DNA binding and showed that DNA binding by this TF is inhibited by 6-phosphogluconate. We also showed that the GntR family TF AkgR (RSP_0981) directly activates genes encoding several TCA cycle enzymes, and we identified conditions where its activity is increased. The properties of single and double ΔCceR and ΔAkgR mutants illustrate that these 2 TFs cooperatively regulate carbon and energy metabolism. Comparative genomic analysis indicates that CceR and AkgR orthologs are found in other alphaproteobacteria, where they are predicted to have a conserved function in regulating central carbon metabolism. Our characterization of CceR and AkgR has provided important new insight into the networks that control central carbon and energy metabolism in alphaproteobacteria that can be exploited to modify or engineer new traits in these widespread and versatile bacteria.

Fumarate induces redox-dependent senescence by modifying glutathione metabolism.

Mutations in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) are associated with a highly malignant form of renal cancer. We combined analytical chemistry and metabolic computational modelling to investigate the metabolic implications of FH loss in immortalized and primary mouse kidney cells. Here, we show that the accumulation of fumarate caused by the inactivation of FH leads to oxidative stress that is mediated by the formation of succinicGSH, a covalent adduct between fumarate and glutathione. Chronic succination of GSH, caused by the loss of FH, or by exogenous fumarate, leads to persistent oxidative stress and cellular senescence in vitro and in vivo. Importantly, the ablation of p21, a key mediator of senescence, in Fh1-deficient mice resulted in the transformation of benign renal cysts into a hyperplastic lesion, suggesting that fumarate-induced senescence needs to be bypassed for the initiation of renal cancers.

Activity assay for plant mitochondrial enzymes.

Mitochondria are sites for respiration to produce chemical energy via oxidative phosphorylation. Their primary role has been viewed as the oxidation of organic acids via the tricarboxylic acid (TCA) cycle and the synthesis of ATP coupled to the transfer of electrons to O2. TCA cycle enzymes are essential for plant carbon metabolism and provide the reductant for the electron transport chain (ETC) enzymes that in turn drives ATP synthesis. The activity of individual enzymes will determine the flux of metabolism and thus the downstream consequences for respiration rate. Measurements of activities of mitochondrial enzymes, such as components of TCA cycle and the ETC, can provide insight into regulation of mitochondrial function. The activities of these enzymes vary between developmental stages, in different tissues, and in response to environmental conditions. In this chapter, methods for enzymatic assay of TCA cycle enzymes and a number of the ETC complex enzymes are described in detail.

Genetic variants in genes of tricarboxylic acid cycle key enzymes are associated with prognosis of patients with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is characterized by poor prognosis and only a few molecular markers may be potentially used to predict the outcome. Metabolic reprogramming is a hallmark of cancer, including the alterations of tricarboxylic acid (TCA) cycle key enzymes. However, the significance of single nucleotide polymorphisms (SNPs) in genes encoding these key enzymes has not been investigated in NSCLC. In this study, we genotyped 18 potentially functional SNPs in 7 genes belonging to 3 TCA cycle enzyme families (SDH, FH and IDH) using Sequenom iPLEX genotyping system in a cohort of 500 NSCLC patients. Multivariate Cox proportional hazards model and Kaplan-Meier curve were used for the survival analysis. Our results showed that SDHC gene: SNP rs12064957, IDH2 gene: SNP rs11540478 and FH gene: SNP rs1414493 were associated with overall survival (OS) and SDHA gene: SNP rs13173911, IDH2 gene: SNP rs4932158 were associated with recurrence-free survival (RFS) of NSCLC patients. Unfavorable genotypes of these SNPs showed a significant cumulative effect on OS and RFS of NSCLC patients (both P<0.001). Furthermore, survival tree analysis indicated that FH: rs1414493 was the primary risk factor contributing to OS of NSCLC patients and the IDH2: rs4932158 was the primary risk factor contributing to RFS of NSCLC patients. Our data suggest that SNPs in TCA cycle key enzyme genes may serve as potential biomarkers to predict the outcomes of NSCLC. Further studies with different ethnicities are needed to validate our findings and generalize their clinical utility.

The deep end of the metabolite pool: influences on epigenetic regulatory mechanisms in cancer.

Epigenetic control of gene expression is mediated by cytosine methylation/demethylation and histone modifications including methylation, acetylation and glycosylation. The epigenetic programme is corrupted in cancer cells to maintain a pattern of gene expression that leads to their de-differentiated, rapidly proliferating phenotype. Enzymes responsible for modifying histones and cytosine are sensitive to the cellular metabolite pool and can be activated by an increase in their substrates or inhibited by an increase in their products or competitors for substrate binding. This review is based on publications identified on PubMed using a literature search of cytosine methylation, histone methylation, acetylation and glycosylation. In cancer, changes in glycolytic enzymes lead to increased production of serine, increasing the pool of S-adenosylmethionine (the major methyl donor for methylation reactions) and UDP-N-acetylglucosamine (a substrate for O-linked glycosylation of histones and cytosine methyltransferases). Mutations in tricarboxylic acid cycle enzymes lead to accumulation of fumarate, succinate and hydroxyglutarate, all of which inhibit demethylation of cytosine and histones. In contrast, proline catabolism produces α-ketoglutarate and reactive oxygen, both of which promote the activity of enzymes that remove methyl groups from cytosine and histones, and the key enzyme in proline catabolism acts as a tumour suppressor. Our emerging understanding of how the epigenetic profiles are metabolically reprogrammed in cancer cells will lead to novel diagnostic and therapeutic targets for treatment of patients.

Histone deacetylase inhibitors reverse manic-like behaviors and protect the rat brain from energetic metabolic alterations induced by ouabain

Studies have revealed alterations in mitochondrial complexes in the brains of bipolar patients. However, few studies have examined changes in the enzymes of the tricarboxylic acid cycle. Several preclinical studies have suggested that histone deacetylase inhibitors may have antimanic effects. The present study aims to investigate the effects of lithium, valproate and sodium butyrate, a histone deacetylase inhibitor, on the activity of tricarboxylic acid cycle enzymes in the brains of rats subjected to an animal model of mania induced by ouabain. Wistar rats received a single intracerebroventricular injection of ouabain or cerebrospinal fluid. Starting on the day following the intracerebroventricular injection, the rats were treated for 7days with intraperitoneal injections of saline, lithium, valproate or sodium butyrate. Risk-taking behavior, locomotor and exploratory activities were measured using the open-field test. Citrate synthase, succinate dehydrogenase, and malate dehydrogenase were examined in the frontal cortex and hippocampus. All treatments reversed ouabain-related risk-taking behavior and hyperactivity in the open-field test. Ouabain inhibited tricarboxylic acid cycle enzymes in the brain, and valproate and sodium butyrate but not lithium reversed this ouabain-induced dysfunction. Thus, protecting the tricarboxylic acid cycle may contribute to the therapeutic effects of histone deacetylase inhibitors.

2-Oxoglutarate: linking TCA cycle function with amino acid, glucosinolate, flavonoid, alkaloid, and gibberellin biosynthesis.

The tricarboxylic acid (TCA) cycle intermediate 2-oxoglutarate (2-OG) is used as an obligatory substrate in a range of oxidative reactions catalyzed by 2-OG-dependent dioxygenases. These enzymes are widespread in nature being involved in several important biochemical processes. We have recently demonstrated that tomato plants in which the TCA cycle enzyme 2-OG dehydrogenase (2-ODD) was antisense inhibited were characterized by early senescence and modified fruit ripening associated with differences in the levels of bioactive gibberellin (GA). Accordingly, there is now compelling evidence that the TCA cycle plays an important role in modulating the rate of flux from 2-OG to amino acid metabolism. Here we discuss recent advances in the biochemistry and molecular biology of 2-OG metabolism occurring in different biological systems indicating the importance of 2-OG and 2-OG dependent dioxygenases not only in glucosinolate, flavonoid and alkaloid metabolism but also in GA and amino acid metabolism. We additionally summarize recent findings regarding the impact of modification of 2-OG metabolism on biosynthetic pathways involving 2-ODDs.

Paraganglioma and pheochromocytoma upon maternal transmission of SDHD mutations.

BackgroundThe SDHD gene encodes a subunit of the mitochondrial tricarboxylic acid cycle enzyme and tumor suppressor, succinate dehydrogenase. Mutations in this gene show a remarkable pattern of parent-of-origin related tumorigenesis, with almost all SDHD-related cases of head and neck paragangliomas and pheochromocytomas attributable to paternally-transmitted mutations.MethodsHere we explore the underlying molecular basis of three cases of paraganglioma or pheochromocytoma that came to our attention due to apparent maternal transmission of an SDHD mutation. We used DNA analysis of family members to establish the mode of inheritance of each mutation. Genetic and immunohistochemical studies of available tumors were then carried out to confirm SDHD-related tumorigenesis.ResultsWe found convincing genetic and immunohistochemical evidence for the maternally-related occurrence of a case of pheochromocytoma, and suggestive evidence in a case of jugular paraganglioma. The third case appears to be a phenocopy, a sporadic paraganglioma in an SDHD mutation carrier with no immunohistochemical or DNA evidence to support a causal link between the mutation and the tumor. Microsatellite analysis in the tumor of patient 1 provided evidence for somatic recombination and loss of the paternal region of chromosome 11 including SDHD and the maternal chromosome including the centromere and the p arm.ConclusionsTransmission of SDHD mutations via the maternal line can, in rare cases, result in tumorigenesis. Despite this finding, the overwhelming majority of carriers of maternally-transmitted mutations will remain tumor-free throughout life.

Evolution and Functional Implications of the Tricarboxylic Acid Cycle as Revealed by Phylogenetic Analysis

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.

Curcumin and hesperidin improve cognition by suppressing mitochondrial dysfunction and apoptosis induced by D-galactose in rat brain

D-galactose, a reducing sugar, induces oxidative stress resulting in alteration in mitochondrial dynamics and apoptosis of neurons. Curcumin and hesperidin are antioxidants possessing multimodal functions; hence, their contribution in minimizing D-galactose induced ageing was assessed in the present study. A week prior to D-galactose treatment (150 mg/kg; s.c. for 56 days), animals were treated with curcumin alone, hesperidin alone and a combination of curcumin (50 and 100 mg/kg; orally) with hesperidin (10 and 25 mg/kg; orally) for 63 days. A naïve control was also maintained. Behavioural studies, tricarboxylic acid cycle enzymes, mitochondrial complexes, protein and lipid oxidation and glutathione levels were assessed in the brain mitochondrial fraction. Western blot analysis of caspase-3, cleaved caspase-3 and histological assessment of the CA1 region of the hippocampus were carried out. D-galactose induced significant cognitive deficits, biochemical changes and histological alterations. Individually, curcumin was more effective than hesperidin in reducing the levels of oxidized lipids, proteins, cleaved caspase-3 expression and mitochondrial enzymes. The combination reduced the expression of cleaved caspase-3, malondialdehyde, improved mitochondrial enzymes and glutathione levels. In combination, curcumin and hesperidin protect the morphological facets and improve biochemical functions of neurons thereby improving cognition.

Inhibition of akt phosphorylation diminishes mitochondrial biogenesis regulators, tricarboxylic acid cycle activity and exacerbates recognition memory deficit in rat model of Alzheimer's disease.

3-Methyladenine (3-MA), as a PI3K inhibitor, is widely used for inhibition of autophagy. Inhibition of PI3K class I leads to inhibition of Akt phosphorylation, a central molecule involved in diverse arrays of intracellular cascades in nervous system. Accordingly, in the present study, we aimed to determine the alterations of specific mitochondrial biogenesis markers and mitochondrial function in 3-MA-injected rats following amyloid beta (Aβ) insult. Our data revealed that inhibition of Akt phosphorylation downregulates master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Our data also showed that decrease in PGC-1α level presumably is due to decrease in the phosphorylation of cAMP-response element binding and AMP-activated kinase, two upstream activators of PGC-1α. As a consequence, the level of some mitochondrial biogenesis factors including nuclear respiratory factor-1, mitochondrial transcription factor A, and Cytochrome c decreased significantly. Also, activities of tricarboxylic acid cycle (TCA) enzymes such as Aconitase, a-ketoglutarate dehydrogenase, and malate dehydrogenase reduced in the presence of 3-MA with or without Aβ insult. Decrease in mitochondrial biogenesis factors and TCA enzyme activity in the rats receiving 3-MA and Aβ were more compared to the rats that received either alone; indicating the additive destructive effects of these two agents. In agreement with our molecular results, data obtained from behavioral test (using novel objective recognition test) indicated that inhibition of Akt phosphorylation with or without Aβ injection impaired novel recognition (non-spatial) memory. Our results suggest that 3-MA amplified deleterious effects of Aβ by targeting central molecule Akt.

Genetic variants in genes of tricarboxylic acid cycle key enzymes predict postsurgical overall survival of patients with hepatocellular carcinoma.

Metabolic reprogramming is a hallmark of cancer, including the alterations of activity and expression in tricarboxylic acid (TCA) cycle key enzymes. However, the significance of single nucleotide polymorphisms (SNPs) in genes encoding these key enzymes has not been investigated in hepatocellular carcinoma (HCC). In this study, 17 SNPs in seven genes encoding three TCA cycle enzyme families (SDH, FH, and IDH) were genotyped in 492 HCC patients with surgical treatment and their association with overall survival (OS) was analyzed. Five SNPs in four genes were identified to be associated with OS in HCC patients. Among them, rs3935401 in the 3' untranslated region of SDHC exhibited the most significant association (P < 0.001). The unfavorable genotype of these five SNPs showed a significant accumulative effect on the prognosis of HCC patients, with a P for trend of <0.001. Furthermore, the haplotype group consisting of wild type in rs4131826 and variant in rs3935401 was significantly associated with increased risk of death in HCC patients. Survival tree analysis indicated that variant genotype of rs3935401 was the primary risk factor contributing to the prediction of OS in HCC patients. SNPs in TCA cycle key enzyme genes may serve as potential biomarkers to predict the OS in HCC patients.

The Oncometabolite Fumarate Promotes Pseudohypoxia Through Noncanonical Activation of NF-κB Signaling

Inactivating mutations of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH) have been linked to an aggressive variant of hereditary kidney cancer (hereditary leiomyomatosis and renal cell cancer). These tumors accumulate markedly elevated levels of fumarate. Fumarate is among a growing list of oncometabolites identified in cancers with mutations of genes involved in intermediary metabolism. FH-deficient tumors are notable for their pronounced accumulation of the transcription factor hypoxia inducible factor-1α (HIF-1α) and aggressive behavior. To date, HIF-1α accumulation in hereditary leiomyomatosis and renal cell cancer tumors is thought to result from fumarate-dependent inhibition of prolyl hydroxylases and subsequent evasion from von Hippel-Lindau-dependent degradation. Here, we demonstrate a novel mechanism by which fumarate promotes HIF-1α mRNA and protein accumulation independent of the von Hippel-Lindau pathway. Here we demonstrate that fumarate promotes p65 phosphorylation and p65 accumulation at the HIF-1α promoter through non-canonical signaling via the upstream Tank binding kinase 1 (TBK1). Consistent with these data, inhibition of the TBK1/p65 axis blocks HIF-1α accumulation in cellular models of FH loss and markedly reduces cell invasion of FH-deficient RCC cancer cells. Collectively, our data demonstrate a novel mechanism by which pseudohypoxia is promoted in FH-deficient tumors and identifies TBK1 as a novel putative therapeutic target for the treatment of aggressive fumarate-driven tumors.

The effect of aspartate on the energy metabolism in the liver of weanling pigs challenged with lipopolysaccharide.

This study was conducted to investigate whether aspartate (Asp) could improve liver energy status in the lipopolysaccharide (LPS)-challenged pigs. Twenty-four weaned pigs were assigned to four treatments: (1) nonchallenged control (control diet and saline-treated); (2) LPS-challenged control (the same control diet and LPS-challenged); (3) LPS + 0.5% Asp treatment (0.5% Asp diet and LPS-challenged); and (4) LPS + 1.0% Asp treatment (a 1.0% Asp diet and LPS-challenged). On d 19, the pigs were injected intraperitoneally with Escherichia coli LPS at 100 μg/kg body weight, and the same volume of 0.9% NaCl solution, respectively. All pigs were slaughtered at 24 h after LPS or saline injection, and the liver was collected for further analysis. Dietary supplementation with Asp improved liver energy status evidenced by the increased ATP concentration and adenylate energy charges, and the decreased AMP concentration and AMP/ATP ratio (p < 0.05). Asp supplementation increased the mRNA expression of key enzymes in hepatic glycolysis and tricarboxylic acid (TCA) cycle, including pyruvate kinase and citrate synthase (p < 0.05), and had a tendency to increase hepatic pyruvate dehydrogenase and isocitrate dehydrogenase β mRNA expression (p < 0.10). In addition, Asp increased the mRNA expressions of hepatic AMP-activated protein kinase (AMPK) α1, AMPKα2, silent information regulator (Sirt1), and proliferator-activated receptor-γ coactivator 1α (PGC1α) (p < 0.05). Moreover, Asp increased AMPKα phosphorylation (p < 0.05). These results indicated that dietary supplementation of Asp could improve energy status in LPS-injured liver, which might result from motivating the metabolism pathway of TCA cycle and glycolysis and stimulating the AMPK signaling pathway.