Intracellular Glutamate Levels Research Articles

Pyroptotic macrophages induce disruption of glutamate metabolism in periodontal ligament stem cells contributing to their compromised osteogenic potential.

Macrophage pyroptosis is of key importance to host defence against pathogen infections and may participate in the progression and recovery of periodontitis. However, the role of pyroptotic macrophages in regulating periodontal ligament stem cells (PDLSCs), the main cell source for periodontium renewal, remains unclear. First, we found that macrophage pyroptosis were enriched in gingiva tissues from periodontitis patients compared with those of healthy people through immunofluorescence. Then the effects of pyroptotic macrophages on the PDLSC osteogenic differentiation were investigated in a conditioned medium (CM)-based coculture system in vitro. CM derived from pyroptotic macrophages inhibited the osteogenic differentiation-related gene and protein levels, ALP activity and mineralized nodule formation of PDLSCs. The osteogenic inhibition of CM was alleviated when pyroptosis was inhibited by VX765. Further, untargeted metabolomics showed that glutamate limitation may be the underlying mechanism. However, exogenous glutamate supplementation aggravated the CM-inhibited osteogenic differentiation of PDLSCs. Moreover, CM increased extracellular glutamate and decreased intracellular glutamate levels of PDLSCs, and enhanced the gene and protein expression levels of system xc - (a cystine/glutamate antiporter). After adding cystine to CM-based incubation, the compromised osteogenic potency of PDLSCs was rescued. Our data suggest that macrophage pyroptosis is related to the inflammatory lesions of periodontitis. Either pharmacological inhibition of macrophage pyroptosis or nutritional supplements to PDLSCs, can rescue the compromised osteogenic potency caused by pyroptotic macrophages.

Disrupted biosynthesis of glycogen and PHB combined with physicochemical factors increased bioproduction of γ-aminobutyric acid and δ-aminolevulinic acid in cyanobacteria

In Synechocystis sp. PCC 6803, redirecting the metabolic flow from upstream substrate for carbon metabolism i.e. glyceraldehyde-3-phosphate (the glycogen synthesis precursor) and poly(3-hydroxybutyrate) (PHB), towards ALA or GABA has not been studied. The PHB synthesis, one of the pathways consuming carbon via glycolysis pathway, was disrupted to generate the strain, ΔP, and another adjacent carbon utilizing pathway that is responsible for glycogen synthesis was also disrupted in the earlier mutant strain to generate ΔGP with the aim to increase carbon flux towards precursor metabolites for GABA and ALA biosynthesis. The ΔP strain showed an increase in intracellular glutamate and ALA levels (∼19.5 and 2.5 ng g−1 DCW, respectively), whereas intracellular GABA levels were found increased (up to 7 ng g−1 DCW) in ΔGP strain as compared to the wild type strain (WT). Modified nutrient conditions including glucose, glutamate and LA supplementation as well as N-deprivation led to several fold increase in GABA and ALA levels in ΔP and ΔGP strains in comparison to those observed in WT, with the maximum GABA and ALA accumulation (∼262 and 48 ng g−1 DCW, respectively) noticed in ΔP. Abiotic stresses including cold and UV irradiation also significantly improved the intracellular GABA and ALA accumulation in mutant strains. Increased biosynthesis of GABA and ALA in mutant strains was found correlated with the increased mRNA levels of genes involved in GABA, ALA and TCA cycle metabolism. Further organic acids profiling revealed that the disruption of two pathways redirects carbon routes to TCA cycle without affecting the viability of cells. This strategy might be applicable in strain development for enhancing other high value bioproducts synthesized by connected metabolic pathways that utilize carbon sources diverted from non-essential metabolism.

Glycogen deficiency enhances carbon partitioning into glutamate for an alternative extracellular metabolic sink in cyanobacteria

Glycogen serves as a metabolic sink in cyanobacteria. Glycogen deficiency causes the extracellular release of distinctive metabolites such as pyruvate and 2-oxoglutarate upon nitrogen depletion; however, the mechanism has not been fully elucidated. This study aimed to elucidate the mechanism of carbon partitioning in glycogen-deficient cyanobacteria. Extracellular and intracellular metabolites in a glycogen-deficient ΔglgC mutant of Synechococcus elongatus PCC 7942 were comprehensively analyzed. In the presence of a nitrogen source, the ΔglgC mutant released extracellular glutamate rather than pyruvate and 2-oxoglutarate, whereas its intracellular glutamate level was lower than that in the wild-type strain. The de novo synthesis of glutamate increased in the ΔglgC mutant, suggesting that glycogen deficiency enhanced carbon partitioning into glutamate and extracellular excretion through an unidentified transport system. This study proposes a model in which glutamate serves as the prime extracellular metabolic sink alternative to glycogen when nitrogen is available.

Comparative transcriptome analysis reveals the contribution of amino acid transporters to acid tolerance in Lactococcus lactis

AbstractAmino acid transporters are promising targets for engineering acid‐tolerant strains of Lactococcus lactis. However, the simple overexpression of transporters alone is insufficient to achieve a highly acid‐resistant phenotype. This study investigated the effects of amino acid transporters on the acid‐stress tolerance of L. lactis. Here, we first verified the contribution of amino acid transporters to acid tolerance by overexpressing the ctrA, glnP, and glnQ genes in L. lactis. Transcriptome analysis revealed that most genes associated with specific amino acid transport, pyrimidine metabolism, and functional proteins, were upregulated in the overexpression strains. Among them, arginine biosynthesis (argG and argH), amino acid transport (yjeM and azlC), and pyrimidine metabolism were considered to be the most important regulatory mechanisms. Importantly, metabolite profiling revealed that the overexpression strains had higher intracellular levels of amino acids, particularly aspartate, glutamate, and arginine at low pH, as well as higher intracellular ATP levels, which was consistent with the corresponding gene‐expression levels. Finally, the simultaneous overexpression of glnP and glnQ led to a further improvement of acid tolerance in L. lactis. This study reveals the regulatory mechanisms of amino acid transporters, and provides a novel strategy for achieving higher acid tolerance via positive tandem expression approaches.

The Role of Serine-Glyoxylate Aminotransferase and Malyl-CoA Lyase in the Metabolism of Methylococcus capsulatus Bath.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin-Benson-Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C1 assimilation pathways in the gammaproteobacterial methanotrophs.

Selective Clearance of Senescent Chondrocytes in Osteoarthritis by Targeting Excitatory Amino Acid Transporter Protein 1 to Induce Ferroptosis.

Aims: This study aimed at exploring the mechanism of ferroptosis (an iron-dependent form of nonapoptotic cell death) resistance in senescent chondrocytes (SenChos). Results: In this study, by utilizing metabolomics and single-cell RNA sequencing, we found that hyperactivation of ferroptosis metabolism was one of the most prominent metabolic features in SenChos. Interestingly, however, SenChos were able to survive in this state and were resistant to ferroptosis-induced cell death. Next, we elucidated that this survival mechanism of SenChos could be primarily attributed to overexpression of the membrane protein excitatory amino acid transporter protein 1 (EAAT1), which can increase intracellular glutamate (Glu) levels and activate the glutathione system to counteract ferroptosis. In addition, 2-amino-5,6,7,8-tetrahydro-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-4H-chromene-3-carbonitrile (UCPH-101) (a specific inhibitor of EAAT1) and siRNA-EAAT1 were able to substantially increase the sensitivity of SenChos to ferroptosis and to induce cell death, with no apparent effects on the normal cells. Administration of an intraarticular injection of UCPH-101 caused inhibition of EAAT1 selectively, cleared SenChos from cartilage, improved the cartilage homeostasis, and significantly delayed the progression of osteoarthritis (OA). Innovation: This work supports a relevant role for EAAT1 in ferroptosis resistance mechanism for SenChos, revealing a potential therapeutic target of OA. Conclusions: EAAT1-Glu-glutathione peroxidase 4 anti-ferroptosis axis is a key survival mechanism for SenChos, and EAAT1 is an effective and specific target for anti-senescence therapy in OA. Antioxid. Redox Signal. 39, 262-277.

Liver X Receptor Inverse Agonist GAC0001E5 Impedes Glutaminolysis and Disrupts Redox Homeostasis in Breast Cancer Cells.

Liver X receptors (LXRs) are members of the nuclear receptor family of ligand-dependent transcription factors which regulate the expression of lipid and cholesterol metabolism genes. Moreover, LXRs and their ligands have been shown to inhibit tumor growth in a variety of cancers. We have previously identified the small molecule compound GAC0001E5 (1E5) as an LXR inverse agonist and a potent inhibitor of pancreatic cancer cells. Transcriptomic and metabolomic studies showed that 1E5 disrupts glutamine metabolism, an essential metabolic pathway commonly reprogrammed during malignant transformation, including in breast cancers. To determine the role of LXRs and potential application of 1E5 in breast cancer, we examined LXR expression in publicly available clinical samples, and found that LXR expression is elevated in breast tumors as compared to normal tissues. In luminal A, endocrine therapy-resistant, and triple-negative breast cancer cells, 1E5 exhibited LXR inverse agonist and "degrader" activity and strongly inhibited cell proliferation and colony formation. Treatments with 1E5 downregulated the transcription of key glutaminolysis genes, and, correspondingly, biochemical assays indicated that 1E5 lowered intracellular glutamate and glutathione levels and increased reactive oxygen species. These results indicate that novel LXR ligand 1E5 is an inhibitor of glutamine metabolism and redox homeostasis in breast cancers and suggest that modulating LXR activity and expression in tumor cells is a promising strategy for targeting metabolic reprogramming in breast cancer therapeutics.

Parvalbumin in the metabolic pathway of glutamate and γ-aminobutyric acid: Influence on expression of GAD65 and GAD67

Parvalbumin-expressing neurons are a type of inhibitory intermediate neuron that play an important role in terminating seizures. The aim of the present study was to use lentiviral construction and packaging technology to overexpress and silence the parvalbumin gene in pheochromocytoma (PC12) cells, and to evaluate how parvalbumin influences the metabolic pathway involving glutamate and γ-aminobutyric acid (GABA). In this work, Immunofluorescence staining was used to verify the differentiation of PC12 cells into neurons after adding nerve growth factor (NGF). Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) were used to confirm lentivirus-mediated knockdown or overexpression of parvalbumin. Expression of parvalbumin, the 65-kDa GAD isoform (GAD65), and the 67-kDa GAD isoform (GAD67) in neuronal cells was examined at the mRNA and protein levels using qRT-PCR, western blotting and immunofluorescence staining, while intracellular glutamate and GABA levels were determined by high performance liquid chromatography (HPLC). We demonstrate that the expression of parvalbumin is associated with GAD65 and GAD67. Interestingly, overexpression of parvalbumin up-regulated GAD65 and GAD67, increased GABA concentration, and decreased glutamate concentration. Silencing of parvalbumin led to the opposite effects. Altogether, parvalbumin affected the expression of GAD65 and GAD67, thereby influencing the metabolic pathway involving glutamate and GABA.

Epstein-Barr virus infection controls the concentration of the intracellular antioxidant glutathione by upregulation of the glutamate transporter EAAT3 in tumor cells.

Epstein-Barr virus or human herpesvirus 4 (EBV/HHV-4) is an omnipresent oncovirus etiologically associated with various B-cell lymphomas and epithelial cancers. The malignant transformation associated with the persistent expression of viral proteins often deregulates the host cellular machinery and EBV infection is coupled to elevated levels of reactive oxygen species. Here, we investigated the role that the glutamate transporter EAAT3 plays in regulating the antioxidant system as a protective mechanism of EBV-infected cells against the virus-induced oxidative stress. Our study demonstrated that the expression of EAAT3 was upregulated and localized to the plasma membrane in EBV latently infected and de novo EBV-infected cells. EAAT3 was regulated by the transcription factor NFAT5 in the infected cells. Membrane localized EAAT3 was found to be involved in the transportation of glutamate from the extracellular space into the cell, as EAAT3 and NFAT5 inhibitors markedly reduced the levels of intracellular glutamate levels in EBV latently infected cells. Additionally, our data demonstrated a notable decrease in the intracellular glutathione levels following treatment with an EAAT3 inhibitor. Collectively, our results suggest that upregulation of the glutamate transporter EAAT3 is an adaptation of EBV-infected cells to maintain cellular redox homeostasis against the virus-induced oxidative stress, and that this cellular balance could be therapeutically destroyed by targeting EAAT3 to impede EBV-associated cancers.

Novel allosteric glutaminase 1 inhibitors with macrocyclic structure activity relationship analysis

Glutamine-addicted cancer metabolism is recently recognized as novel cancer target especially for KRAS and KEAP1 co-occurring mutations. Selective glutaminase1 (GLS1) inhibition was reported using BPTES which has novel mode of allosteric inhibition. However, BPTES is a highly hydrophobic and symmetric molecule with very poor solubility which results in suboptimal pharmacokinetic parameters and hinders its further development. As an ongoing effort to identify more drug-like GLS1 inhibitors via systematic structure − activity relationship (SAR) analysis of BPTES analogs, we disclose our novel macrocycles for GLS1 inhibition with conclusive SAR analysis on the core, core linker, and wing linker, respectively. Selected molecules resulted in reduction in intracellular glutamate levels in LR (LDK378-resistant) cells which is consistent to cell viability result. Finally, compounds 13 selectively reduced the growth of A549 and H460 cells which have co-occurring mutations including KRAS and KEAP1.

Regulation of Cardiac Fibroblast GLS1 Expression by Scleraxis.

Fibrosis is an energy-intensive process requiring the activation of fibroblasts to myofibroblasts, resulting in the increased synthesis of extracellular matrix proteins. Little is known about the transcriptional control of energy metabolism in cardiac fibroblast activation, but glutaminolysis has been implicated in liver and lung fibrosis. Here we explored how pro-fibrotic TGFβ and its effector scleraxis, which drive cardiac fibroblast activation, regulate genes involved in glutaminolysis, particularly the rate-limiting enzyme glutaminase (GLS1). The GLS1 inhibitor CB-839 attenuated TGFβ-induced fibroblast activation. Cardiac fibroblast activation to myofibroblasts by scleraxis overexpression increased glutaminolysis gene expression, including GLS1, while cardiac fibroblasts from scleraxis-null mice showed reduced expression. TGFβ induced GLS1 expression and increased intracellular glutamine and glutamate levels, indicative of increased glutaminolysis, but in scleraxis knockout cells, these measures were attenuated, and the response to TGFβ was lost. The knockdown of scleraxis in activated cardiac fibroblasts reduced GLS1 expression by 75%. Scleraxis transactivated the human GLS1 promoter in luciferase reporter assays, and this effect was dependent on a key scleraxis-binding E-box motif. These results implicate scleraxis-mediated GLS1 expression as a key regulator of glutaminolysis in cardiac fibroblast activation, and blocking scleraxis in this process may provide a means of starving fibroblasts of the energy required for fibrosis.

The solute carrier family 7 member 11 (SLC7A11) is regulated by LH/androgen and required for cystine/glutathione homeostasis in mouse Sertoli cells

Luteinizing hormone (LH) stimulates testosterone production from Leydig cells. Both LH and testosterone play important roles in spermatogenesis and male fertility. To identify LH - and testosterone - responsive transporter genes that play key roles in spermatogenesis, we performed large-scale gene expression analyses on testes obtained from adult control and Lhb knockout mice. We found a significant reduction in cystine/glutamate transporter encoding Slc7a11 mRNA in testes of Lhb null mice. We observed that Slc7a11/SLC7A11 expression was initiated pre-pubertally and developmentally regulated in mouse testis. Immunolocalization studies confirmed that SLC7A11 was mostly expressed in Sertoli cells in testes of control and germ cell-deficient mice. Western blot analyses indicated that SLC7A11 was significantly reduced in testes of mutant mice lacking either LH or androgen receptor selectively in Sertoli cells. Genetic and pharmacological rescue of Lhb knockout mice restored the testicular expression of Slc7a11 comparable to that observed in controls. Additionally, Slc7a11 mRNA was significantly suppressed upon Sertoli cell/testicular damage induced in mice by cadmium treatment. Knockdown of Slc7a11 in vitro in TM4 Sertoli cells or treatment of mice with sulfasalazine, a SLC7A11 inhibitor caused a significant reduction in intracellular cysteine and glutathione levels but glutamate content remained unchanged as determined by metabolomic analysis. Knockdown of Slc7a11 resulted in compensatory upregulation of other glutamate transporters belonging to the Slc1a family presumably to maintain intracellular glutamate levels. Collectively, our studies identified that SLC7A11 is an LH/testosterone-regulated transporter that is required for cysteine/glutathione but not glutamate homeostasis in mouse Sertoli cells.

Degradation of HIF-1α induced by curcumol blocks glutaminolysis and inhibits epithelial-mesenchymal transition and invasion in colorectal cancer cells.

Colorectal cancer (CRC) has high morbidity and mortality. Epithelial-mesenchymal transition (EMT) is associated with CRC progression and metastasis. Glutaminolysis is essential for malignancy of cancer cells. Here, we examined the effects of curcumol on CRC EMT. We observed that curcumol suppressed invasion and migration in human CRC cells associated with upregulation of epithelial markers E-cadherin and Zonula occludens 1 and downregulation of mesenchymal markers N-cadherin and Vimentin as well as EMT-related transcription factors Snail and Twist. Curcumol increased intracellular levels of glutamine but decreased intracellular levels of glutamate, α-ketoglutarate, ATP, glutathione, and tricarboxylic acid cycle metabolites, suggesting interruption of glutaminolysis. Next, curcumol repressed glutaminase 1 (Gls1) mRNA and protein expression, and overexpression of Gls1 promoted EMT and abolished curcumol effects on CRC cell EMT. Molecular examinations showed that curcumol stimulated protein degradation of hypoxia-inducible factor-1α (HIF-1α) and prevented its nuclear accumulation in CRC cells. HIF-1α agonist deferoxamine (DFO) promoted HIF-1α binding to Gls1 promoter and increased Gls1 expression but abolished curcumol's inhibitory effects on Gls1 expression. DFO also enhanced EMT and invasion and migration in CRC cells and eliminated curcumol effects. Furthermore, mouse CRC models were established with in vivo overexpression of HIF-1α and Gls1. Curcumol effectively inhibited CRC growth, metastasis, and EMT in mice, which was abrogated by overexpression of HIF-1α or Gls1. Altogether, stimulation of HIF-1α degradation was required for curcumol to disrupt EMT and repress invasion and migration in CRC cells through inhibiting Gls1-mediated glutaminolysis. Curcumol could be a promising candidate for intervention of CRC metastasis. • Curcumol inhibits EMT and blocks glutaminolysis in CRC cells. • Inhibition of Gls1 is required for curcumol blockade of glutaminolysis and EMT. • Curcumol induces HIF-1α degradation leading to inhibition of Gls1 and blockade of glutaminolysis and EMT. • Curcumol suppresses CRC growth and metastasis via inhibiting HIF-1α, glutaminolysis and EMT in mice.

Effects of in vitro exposure of perfluorooctanoic acid and monocrotophos on astroglia SVG p12 cells

Glia cells provide supportive functions to the central nervous system and can be compromised by environmental contaminants. The primary objective of this study was to characterize the effects of in vitro exposure to perfluorooctanoic acid, a persistent environmental contaminant and/or monocrotophos (MCP), a neurotoxic organophosphate that is rapidly metabolized, to astroglia SVG p12 cells. The endpoints evaluated include cell viability, intracellular glutamate levels as a marker of astrocyte homeostasis function, differential gene expression for selected proteins, which include inflammatory markers (tachykinin), astrocytosis (nestin), S100B, and metabolism enzymes (CYP1A1). The results from cell viability revealed significant differences from the controls at some of the concentrations tested. Also, intracellular glutamate levels were elevated at the 10-μM concentration for perfluorooctanoic acid (PFOA) as well as the 10-μM PFOA/5-μM MCP concentration. Gene expression results at 80-μM PFOA concentration revealed a significant increase in the expression of S100B, tachykinin and CYP1A1. A combination of 10-μM PFOA/20-μM MCP caused a significant decrease in the expression of tachykinin. Gene expression for MCP exposures produced a decrease at the 20-μM MCP concentration. Immunofluorescence results indicated an increase in nestin protein expression for the 20-μM concentration of MCP, which contradicted the gene expression at the same concentration tested. The results indicate that toxicity to glia cells can compromise critical glia functions and could be implicated in neurodegenerative diseases.

Inhibiting spinal secretory phospholipase A2 after painful nerve root injury attenuates established pain and spinal neuronal hyperexcitability by altering spinal glutamatergic signaling.

Neuropathic injury is accompanied by chronic inflammation contributing to the onset and maintenance of pain after an initial insult. In addition to their roles in promoting immune cell activation, inflammatory mediators like secretory phospholipase A2 (sPLA2) modulate nociceptive and excitatory neuronal signaling during the initiation of pain through hydrolytic activity. Despite having a known role in glial activation and cytokine release, it is unknown if sPLA2 contributes to the maintenance of painful neuropathy and spinal hyperexcitability later after neural injury. Using a well-established model of painful nerve root compression, this study investigated if inhibiting spinal sPLA2 7 days after painful injury modulates the behavioral sensitivity and/or spinal dorsal horn excitability that is typically evident. The effects of sPLA2 inhibition on altered spinal glutamatergic signaling was also probed by measuring spinal intracellular glutamate levels and spinal glutamate transporter (GLAST and GLT1) and receptor (mGluR5, GluR1, and NR1) expression. Spinal sPLA2 inhibition at day 7 abolishes behavioral sensitivity, reduces both evoked and spontaneous neuronal firing in the spinal cord, and restores the distribution of neuronal phenotypes to those of control conditions. Inhibiting spinal sPLA2 also increases intracellular glutamate concentrations and restores spinal expression of GLAST, GLT1, mGluR5, and GluR1 to uninjured expression with no effect on NR1. These findings establish a role for spinal sPLA2 in maintaining pain and central sensitization after neural injury and suggest this may be via exacerbating glutamate excitotoxicity in the spinal cord.

Critical role of glutamine metabolism in cardiomyocytes under oxidative stress

BackgroundMetabolic remodeling in cardiomyocytes is deeply associated with the pathogenesis of heart failure (HF). Glutaminolysis is an anaplerotic pathway that incorporates α-ketoglutarate (αKG) derived from glutamine into the tricarboxylic acid (TCA) cycle. It is well known that cancer cells depend on glutamine for their increased energy demand and proliferation; however, the physiological roles of glutamine metabolism in failing hearts remain unclear. ObjectiveTo investigate the regulatory mechanisms and biological effects of glutamine metabolism in oxidative stress-induced failing myocardium. Methods and resultsThe intracellular levels of glutamine, glutamate, and αKG were significantly decreased by H2O2 stimulation in rat neonatal cardiomyocytes (RNCMs). To better understand the metabolic flux in failing myocardium, we performed a stable isotope tracing study and found that glutaminolysis was upregulated in RNCMs under oxidative stress. Consistent with this, the enzymatic activity of glutaminase (Gls), which converts glutamine to glutamate, was augmented in RNCMs treated with H2O2. These findings suggest that glutamine anaplerosis is enhanced in cardiomyocytes under oxidative stress to compensate for the reduction of αKG. Furthermore, the inhibition of Gls reduced cardiac cell viability, ATP production, and glutathione (GSH) synthesis in RNCMs with H2O2 stimulation. Finally, we evaluated the effects of αKG on failing myocardium and observed that dimethyl α-ketoglutarate (DMKG) suppressed oxidative stress-induced cell death likely due to the enhancement of intracellular ATP and GSH levels. ConclusionOur study demonstrates that under oxidative stress, glutaminolysis is upregulated to compensate for the loss of αKG and its replenishment into the TCA cycle, thereby exerting cardioprotective effects by maintaining ATP and GSH levels. Modulation of glutamine metabolism in failing hearts might provide a new therapeutic strategy for HF.

Branched-chain amino acid aminotransferase 2 regulates ferroptotic cell death in cancer cells

Ferroptosis, a form of iron-dependent cell death driven by cellular metabolism and iron-dependent lipid peroxidation, has been implicated as a tumor-suppressor function for cancer therapy. Recent advance revealed that the sensitivity to ferroptosis is tightly linked to numerous biological processes, including metabolism of amino acid and the biosynthesis of glutathione. Here, by using a high-throughput CRISPR/Cas9-based genetic screen in HepG2 hepatocellular carcinoma cells to search for metabolic proteins inhibiting ferroptosis, we identified a branched-chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib, and sulfasalazine) activated AMPK/SREBP1 signaling pathway through iron-dependent ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 as the key enzyme mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc– inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. On the contrary, direct inhibition of BCAT2 by RNA interference, or indirect inhibition by blocking system Xc– activity, triggers ferroptosis. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

Adenosine Receptor A1-A2a Heteromers Regulate EAAT2 Expression and Glutamate Uptake via YY1-Induced Repression of PPARγ Transcription.

Adenosine receptors A1 (A1AR) and A2a (A2aAR) play an important role in regulating glutamate uptake to avoid glutamate accumulation that causes excitotoxicity in the brain; however, the precise mechanism of the effects of A1AR and A2aAR is unclear. Herein, we report that expression of the A1AR protein in the astrocyte membrane and the level of intracellular glutamate were decreased, while expression of the A2aR protein was elevated in cells exposed to oxygen-glucose deprivation (OGD) conditions. Coimmunoprecipitation (Co-IP) experiments showed that A1AR interacts with A2aAR under OGD conditions. The activation of A1AR and inactivation of A2aAR by 2-chloro-N6-cyclopentyladenosine (CCPA) and SCH58251, respectively, partly reversed OGD-mediated glutamate uptake dysfunction, elevated EAAT2, and PPARγ protein levels, and suppressed the expression of Ying Yang 1 (YY1). Both the silencing of YY1 and the activation of PPARγ upregulated EAAT2 expression. Moreover, YY1 silencing elevated the PPARγ level under both normal and OGD conditions. Histone deacetylase (HDAC)1 was found to interact with YY1, and HDAC1 silencing improved PPARγ promoter activity. Taken together, our findings suggest that A1AR-A2aAR heteromers regulate EAAT2 expression and glutamate uptake through the YY1-mediated recruitment of HDAC1 to the PPARγ promoter region.

Metabolomic profiling to evaluate the efficacy of proxalutamide, a novel androgen receptor antagonist, in prostate cancer cells

Proxalutamide is a newly developed androgen receptor (AR) antagonist for the treatment of castration-resistant prostate cancer (PCa) that has entered phase III clinical trials. In the present study, we intended to elucidate the antitumor efficacy of proxalutamide through the metabolomic profiling of PCa cells. Two AR-positive PCa cell lines and two AR-negative PCa cell lines were investigated. Cell viability assays based on ATP quantitation were conducted. LC-Q/TOF-MS was used to analyze intracellular metabolites before or after the administration of proxalutamide and two other clinical AR antagonists (bicalutamide and enzalutamide). The results of this study showed that the inhibitory effect of proxalutamide on PCa cell proliferation was better than that of bicalutamide and enzalutamide, and proxalutamide preferentially affected AR-positive PCa cells over AR-negative cells. The metabolic composition of PCa cells changed significantly after proxalutamide administration, and these changes in response to proxalutamide were significantly different from those in the presence of the two other AR antagonists. In AR-positive cells, proxalutamide significantly decreased the intracellular levels of glutamine, glutamate, glutathione, cysteine, glycine, aspartate, uridine, cytidine and thymidine. However, the effects of the two other antagonists on these discriminant metabolites were ambiguous, and no changes in these metabolites were found in AR-negative cells. Our findings indicate that proxalutamide has inhibitory effects on glutamine metabolism, redox homeostasis and de novo pyrimidine synthesis in AR-positive PCa cells that enhance the cellular sensitivity to proxalutamide.

Less biomass and intracellular glutamate in anodic biofilms lead to efficient electricity generation by microbial fuel cells

BackgroundUsing a microbial fuel cell (MFC), we observed that a complex microbial community decomposed starch and transferred electrons to a graphite felt anode to generate current. In spite of the same reactor configuration, inoculum, substrate, temperature, and pH, MFCs produced different current and power density. To understand which factor(s) affected electricity generation, here, we analyzed a complex microbial community in an anodic biofilm and fermentation broth using Illumina MiSeq sequencing and metabolomics.ResultsMicrobial biomass on the anode was lower in MFCs generating more electricity (0.09–0.16 mg cm−2-anode) than in those generating less electricity (0.60–2.80 mg cm−2-anode), while being equal (3890–4196 mg L−1-broth) in the fermentation broth over the same operational period. Chemical oxygen demand removal and acetate concentration were also similar in fermentation broths. MFCs generating more electricity had relatively more exoelectrogenic bacteria, such as Geobacter sp., but fewer acetate-utilizing Methanosarcina sp. and/or Lactococcus sp. in anodic biofilms. Accordingly, anodic biofilms generating more electricity presented higher levels of most intracellular metabolites related to the tricarboxylic acid cycle and a higher intracellular ATP/ADP ratio, but a lower intracellular NADH/NAD+ ratio. Moreover, the level of intracellular glutamate, an essential metabolite for microbial anabolic reactions, correlated negatively with current density.ConclusionMicrobial growth on the anode and intracellular glutamate levels negatively affect electricity generation by MFCs. Reduced formation of anodic biofilm, in which intracellular glutamate concentration is 33.9 μmol g-cell−1 or less, favors the growth of acetate-utilizing Geobacter sp. on the anode and improves current generation.