A paradigm shift in cancer research based on integrative multi-omics approaches: glutaminase serves as a pioneering cuproptosis-related gene in pan-cancer.

BACKGROUND: Cuproptosis is a newly identified form of unprogrammed cell death. As a pivotal metabolic regulator, glutaminase (GLS) has recently been discovered to be linked to cuproptosis. Despite this discovery, the oncogenic functions and mechanisms of GLS in various cancers are still not fully understood. METHODS: In this study, a comprehensive omics analysis was performed to investigate the differential expression levels, diagnostic and prognostic potential, correlation with tumor immune infiltration, genetic alterations, and drug sensitivity of GLS across multiple malignancies. RESULTS: Our findings revealed unique expression patterns of GLS across various cancer types and molecular subtypes of carcinomas, underscoring its pivotal role primarily in energy and nutrition metabolism. Additionally, GLS showed remarkable diagnostic and prognostic performance in specific cancers, suggesting its potential as a promising biomarker for cancer detection and prognosis. Furthermore, we focused on uterine corpus endometrial carcinoma (UCEC) and developed a novel prognostic model associated with GLS, indicating a close correlation between GLS and UCEC. Moreover, our exploration into immune infiltration, genetic heterogeneity, tumor stemness, and drug sensitivity provided novel insights and directions for future research and laid the foundation for high-quality verification. CONCLUSION: Collectively, our study is the first comprehensive investigation of the biological and clinical significance of GLS in pan-cancer. In our study, GLS was identified as a promising biomarker for UCEC, providing valuable evidence and a potential target for anti-tumor therapy. Overall, our findings shed light on the multifaceted functions of GLS in cancer and offer new avenues for further research.

CircCOL1A1 promotes proliferation, migration, and invasion of colorectal cancer (CRC) cells and glutamine metabolism through GLS1 up-regulation by sponging miR-214-3p.

BACKGROUND: Circular ribose nucleic acids (circRNAs), an abundant type of noncoding RNAs, are widely expressed in eukaryotic cells and exert a significant impact on the initiation and progression of various disorders, including different types of cancer. However, the specific role of various circRNAs in colorectal cancer (CRC) pathology is still not fully understood. METHODS: The initial step involved the use of quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of circRNAs and messenger RNA (mRNA) in CRC cell lines and tissues. Subsequently, functional analyses of circCOL1A1 knockdown were conducted in vitro and in vivo through cell counting kit (CCK)-8, colony formation and transwell assays, as well as xenograft mouse model of tumor formation. Molecular expression and interactions were investigated using luciferase reporter assays, Western blot analysis, RNA immunoprecipitation (RIP), and immunohistochemical staining. RESULTS: The RT-qPCR results revealed elevated levels of circCOL1A1 expressions in CRC tissues and cell lines as compared to the normal counterparts. In addition, circCOL1A1 expression level was found to be correlated with TNM stage, lymph node metastases, distant metastases, and invasion. Knockdown of circCOL1A1 resulted in impaired invasion, migration, and proliferation of CRC cells, and suppressed tumor generation in the animal model. We further demonstrated that circCOL1A1 could act as a sponge for miR-214-3p, suppressing miR-214-3p activity and leading to the upregulation of GLS1 protein to promote glutamine metabolism. CONCLUSION: These findings suggest that circCOL1A1 functions as an oncogenic molecule to promote CRC progression via miR-214-3p/GLS1 axis, hinting on the potential of circCOL1A1 as a therapeutic target for CRC.

ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression.

Many of the biological processes of the cell, from its structure to signal transduction, involve protein-protein interactions. On this basis, our aim was to identify cellular proteins that interact with ERK5, a serine/threonine protein kinase with a key role in tumor genesis and progression and a promising therapeutic target in many tumor types. Using affinity chromatography, immunoprecipitation, and mass spectrometry techniques, we unveiled an interaction between ERK5 and the mitochondrial glutaminase GLS in pancreatic tumor cells. Subsequent co-immunoprecipitation and immunofluorescence studies supported this interaction in breast and lung tumor cells as well. Genetic approaches using RNA interference techniques and CRISPR/Cas9 technology demonstrated that the loss of ERK5 function led to increased protein levels of GLS isoforms (KGA/GAC) and a concomitant increase in their activity in tumor cells. It is well known that the tumor cell reprograms its intermediary metabolism to meet its increased metabolic needs. In this sense, mitochondrial GLS is involved in the first step of glutamine catabolism, one of the main energy sources in the context of cancer. Our data suggest that ERK5 contributes to the regulation of tumor cell energy metabolism via glutaminolysis.

Metabolic reprogramming of cancer cells by JMJD6-mediated pre-mRNA splicing associated with therapeutic response to splicing inhibitor.

Dysregulated pre-mRNA splicing and metabolism are two hallmarks of MYC-driven cancers. Pharmacological inhibition of both processes has been extensively investigated as potential therapeutic avenues in preclinical and clinical studies. However, how pre-mRNA splicing and metabolism are orchestrated in response to oncogenic stress and therapies is poorly understood. Here, we demonstrate that jumonji domain containing 6, arginine demethylase, and lysine hydroxylase, JMJD6, acts as a hub connecting splicing and metabolism in MYC-driven human neuroblastoma. JMJD6 cooperates with MYC in cellular transformation of murine neural crest cells by physically interacting with RNA binding proteins involved in pre-mRNA splicing and protein homeostasis. Notably, JMJD6 controls the alternative splicing of two isoforms of glutaminase (GLS), namely kidney-type glutaminase (KGA) and glutaminase C (GAC), which are rate-limiting enzymes of glutaminolysis in the central carbon metabolism in neuroblastoma. Further, we show that JMJD6 is correlated with the anti-cancer activity of indisulam, a 'molecular glue' that degrades splicing factor RBM39, which complexes with JMJD6. The indisulam-mediated cancer cell killing is at least partly dependent on the glutamine-related metabolic pathway mediated by JMJD6. Our findings reveal a cancer-promoting metabolic program is associated with alternative pre-mRNA splicing through JMJD6, providing a rationale to target JMJD6 as a therapeutic avenue for treating MYC-driven cancers.

A novel method for high level production of protein glutaminase by sfGFP tag in Bacillus subtilis.

Protein glutaminase (PG; EC 3.5.1.44) is a novel deamidase that helps to improve functional properties of food proteins. Currently, the highest activated PG enzyme activity was 26 U/mg when recombinantly expressed via the twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum. In this study, superfolder green fluorescent protein (sfGFP) was used to replace traditional signal peptides to facilitate efficient heterologous expression and secretion of Propeptide-Protein glutaminase (PP) in Bacillus subtilis. The fusion protein, sfGFP-PP, was secreted from 12 h of fermentation and reached its highest extracellular expression at 28 h, with a secretion efficiency of about 93 %. Moreover, when fusing sfGFP with PP at the N-terminus, it significantly enhances PG expression up to 26 U/mL by approximately 2.2-fold compared to conventional signal-peptides- guided PP with 11.9 U/mL. Finally, the PG enzyme activity increased from 26 U/mL to 36.9 U/mL after promoter and RBS optimization. This strategy not only provides a new approach to increase PG production as well as extracellular secretion but also offers sfGFP as an effective N-terminal tag for increased secreted production of difficult-to-express proteins.

LncRNA SNHG11 reprograms glutaminolysis in hepatic stellate cells via Wnt/ß-catenin/GLS axis.

Long non-coding RNAs (lncRNAs) have been identified as decisive regulators of liver fibrosis. Hepatic stellate cells (HSCs), major hepatic cells contributing to liver fibrosis, undergo metabolic reprogramming for transdifferentiation and activation maintenance. As a crucial part of metabolic reprogramming, glutaminolysis fuels the tricyclic acid (TCA) cycle that renders HSCs addicted to glutamine. However, how lncRNAs reprogram glutamine metabolism in HSCs is unknown. For this research, we characterized the pro-fibrogenic function of small nucleolar host gene 11 (SNHG11). Our data showed that in carbon tetrachloride (CCl4, 7 µL/g, intraperitoneally) treated C57BL/6J mice, SNHG11 expression was highly up-regulated in fibrotic livers and activated primary HSCs. SNHG11 knockdown attenuated the accumulation of fibrotic markers α-SMA and Col1A1 in liver fibrosis tissues and activated HSCs. Western blot and qRT-PCR assays demonstrated that glutaminase (GLS), the rate-limiting enzyme for glutaminolysis, was a downstream target of SNHG11. Furthermore, SNHG11 upregulated glutaminolysis in HSCs through the activation of the Wnt/ß-catenin signaling pathway. The results highlighted that SNHG11 is a glutaminolysis-regulated lncRNA that promotes liver fibrosis. A novel insight into the metabolic mechanism that reprograms glutaminolysis in HSCs could be exploited as anti-fibrotic targets.

LRPPRC promotes glycolysis by stabilising LDHA mRNA and its knockdown plus glutamine inhibitor induces synthetic lethality via m6 A modification in triple-negative breast cancer.

BACKGROUND: Targeted therapy for triple-negative breast cancer (TNBC) remains a challenge. N6-methyladenosine (m6 A) is the most abundant internal mRNA modification in eukaryotes, and it regulates the homeostasis and function of modified RNA transcripts in cancer. However, the role of leucine-rich pentatricopeptide repeat containing protein (LRPPRC) as an m6 A reader in TNBC remains poorly understood. METHODS: Western blotting, reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry were used to investigate LRPPRC expression levels. Dot blotting and colorimetric enzyme linked immunosorbent assay (ELISA) were employed to detect m6 A levels. In vitro functional assays and in vivo xenograft mouse model were utilised to examine the role of LRPPRC in TNBC progression. Liquid chromatography-mass spectrometry/mass spectrometry and Seahorse assays were conducted to verify the effect of LRPPRC on glycolysis. MeRIP-sequencing, RNA-sequencing, MeRIP assays, RNA immunoprecipitation assays, RNA pull-down assays and RNA stability assays were used to identify the target genes of LRPPRC. Patient-derived xenografts and organoids were employed to substantiate the synthetic lethality induced by LRPPRC knockdown plus glutaminase inhibition. RESULTS: The expressions of LRPPRC and m6 A RNA were elevated in TNBC, and the m6 A modification site could be recognised by LRPPRC. LRPPRC promoted the proliferation, metastasis and glycolysis of TNBC cells both in vivo and in vitro. We identified lactate dehydrogenase A (LDHA) as a novel direct target of LRPPRC, which recognised the m6 A site of LDHA mRNA and enhanced the stability of LDHA mRNA to promote glycolysis. Furthermore, while LRPPRC knockdown reduced glycolysis, glutaminolysis was enhanced. Moreover, the effect of LRPPRC on WD40 repeat domain-containing protein 76 (WDR76) mRNA stability was impaired in an m6 A-dependent manner. Then, LRPPRC knockdown plus a glutaminase inhibition led to synthetic lethality. CONCLUSIONS: Our study demonstrated that LRPPRC promoted TNBC progression by regulating metabolic reprogramming via m6 A modification. These characteristics shed light on the novel combination targeted therapy strategies to combat TNBC.

Glutaminase potentiates the glycolysis in esophageal squamous cell carcinoma by interacting with PDK1.

Increasing evidence has demonstrated that glutaminase (GLS) as a key mitochondrial enzyme plays a pivotal role in glutaminolysis, which widely participates in glutamine metabolism serving as main energy sources and building blocks for tumor growth. However, the roles and molecular mechanisms of GLS in esophageal squamous cell carcinoma (ESCC) remains unknown. Here, we found that GLS was highly expressed in ESCC tissues and cells. GLS inhibitor CB-839 significantly suppressed cell proliferation, colony formation, migration and invasion of ESCC cells, whereas GLS overexpression displayed the opposite effects. In addition, CB-839 markedly suppressed glucose consumption and lactate production, coupled with the downregulation of glycolysis-related proteins HK2, PFKM, PKM2 and LDHA, whereas GLS overexpression exhibited the adverse results. In vivo animal experiment revealed that CB-839 dramatically suppressed tumor growth, whereas GLS overexpression promoted tumor growth in ESCC cells xenografted nude mice. Mechanistically, GLS was localized in mitochondria of ESCC cells, which interacted with PDK1 protein. CB-839 attenuated the interaction of GLS and PDK1 in ESCC cells by suppressing PDK1 expression, which further evoked the downregulation of p-PDHA1 (s293), however, GLS overexpression markedly enhanced the level of p-PDHA1 (s293). These findings suggest that interaction of GLS with PDK1 accelerates the glycolysis of ESCC cells by inactivating PDH enzyme, and thus targeting GLS may be a novel therapeutic approach for ESCC patients.

Biochemical characterization of glutaminase-free L-asparaginases from Himalayan Pseudomonas and Rahnella spp. for acrylamide mitigation.

L-asparaginase having low glutaminase activity is important in clinical and food applications. Herein, glutaminase-free L-asparaginase (type I) coding genes from Pseudomonas sp. PCH182 (Ps-ASNase I) and Rahnella sp. PCH162 (Rs-ASNase I) was amplified using gene-specific primers, cloned into a pET-47b(+) vector, and plasmids were transformed into Escherichia coli (E. coli). Further, affinity chromatography purified recombinant proteins to homogeneity with monomer sizes of ~37.0 kDa. Purified Ps-ASNase I and Rs-ASNase I were active at wide pHs and temperatures with optimum activity at 50 °C (492 ± 5 U/mg) and 37 °C (308 ± 4 U/mg), respectively. Kinetic constant Km and Vmax for L-asparagine (Asn) were 2.7 ± 0.06 mM and 526.31 ± 4.0 U/mg for Ps-ASNase I, and 4.43 ± 1.06 mM and 434.78 ± 4.0 U/mg for Rs-ASNase I. Circular dichroism study revealed 29.3 % and 24.12 % α-helix structures in Ps-ASNase I and Rs-ASNase I, respectively. Upon their evaluation to mitigate acrylamide formation, 43 % and 34 % acrylamide (AA) reduction were achieved after pre-treatment of raw potato slices, consistent with 65 % and 59 % Asn reduction for Ps-ASNase I and Rs-ASNase I, respectively. Current findings suggested the potential of less explored intracellular L-asparaginase in AA mitigation for food safety.

Inhibition of glutaminase-1 in DLBCL potentiates venetoclax-induced antitumor activity by promoting oxidative stress.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in adults, but first-line immunochemotherapy fails to produce a durable response in about one-third of the patients. Because tumor cells often reprogram their metabolism, we investigated the importance of glutaminolysis, a pathway converting glutamine to generate energy and various metabolites, for the growth of DLBCL cells. Glutaminase-1 (GLS1) expression was robustly detected in DLBCL biopsy samples and cell lines. Both pharmacological inhibition and genetic knockdown of GLS1 induced cell death in DLBCL cells regardless of their subtype classification, whereas primary B cells remained unaffected. Interestingly, GLS1 inhibition resulted not only in reduced levels of intermediates of the tricarboxylic acid cycle but also in a strong mitochondrial accumulation of reactive oxygen species. Supplementation of DLBCL cells with α-ketoglutarate or with the antioxidant α-tocopherol mitigated oxidative stress and abrogated cell death upon GLS1 inhibition, indicating an essential role of glutaminolysis in the protection from oxidative stress. Furthermore, the combination of the GLS1 inhibitor CB-839 with the therapeutic BCL2 inhibitor ABT-199 not only induced massive reactive oxygen species (ROS) production but also exhibited highly synergistic cytotoxicity, suggesting that simultaneous targeting of GLS1 and BCL2 could represent a novel therapeutic strategy for patients with DLBCL.

Glutaminase (GLS1) gene expression in primary breast cancer.

BACKGROUND: Tumor growth is mediated in part by glutamine, and glutaminase is an enzyme necessary for glutamine catabolism. We studied glutaminase (GLS1) gene expression in primary breast cancer to determine correlations with clinical and tumor characteristics, and gene associations in publicly available databases. A better understanding of glutaminase gene expression may help guide further exploration of glutaminase inhibitors in breast cancer. METHODS: GLS1 mRNA levels were evaluated in The Cancer Genome Atlas (n = 817) and METABRIC (n = 1992) datasets. Associations between GLS1 and tumor subtype (ANOVA followed by post-hoc Tukey test for pairwise comparisons) and selected genes involved in the pathogenesis of breast cancer (Pearson's correlations) were determined in both datasets. In METABRIC, associations with overall survival (Cox proportional hazard model) were determined. For all analyses, p < 0.05 was the threshold for statistical significance. RESULTS: GLS1 expression was significantly higher in triple negative breast cancer (TNBC) than hormone receptor (HR) +/HER2- and HER2+ breast cancer (p < 0.001) and basal versus luminal A, luminal B, and HER2 enriched breast cancer (p < 0.001) in both datasets. In METABRIC, higher GLS1 expression was associated with improved overall survival (HR 0.91, 95% CI: 0.85-0.97, p = 0.005) and this association remained significant in the TNBC subset (HR 0.83, 95% CI: 0.71-0.98, p = 0.032). GLS1 had significant positive gene correlations with immune, proliferative, and basal genes, and inverse correlations with luminal genes and genes involved in metabolism. CONCLUSION: GLS1 expression is highest in TNBC and basal breast cancer, supporting ongoing clinical investigation of GLS1 inhibition in TNBC. GLS1 may have prognostic implications but further research is needed to validate this finding. GLS1 had significant positive gene correlations with immune genes, which may have implications for potential combinations of glutaminase inhibition and immunotherapy.

SIRT4 protects against intestinal fibrosis by facilitating GLS1 degradation.

Intestinal fibrosis is a prevalent complication of Crohn's disease (CD), characterized by excessive deposition of extracellular matrix (ECM), and no approved drugs are currently available for its treatment. Sirtuin 4 (SIRT4), a potent anti-fibrosis factor in mitochondria, has an unclear role in intestinal fibrosis. In this study, fibroblasts isolated from biopsies of stenotic ileal mucosa in CD patients were analyzed to identify the most down-regulated protein among SIRT1-7, and SIRT4 was found to be the most affected. Moreover, in vivo and in vitro models of intestinal fibrosis, SIRT4 expression was significantly decreased in a TGF-ß dependent manner, and its decrease was negatively associated with disease severity. SIRT4 impeded ECM deposition by inhibiting glutaminolysis, but not glycolysis, and α-ketoglutarate (α-KG) was identified as the key metabolite. Specifically, SIRT4 hinders SIRT5's stabilizing interaction with glutaminase 1 (GLS1), thereby facilitating the degradation of GLS1. KDM6, rather than KDM4, is a potential mediator for α-KG-induced transcription of ECM components, and SIRT4 enhances the enrichment of H3K27me3 on their promotors and enhancers. These findings indicate that the activation of TGF-ß signals decreases the expression of SIRT4 in intestinal fibrosis, and SIRT4 can facilitate GLS1 degradation, thereby resisting glutaminolysis and alleviating intestinal fibrosis, providing a novel therapeutic target for intestinal fibrosis.

Optimization of L-glutaminase production by Monascus ruber URM 8542 isolated from ice cream industrial effluent.

L-glutaminase is a hydrolytic enzyme with wide biotechnological applications. Mostly, these enzymes are employed in the feed industry for flavor enhancement and acrylamide mitigation. Also, L-glutaminase may have antiviral and antineoplastic effects making it a good choice for pharmaceutical applications. In this study, the strain Monascus ruber URM 8542 was identified through classical and molecular taxonomy using partial sequencing of ß-tubulin and calmodulin genes. Subsequently, the optimal culture conditions were evaluated by submerged fermentation (L-glutamine 10 g.L- 1) for L-glutaminase excretion. The isolate was identified as M. ruber URM 8542 which showed significant extracellular enzyme production with a yield of 11.4 times in relation to the specific activity of intracellular L-glutaminase. Regarding the optimization experiments, several factors such as L-glutamine concentration, temperature, and pH were compared using a full factorial design (23). The concentrations greater than 1% proved to be significantly better for glutaminase production (R2 = 0.9077). Additionally, the L-glutaminase was optimally active at pH 7.0 and 30 ºC. The L-glutaminase was remarkably stable across an alkaline pH range (7.0-8.0) and had a thermal stability ranging from 30 ºC to 60 ºC for 1 h. Taken together, these findings suggest that the L-glutaminase produced by M. ruber is a promising candidate for pharmacological application, although further studies need to be performed. To the best of our knowledge, this is the first report of L-glutaminase production by Monascus ruber.

m5C-methylated lncRNA NR_033928 promotes gastric cancer proliferation by stabilizing GLS mRNA to promote glutamine metabolism reprogramming.

Abnormal 5-methylcytosine (m5C) methylation has been proved to be closely related to gastric carcinogenesis, progression, and prognosis. Dysregulated long noncoding RNAs (lncRNAs) participate in a variety of biological processes in cancer. However, to date, m5C-methylated lncRNAs are rarely researched in gastric cancer (GC). Here, we found that RNA cytosine-C(5)-methyltransferase (NSUN2) was upregulated in GC and high NSUN2 expression was associated with poor prognosis. NR_033928 was identified as an NSUN2-methylated and upregulated lncRNA in GC. Functionally, NR_033928 upregulated the expression of glutaminase (GLS) by interacting with IGF2BP3/HUR complex to promote GLS mRNA stability. Increased glutamine metabolite, α-KG, upregulated NR_033928 expression by enhancing its promoter 5-hydroxymethylcytosine (hm5C) demethylation. In conclusion, our results revealed that NSUN2-methylated NR_033928 promoted GC progression and might be a potential prognostic and therapeutic target for GC.

Targeting Glutamine Metabolism through Glutaminase Inhibition Suppresses Cell Proliferation and Progression in Nasopharyngeal Carcinoma.

BACKGROUND: Glutaminase (GLS), the key enzyme involved in glutamine metabolism, has been identified as a critical player in tumor growth and progression. The GLS inhibitor CB-839 has entered several clinical trials against a variety of tumors. OBJECTIVE: Our study aimed to investigate the role and underlying mechanism of GLS and its inhibitor CB-839 in nasopharyngeal carcinoma (NPC). METHODS: The expression, downstream genes, and signaling pathways of GLS in NPC were determined by real-time polymerase chain reaction (RT-PCR), PCR array, western blotting (WB), and immunohistochemical staining (IHC), and the phenotype of GLS was confirmed by in vivo experiments of subcutaneous tumor formation in mice and in vitro experiments of functional biology, including Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, transwell migration, and Boyden invasion assay. Finally, it was also verified whether the treatment of NPC cells by GLS inhibitor CB-839 can change various biological functions and protein expression to achieve the purpose of blocking tumor progression. RESULTS: GLS was remarkably overexpressed in NPC cells and tissues, predicting a poor overall survival of NPC patients. GLS promoted cell cycle, proliferation, colony formation, migratory, and invasive capacities by regulating Cyclin D2 (CCND2) via PI3K/AKT/mTOR pathway in NPC in vitro and in vivo. Notably, CB-839 showed an effective anti-NPC tumor effect by blocking the biological functions of the tumor. CONCLUSION: The first innovative proof is that GLS promotes cell proliferation by regulating CCND2 via PI3K/AKT/mTOR pathway in NPC, and GLS inhibitor CB-839 may serve as a new potential therapeutic target for NPC treatment.

LncRNA PVT1 Confers Cisplatin Resistance of Esophageal Cancer Cells through Modulating the miR-181a-5p-Glutaminase (GLS) Axis.

Esophageal carcinoma (ESCA) is one of the prevalent malignancies worldwide. Cisplatin (CDDP) is a conventional chemotherapy drug. However, the acquired cisplatin resistance limits its extensively clinical applications. In this study, the roles and underlying mechanisms of lncRNA PVT1 in cisplatin-resistant ESCA are investigated. PVT1 was significantly upregulated in ESCA patient specimens and cell lines. Higher PVT1 level was associated with a poor survival rate of ESCA patients. Silencing PVT1 effectively increased cisplatin sensitivity of ESCA cells. We established cisplatin-resistant ESCA cell line (EC109 CDDP Res) and detected that PVT1 and glutamine metabolism were remarkedly elevated in CDDP-resistant esophageal cancer cells. Bioinformatical analysis and luciferase assay illustrated that PVT1 sponged miR-181a-5p to form a ceRNA network, resulting in the downregulation of miR-181a-5p expression in ESCA cells. Glutaminase (GLS), which is a key enzyme in the glutamine metabolism, was identified and validated as a direct target of miR-181-5p in ESCA cells. Inhibiting glutamine metabolism effectively re-sensitized CDDP-resistant cells. Rescue experiments demonstrated that restoration of miR-181a-5p in PVT1-overexpressing CDDP-resistant ESCA cells successfully overcame the PVT1-promoted cisplatin resistance through targeting GLS. Summarily, our study revealed molecular mechanisms of the lncRNA PVT1-promoted cisplatin resistance in ESCA by modulating the miR-181a-5p-GLS axis.

Glutaminase 1 deficiency confined in forebrain neurons causes autism spectrum disorder-like behaviors.

An abnormal glutamate signaling pathway has been proposed in the mechanisms of autism spectrum disorder (ASD). However, less is known about the involvement of alterations of glutaminase 1 (GLS1) in the pathophysiology of ASD. We show that the transcript level of GLS1 is significantly decreased in the postmortem frontal cortex and peripheral blood of ASD subjects. Mice lacking Gls1 in CamKIIα-positive neurons display a series of ASD-like behaviors, synaptic excitatory and inhibitory (E/I) imbalance, higher spine density, and glutamate receptor expression in the prefrontal cortex, as well as a compromised expression pattern of genes involved in synapse pruning and less engulfed synaptic puncta in microglia. A low dose of lipopolysaccharide treatment restores microglial synapse pruning, corrects synaptic neurotransmission, and rescues behavioral deficits in these mice. In summary, these findings provide mechanistic insights into Gls1 loss in ASD symptoms and identify Gls1 as a target for the treatment of ASD.

Exosomal MiR-381 from M2-polarized macrophages attenuates urethral fibroblasts activation through YAP/GLS1-regulated glutaminolysis.

OBJECTIVE AND DESIGN: Post-traumatic urethral stricture is a clinical challenge for both patients and clinicians. Targeting glutamine metabolism to suppress excessive activation of urethral fibroblasts (UFBs) is assumed to be a potent and attractive strategy for preventing urethral scarring and stricture. MATERIAL OR SUBJECTS: In cellular experiments, we explored whether glutaminolysis meets the bioenergetic and biosynthetic demands of quiescent UFBs converted into myofibroblasts. At the same time, we examined the specific effects of M2-polarized macrophages on glutaminolysis and activation of UFBs, as well as the mechanism of intercellular signaling. In addition, findings were further verified in vivo in New Zealand rabbits. RESULTS: It revealed that glutamine deprivation or knockdown of glutaminase 1 (GLS1) significantly inhibited UFB activation, proliferation, biosynthesis, and energy metabolism; however, these effects were rescued by cell-permeable dimethyl α-ketoglutarate. Moreover, we found that exosomal miR-381 derived from M2-polarized macrophages could be ingested by UFBs and inhibited GLS1-dependent glutaminolysis, thereby preventing excessive activation of UFBs. Mechanistically, miR-381 directly targets the 3'UTR of Yes-associated protein (YAP) mRNA to reduce its stability at the transcriptional level, ultimately downregulating expression of YAP, and GLS1. In vivo experiments revealed that treatment with either verteporfin or exosomes derived from M2-polarized macrophages significantly reduced urethral stricture in New Zealand rabbits after urethral trauma. CONCLUSION: Collectively, this study demonstrates that exosomal miR-381 from M2-polarized macrophages reduces myofibroblast formation of UFBs and urethral scarring and stricture by inhibiting YAP/GLS1-dependent glutaminolysis.

Pancreatic ß-cell glutaminase 2 maintains glucose homeostasis under the condition of hyperglycaemia.

Glutaminase 2 (GLS2), a master regulator of glutaminolysis that is induced by p53 and converts glutamine to glutamate, is abundant in the liver but also exists in pancreatic ß-cells. However, the roles of GLS2 in islets associated with glucose metabolism are unknown, presenting a critical issue. To investigate the roles of GLS2 in pancreatic ß-cells in vivo, we generated ß-cell-specific Gls2 conditional knockout mice (Gls2 CKO), examined their glucose homeostasis, and validated the findings using a human islet single-cell analysis database. GLS2 expression markedly increased along with p53 in ß-cells from control (RIP-Cre) mice fed a high-fat diet. Furthermore, Gls2 CKO exhibited significant diabetes mellitus with gluconeogenesis and insulin resistance when fed a high-fat diet. Despite marked hyperglycaemia, impaired insulin secretion and paradoxical glucagon elevation were observed in high-fat diet-fed Gls2 CKO mice. GLS2 silencing in the pancreatic ß-cell line MIN6 revealed downregulation of insulin secretion and intracellular ATP levels, which were closely related to glucose-stimulated insulin secretion. Additionally, analysis of single-cell RNA-sequencing data from human pancreatic islet cells also revealed that GLS2 expression was elevated in ß-cells from diabetic donors compared to nondiabetic donors. Consistent with the results of Gls2 CKO, downregulated GLS2 expression in human pancreatic ß-cells from diabetic donors was associated with significantly lower insulin gene expression as well as lower expression of members of the insulin secretion pathway, including ATPase and several molecules that signal to insulin secretory granules, in ß-cells but higher glucagon gene expression in α-cells. Although the exact mechanism by which ß-cell-specific GLS2 regulates insulin and glucagon requires further study, our data indicate that GLS2 in pancreatic ß-cells maintains glucose homeostasis under the condition of hyperglycaemia.