Glutamine-mediated epigenetic regulation of cFLIP underlies resistance to TRAIL in pancreatic cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it kills cancer cells while sparing normal cells. However, many cancers, including pancreatic ductal adenocarcinoma (PDAC), exhibit intrinsic or acquired resistance to TRAIL, and the molecular mechanisms underlying TRAIL resistance in cancers, particularly in PDAC, remain unclear. In this study, we demonstrated that glutamine (Gln) endows PDAC cells with resistance to TRAIL through KDM4C-mediated epigenetic regulation of cFLIP. Inhibition of glutaminolysis significantly reduced the cFLIP level, leading to TRAIL-mediated formation of death-inducing signaling complexes. Overexpression of cFLIP dramatically rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Alpha-Ketoglutarate (aKG) supplementation significantly reversed the decrease in the cFLIP level induced by glutaminolysis inhibition and rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Knockdown of glutamic-oxaloacetic transaminase 2, which facilitates the conversion of oxaloacetate and glutamate into aspartate and aKG, decreased aKG production and the cFLIP level and activated TRAIL-induced apoptosis. AKG-mediated epigenetic regulation was necessary for maintaining a high level of cFLIP. Glutaminolysis inhibition increased the abundance of H3K9me3 in the cFLIP promoter, indicating that Gln-derived aKG production is important for Jumonji-domain histone demethylase (JHDM)-mediated cFLIP regulation. The JHDM KDM4C regulated cFLIP expression by binding to its promoter, and KDM4C knockdown sensitized PDAC cells to TRAIL-induced apoptosis. The present findings suggest that Gln-derived aKG production is required for KDM4C-mediated epigenetic regulation of cFLIP, which leads to resistance to TRAIL.

System analysis based on glutamine catabolic-related enzymes identifies GPT2 as a novel immunotherapy target for lung adenocarcinoma.

BACKGROUND: In recent years, targeting glutamine metabolism has gained attention as a promising therapeutic approach. Glutamine catabolic-related enzymes play a crucial role in modulating glutamine metabolism and influencing immune responses in the tumor immune microenvironment (TME). However, current literature on the function of glutamine catabolic enzymes in lung adenocarcinoma (LUAD) is limited. METHODS: We validated the glutamine dependency of LUAD cells in vitro, followed by transcriptome data to identify differentially expressed genes (DEGs), with transcriptome and single-cell data analysis utilized to explore the role of such genes within the tumor immune microenvironment. We performed employed subcutaneous injection of lewis lung carcinoma cells in C57BL/6 mice to confirm the role of candidate genes in tumor growth and anti-tumor immunity. RESULTS: Our study revealed that glutamine is essential for the growth of LUAD cells. Subsequently, we identified four DEGs - glutamate pyruvate transaminase 1 (GPT1), glutamate pyruvate transaminase 2 (GPT2), glutamic-oxaloacetic transaminase 1 (GOT1), and glutamic-oxaloacetic transaminase 2 (GOT2) - in LUAD patients, which were highly expressed in tumor tissue and associated with an immunosuppressive TME. Single-cell sequencing analysis detected high expression levels of GOT1 and GOT2 in immune and stromal cell subpopulations, while GPT1 and GPT2 showed relatively lower expression. Based on the lower immune score and lower expression in immune and stromal cells, we validated the role of GPT2 in vivo for modulating the TME and tumor growth. Inhibition of GPT2 resulted in suppressed tumor growth and increased the expression of CD4 and CD8. Additionally, GPT2 inhibitors induced a stronger antitumor immunity when used in combination with anti-programmed cell death ligand 1. CONCLUSION: This study is the first to show the critical role of glutamine catabolic-related enzymes in the TME, and identified GPT2 as a promising therapeutic target for inhibiting tumor growth and improving anti-tumour immune responses for LUAD. Additional studies will be required to define the roles glutamine catabolic-related enzymes play in LUAD.

c-Myc protects hepatocellular carcinoma cell from ferroptosis induced by glutamine deprivation via upregulating GOT1 and Nrf2.

BACKGROUND: Glutamine metabolism is critical for development of hepatocellular carcinoma (HCC), which makes it a novel promising treatment target. However, clinical evidence suggested glutamine withdrawal therapy does not achieved the desired tumor suppression. Therefore, it is valuable to investigate the survival mechanisms of tumors with glutamine deprivation. METHODS: The HCC cells were cultured in glutamine-free medium or supplemented with glutamine metabolites or ferroptosis inhibitors. The parameters related to ferroptosis and the activity of GSH synthesis-related enzymes of the HCC cells were detected by corresponding kits. The expressions of glutamate oxaloacetate transaminase 1 (GOT1), c-Myc and Nrf2 were detected by western blot and qRT-PCR. The chromatin immunoprecipitation and luciferase reporter assays were performed to investigate the correlation between c-Myc and GOT1. The siRNAs of c-Myc and GOT1 were used to explore their roles in GSH (GSH) synthesis and ferroptosis in vitro and in vivo. RESULTS: Glutamine deprivation-induced ferroptosis did not completely inhibit HCC cells proliferation. Glutamine deprivation activated the expression of c-Myc, which promoted the transcription of GOT1 and Nrf2, consequently maintaining the GSH synthesis and inhibiting ferroptosis. In addition, combined inhibition of GOT1 with glutamine deprivation could result in better inhibition of HCC in vitro and in vivo. CONCLUSIONS: In our work, the results indicate that GOT1 induced by c-Myc may play an important role in combating ferroptosis due to glutamine deprivation, making it a significant target in glutamine withdrawal therapy. This study provides a theoretical foundation for the clinical targeted therapy for HCC.

Co-administration with A1M does not influence apoptotic response of 177Lu-octreotate in GOT1 neuroendocrine tumors.

Recombinant α1-microglobulin (A1M) is a proposed radioprotector during 177Lu-octreotate therapy of neuroendocrine tumors (NETs). To ensure a maintained therapeutic effect, we previously demonstrated that A1M does not affect the 177Lu-octreotate induced decrease in GOT1 tumor volume. However, the underlying biological events of these findings are still unknown. The aim of this work was to examine the regulation of apoptosis-related genes in GOT1 tumors short-time after i.v. administration of 177Lu-octreotate with and without A1M or A1M alone. Human GOT1 tumor-bearing mice received 30 MBq 177Lu-octreotate or 5 mg/kg A1M or co-treatment with both. Animals were sacrificed after 1 or 7 days. Gene expression analysis of apoptosis-related genes in GOT1 tissue was performed with RT-PCR. In general, similar expression patterns of pro- and anti-apoptotic genes were found after 177Lu-octreotate exposure with or without co-administration of A1M. The highest regulated genes in both irradiated groups compared to untreated controls were FAS and TNFSFRS10B. Administration of A1M alone only resulted in significantly regulated genes after 7 days. Co-administration of A1M did not negatively affect the transcriptional apoptotic response of 177Lu-octreotate in GOT1 tumors.

Tumor Cell Derived Exosomal GOT1 Suppresses Tumor Cell Ferroptosis to Accelerate Pancreatic Cancer Progression by Activating Nrf2/HO-1 Axis via Upregulating CCR2 Expression.

Recently, evidence has shown that GOT1 expression is upregulated in pancreatic cancer tissues and promotes cancer development, but the specific mechanism remains unclear. We found that GOT1 expression was upregulated in pancreatic cancer cell-derived exosomes. When PANC-1 cells were incubated with exosomes alone or transfected together with si-GOT1, we found that exosomes enhanced cell proliferation, invasion and migration, promoted ferroptosis, and si-GOT1 reversed the effects of exosomes. The results of online bioinformatics database analysis indicated that CCR2 was a potential binding protein of GOT1 and is highly expressed in pancreatic cancer tissues. PANC-1 cells were transfected with pcDNA-CCR2 or si-CCR2, and it was found that pcDNA-CCR2 enhanced cell proliferation, invasion and migration, promoted ferroptosis, and si-CCR2 had an opposite effect. Next, exosome-treated cells were transfected with si-GOT1 alone or together with pcDNA-CCR2, and we found that exosomes promoted CCR2 expression, promoted cell proliferation and invasion, and inhibited ferroptosis, the transfection of si-GOT1 abolished the effect of exosomes, and the transfection of pcDNA-CCR2 again reversed the effect of si-GOT1. Furthermore, when exosome-treated cells were transfected with si-GOT1 alone or co-incubated with Nrf2 activator NK-252, we found that si-GOT1 reversed the promoting effect of exosomes on Nrf2 and HO-1 expression, as well as its inhibitory effect on ferroptosis, but this effect was abrogated by NK-252. In vivo studies showed that knockdown of GOT1 expression inhibited tumor formation compared with tumor tissues formed upon exosome induction, which was mediated by promoting ferroptosis via suppressing the protein expression of GOT1, CCR2, Nrf2 and HO-1 in tumor tissues.

The discovery of a non-competitive GOT1 inhibitor, hydralazine hydrochloride, via a coupling reaction-based high-throughput screening assay.

Glutamate oxaloacetate transaminase 1 (GOT1) plays a key role in aberrant glutamine metabolism. GOT1 suppression can arrest tumor growth and prevent the development of cancer, indicating GOT1 as a potential anticancer target. Reported GOT1 inhibitors, on the other hand, are quite restricted. Here, we developed and optimized a coupling reaction-based high-throughput screening assay for the discovery of GOT1 inhibitors. By using this screening assay, we found that the cardiovascular drug hydralazine hydrochloride inhibited GOT1 catalytic activity, with an IC50 of 26.62 ± 7.45 µM, in a non-competitive and partial-reversible manner. In addition, we determined the binding affinity of hydralazine hydrochloride to GOT1, with a Kd of 16.54 ± 8.59 µM, using a microscale thermophoresis assay. According to structure-activity relationship analysis, the inhibitory activity of hydralazine hydrochloride is mainly derived from its hydrazine group. Furthermore, it inhibits the proliferation of cancer cells MCF-7 and MDA-MB-468 with a slight inhibitory effect compared to other tested cancer cells, highlighting GOT1 as a promising therapeutic target for the treatment of breast cancer.

Osteoblasts induce glucose-derived ATP perturbations in chondrocytes through noncontact communication.

Cartilage and subchondral bone communicate with each other through material and signal exchanges. However, direct evidence provided by experimental studies on their interactions is insufficient. In the present study, we establish a noncontact co-culture model with a transwell chamber to explore the energetic perturbations in chondrocytes influenced by osteoblasts. Our results indicate that osteoblasts induce more ATP generation in chondrocytes through an energetic shift characterized by enhanced glycolysis and impaired mitochondrial tricarboxylic acid cycle. Enhanced glycolysis is shown by an increase of secreted lactate and the upregulation of glycolytic enzymes, including glucose-6-phosphate isomerase (Gpi), liver type ATP-dependent 6-phosphofructokinase (Pfkl), fructose-bisphosphate aldolase C (Aldoc), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), triosephosphate isomerase (Tpi1), and phosphoglycerate kinase 1 (Pgk1). Impaired mitochondrial tricarboxylic acid cycle is characterized by the downregulation of cytoplasmic aspartate aminotransferase (Got1) and mitochondrial citrate synthase (Cs). Osteoblasts induce the activation of Akt and P38 signaling to mediate ATP perturbations in chondrocytes. This study may deepen our understanding of the maintenance of metabolic homeostasis in the bone-cartilage unit.

Ziprasidone suppresses pancreatic adenocarcinoma cell proliferation by targeting GOT1 to trigger glutamine metabolism reprogramming.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant tumor whose effective treatment has not been found. The redox state and proliferative activity of PDAC cells are maintained by the conversion of aspartic acid in the cytoplasm into oxaloacetate though aspartate aminotransferase 1 (GOT1). Therefore, GOT1 inhibitors as a potential approach for treating PDAC have attracted more attention of researchers. Ziprasidone effectively inhibited GOT1 in a non-competitive manner. The potential cytotoxicity and anti-proliferation effects of ziprasidone against PDAC cells in vitro and in vivo were evaluated. Ziprasidone can induce glutamine metabolism disorder and redox state imbalance of PDAC cells by targeting GOT1, thereby inhibiting proliferation, preventing migration, and inducing apoptosis. Ziprasidone displayed significant in vivo antitumor efficacy in SW1990 cell-derived xenografts. What's more, knockdown of GOT1 in SW1990 reduced the anti-proliferative effects of ziprasidone. As a novel GOT1 inhibitor, ziprasidone may be a lead compound for the treatment of PDAC. KEY MESSAGES: Small molecule inhibitors targeting GOT1 may provide a therapeutic target in PDAC. Ziprasidone effectively inhibited GOT1 enzyme in a non-competitive manner. Ziprasidone repressed glutamine metabolism and inhibited the growth of tumor in vivo. Knockdown of GOT1 decreased the anti-proliferative effects of ziprasidone.

Glutamic oxaloacetic transaminase 1 as a potential target in human cancer.

Glutamic Oxaloacetic Transaminase 1 (GOT1) is one distinct isoenzyme of glutamic oxaloacetic transaminase in eukaryotic cells, which is located in the cytoplasm. To date, several studies have shown that GOT1 plays a critical role in regulating cell proliferation by participating in amino acid metabolism, especially in glutamine metabolism. In addition, GOT1 is overexpressed in many cancer, so GOT1 has been identified as a potentially therapeutic target. Herein, this review summarizes the structure and function of GOT1 and the important roles of GOT1 in some tumor progress, as well as the characterization of GOT1 inhibitors. It may provide new insight into the discovery of small compounds as potential anti-GOT1 drugs for treatment of cancer.

CircGOT1 promotes cell proliferation, mobility, and glycolysis-mediated cisplatin resistance via inhibiting its host gene GOT1 in esophageal squamous cell cancer.

Esophageal squamous cell cancer (ESCC) is a prevalent malignant cancer with high incidence and fatality rate. Surging evidences have revealed that circular RNAs (circRNAs) act key role in ESCC tumorigenesis and progression. Therefore, the purpose of this study is to explore the role and regulatory mechanism of a novel circGOT1 in ESCC. In the present study, the transcriptional expression of circGOT1, miR-606 and GOT1, and the epithelial-mesenchymal transition (EMT) and apoptosis-related markers were examined by quantitative PCR. The protein levels of GOT1 and glycolysis-related proteins were detected by Western blotting. In addition, the glycolytic levels were determined via measuring glucose uptake, lactate production, and ATP levels. Then, the function experiments and rescue experiments were used to investigate the function and mechanism of circGOT1 in ESCC. In addition, RNA immunoprecipitation, pull-down, and luciferase activity reporter gene assays were used to analyze the circGOT1/miR-606/GOT1 axis. The xenograft mouse mode was used to determine the function of circGOT1 in vivo. Here, we identified that circGOT1 and GOT1 upregulate, whereas miR-606 was reduced in ESCC tissues and cell lines. High circGOT1 and GOT1 expression associated with poor survival and worse prognosis of ESCC patients, but miR-606 revealed opposite traits. Mechanically, circGOT1 sponged miR-606 to promote GOT1, which induced cell proliferation, migration, aerobic glycolysis, and cisplatin resistance. The tumor growth was reduced by circGOT1 inhibition in xenograft mouse. Our results indicate the oncogene role of circGOT1 in ESCC via an endogenous competition RNA (ceRNA) mechanism to promote GOT1 expression via sponging miR-606.

Discovery of GOT1 Inhibitors from a Marine-Derived Aspergillus terreus That Act against Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating digestive system carcinoma with high incidence and death rates. PDAC cells are dependent on the Gln metabolism, which can preferentially utilize glutamic oxaloacetate transaminase 1 (GOT1) to maintain the redox homeostasis of cancer cells. Therefore, small molecule inhibitors targeting GOT1 can be used as a new strategy for developing cancer therapies. In this study, 18 butyrolactone derivatives (1-18) were isolated from a marine-derived Aspergillus terreus, and asperteretone B (5), aspulvinone H (AH, 6), and (+)-3',3'-di-(dimethylallyl)-butyrolactone II (12) were discovered to possess significant GOT1-inhibitory activities in vitro, with IC50 values of (19.16 ± 0.15), (5.91 ± 0.04), and (26.38 ± 0.1) µM, respectively. Significantly, the molecular mechanism of the crystal structure of GOT1-AH was elucidated, wherein AH and the cofactor pyrido-aldehyde 5-phosphate competitively bound to the active sites of GOT1. More importantly, although the crystal structure of GOT1 has been discovered, the complex structure of GOT1 and its inhibitors has never been obtained, and the crystal structure of GOT1-AH is the first reported complex structure of GOT1/inhibitor. Further in vitro biological study indicated that AH could suppress glutamine metabolism, making PDAC cells sensitive to oxidative stress and inhibiting cell proliferation. More significantly, AH exhibited potent in vivo antitumor activity in an SW1990-cell-induced xenograft model. These findings suggest that AH could be considered as a promising lead molecule for the development of anti-PDAC agents.

Structural and functional analysis of disease-associated mutations in GOT1 gene: An in silico study.

Disease-associated single nucleotide polymorphisms (SNPs) alter the natural functioning and the structure of proteins. Glutamic-oxaloacetic transaminase 1 (GOT1) is a gene associated with multiple cancers and neurodegenerative diseases which codes for aspartate aminotransferase. The present study involved a comprehensive in-silico analysis of the disease-associated SNPs of human GOT1. Four highly deleterious nsSNPs (L36R, Y159C, W162C and L345P) were identified through SNP screening using several sequence-based and structure-based tools. Conservation analysis and oncogenic analysis showed that most of the nsSNPs are at highly conserved residues, oncogenic in nature and cancer drivers. Molecular dynamics simulations (MDS) analysis was performed to understand the dynamic behaviour of native and mutant proteins. PTM analysis revealed that the nsSNP Y159C is at a PTM site and will mostly affect phosphorylation at that site. Based on the overall analyses carried out in this study, L36R is the most deleterious mutation amongst the aforementioned deleterious mutations of GOT1.

GOT1 inhibition promotes pancreatic cancer cell death by ferroptosis.

Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis.

Blood glutamate scavengers increase pro-apoptotic signaling and reduce metastatic melanoma growth in-vivo.

Inhibition of extracellular glutamate (Glu) release decreases proliferation and invasion, induces apoptosis, and inhibits melanoma metastatic abilities. Previous studies have shown that Blood-glutamate scavenging (BGS), a novel treatment approach, has been found to be beneficial in attenuating glioblastoma progression by reducing brain Glu levels. Therefore, in this study we evaluated the ability of BGS treatment to inhibit brain metastatic melanoma progression in-vivo. RET melanoma cells were implanted in C56BL/6J mice to induce brain melanoma tumors followed by treatment with BGS or vehicle administered for fourteen days. Bioluminescent imaging was conducted to evaluate tumor growth, and plasma/CSF Glu levels were monitored throughout. Immunofluorescence staining of Ki67 and 53BP1 was used to analyze tumor cell proliferation and DNA double-strand breaks. In addition, we analyzed CD8, CD68, CD206, p-STAT1 and iNOS expression to evaluate alterations in tumor micro-environment and anti-tumor immune response due to treatment. Our results show that BGS treatment reduces CSF Glu concentration and consequently melanoma growth in-vivo by decreasing tumor cell proliferation and increasing pro-apoptotic signaling in C56BL/6J mice. Furthermore, BGS treatment supported CD8+ cell recruitment and CD68+ macrophage invasion. These findings suggest that BGS can be of potential therapeutic relevance in the treatment of metastatic melanoma.

Metabolic Rewiring by Loss of Sirt5 Promotes Kras-Induced Pancreatic Cancer Progression.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was used for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in patients with PDAC. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited antitumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased noncanonic use of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC.

Glutamate-Oxaloacetate Transaminase 1 Impairs Glycolysis by Interacting with Pyruvate Carboxylase and Further Inhibits the Malignant Phenotypes of Glioblastoma Cells.

BACKGROUND: Glycolysis is an important metabolic manner in glioblastoma multiforme (GBM)'s rapid growth. It has been reported that glutamate-oxaloacetate transaminase 1 (GOT1) is low-expressed in GBM and patients with high-expressed GOT1 have better prognosis. However, the effect and mechanism of GOT1 on glycolysis and malignant phenotypes of GBM cells are still unclear. METHODS: The expression differences of GOT1 between GBM parenchyma and adjacent tissues were detected. The prognosis and clinical data with different levels of GOT1 were also analyzed. The glucose consumption, production of lactate and pyruvate were measured after GOT1 was knocked down or overexpressed. The effects of GOT1 on GBM cell's malignant phenotypes were analyzed by Western blot, CCK-8 assay, and flow cytometry. The relationship between GOT1 and pyruvate carboxylase (PC) was examined by immunoprecipitation and immunofluorescence. RESULTS: GOT1 was expressed little in GBM, and patients with highly expressed GOT1 had longer survival periods. Overexpressed GOT1 inhibited the glycolysis and malignant phenotypes of GBM cells. 2-DG treatment could partially reverse the enhancement of malignant phenotypes caused by knockdown of GOT1. The expression of GOT1 was positively correlated with PC. The inhibitory effect of GOT1 on glycolysis could be partially reversed by PC's knockdown. CONCLUSIONS: GOT1 could impair glycolysis by interacting with PC and further inhibit the malignant phenotypes of GBM cells.

Medioresinol as a novel PGC-1α activator prevents pyroptosis of endothelial cells in ischemic stroke through PPARα-GOT1 axis.

AIM: Brain microvascular endothelial cells (BMVECs), as the important structure of blood-brain barrier (BBB), play a vital role in ischemic stroke. Pyroptosis of different cells in the brain may aggravate cerebral ischemic injury, and PGC-1α plays a major role in pyroptosis. However, it is not known whether BMVECs undergo pyroptosis after ischemic stroke and whether PGC-1α activator Medioresinol (MDN) we discovered may be useful against pyroptosis of endothelial cells and ischemic brain injury. METHODS: For in vitro experiments, the bEnd.3 cells and BMVECs under oxygen and glucose-deprivation (OGD) were treated with or without MDN, and the LDH release, tight junction protein degradation, GSDMD-NT membrane location and pyroptosis-associated proteins were evaluated. For in vivo experiments, mice underwent transient middle cerebral artery occlusion (tMCAO) for ischemia model, and the neuroprotective effects of MDN were measured by infarct volume, the permeability of BBB and pyroptosis of BMVECs. For mechanistic study, effects of MDN on the accumulation of phenylalanine, mitochondrial reactive oxygen species (mtROS) were tested by untargeted metabolomics and MitoSOX Red probe, respectively. RESULTS: BMVECs underwent pyroptosis after ischemia. MDN dose-dependently activated PGC-1α, significantly reduced pyroptosis, mtROS and the expressions of pyroptosis-associated proteins (NLRP3, ASC, cleaved caspase-1, IL-1ß, GSDMD-NT), and increased ZO-1 and Occludin protein expressions in BMVECs. In tMCAO mice, MDN remarkably reduced brain infarct volume and the permeability of BBB, inhibited pyroptosis of BMVECs, and promoted long-term neurobehavioral functional recovery. Mechanistically, MDN promoted the interaction of PGC-1α with PPARα to increase PPARα nuclear translocation and transcription activity, further increased the expression of GOT1 and PAH, resulting in enhanced phenylalanine metabolism to reduce the ischemia-caused phenylalanine accumulation and mtROS and further ameliorate pyroptosis of BMVECs. CONCLUSION: In this study, we for the first time discovered that pyroptosis of BMVECs was involved in the pathogenesis of ischemic stroke and MDN as a novel PGC-1α activator could ameliorate the pyroptosis of endothelial cells and ischemic brain injury, which might attribute to reduction of mtROS through PPARα/GOT1 axis in BMVECs. Taken together, targeting endothelial pyroptosis by MDN may provide alternative therapeutics for brain ischemic stroke.

Glutamate is an essential mediator in glutamine-amplified insulin secretion.

AIMS/INTRODUCTION: Glutamine is the most abundant amino acid in the circulation. In this study, we investigated cell signaling in the amplification of insulin secretion by glutamine. MATERIALS AND METHODS: Clonal pancreatic ß-cells MIN6-K8, wild-type B6 mouse islets, glutamate dehydrogenase (GDH) knockout clonal ß-cells (Glud1KOßCL), and glutamate-oxaloacetate transaminase 1 (GOT1) knockout clonal ß-cells (Got1KOßCL) were studied. Insulin secretion from these cells and islets was examined under various conditions, and intracellular glutamine metabolism was assessed by metabolic flux analysis. Intracellular Ca2+ concentration ([Ca2+ ]i ) was also measured. RESULTS: Glutamine dose-dependently amplified insulin secretion in the presence of high glucose in both MIN6-K8 cells and Glud1KOßCL. Inhibition of glutaminases, the enzymes that convert glutamine to glutamate, dramatically reduced the glutamine-amplifying effect on insulin secretion. A substantial amount of glutamate was produced from glutamine through direct conversion by glutaminases. Glutamine also increased [Ca2+ ]i at high glucose, which was abolished by inhibition of glutaminases. Glutamic acid dimethylester (dm-Glu), a membrane permeable glutamate precursor that is converted to glutamate in cells, increased [Ca2+ ]i as well as induced insulin secretion at high glucose. These effects of glutamine and dm-Glu were dependent on calcium influx. Glutamine also induced insulin secretion in clonal ß-cells MIN6-m14, which otherwise exhibit no insulin secretory response to glucose. CONCLUSIONS: Glutamate converted from glutamine is an essential mediator that enhances calcium signaling in the glutamine-amplifying effect on insulin secretion. Our data also suggest that glutamine exerts a permissive effect on glucose-induced insulin secretion.

Inhibition of endogenous blood glutamate oxaloacetate transaminase enhances the ischemic damage.

Glutamate oxaloacetate transaminase 1 (GOT1) enzyme plays a critical role in the cell metabolism by participating in the carbohydrate and amino acid metabolism. In ischemic stroke, we have demonstrated that recombinant GOT1 acts as a novel neuroprotective treatment against the excess of extracellular glutamate that accumulates in the brain following ischemic stroke. In this study, we investigated the inhibitory effect of GOT1 on brain metabolism and on the ischemic damage in a rat model of ischemic stroke by means of a specific antibody developed against this enzyme. Inhibition of GOT1 caused higher brain glutamate and lactate levels and this response was associated with larger ischemic lesion. This study represents the first demonstration that the inhibition of the blood GOT1 activity leads to more severe ischemic damage and poorer outcome and supports the protective role of GOT1 against ischemic insults.