Glutamine Synthetase Expression Research Articles

Curcumin synergistically enhances the efficacy of gemcitabine against gemcitabine-resistant cholangiocarcinoma via the targeting LAT2/glutamine pathway

Cholangiocarcinoma (CCA) is often diagnosed late, leading to incomplete tumor removal, drug resistance and reduced chemotherapy efficacy. Curcumin has the potential for anti-cancer activity through various therapeutic properties and can improve the efficacy of chemotherapy. We aimed to investigate the synergistic effect of a combination of curcumin and gemcitabine against CCA, targeting the LAT2/glutamine pathway. This combination synergistically suppressed proliferation in gemcitabine-resistant CCA cells (KKU-213BGemR). It also resulted in a remarkable degree of CCA cell apoptosis and cell cycle arrest, characterized by a high proportion of cells in the S and G2/M phases. Knockdown of SLC7A8 decreased the expressions of glutaminase and glutamine synthetase, resulting in inhibited cell proliferation and sensitized CCA cells to gemcitabine treatment. Moreover, in vivo experiments showed that a combination curcumin and gemcitabine significantly reduced tumor size, tumor growth rate and LAT2 expression in a gemcitabine-resistant CCA xenograft mouse model. Suppression of tumor progression in an orthotopic CCA hamster model provided strong support for clinical application. In conclusion, curcumin synergistically enhances gemcitabine efficacy against gemcitabine-resistant CCA by induction of apoptosis, partly via inhibiting LAT2/glutamine pathway. This approach may be an alternative strategy for the treatment of gemcitabine-resistant in CCA patients.

Long non-coding RNA PXN-AS1 promotes glutamine synthetase-mediated chronic myeloid leukemia BCR::ABL1-independent resistance to Imatinib via cell cycle signaling pathway

BackgroundChronic myeloid leukemia (CML) is a common hematological malignancy, and tyrosine kinase inhibitors (TKIs) represent the primary therapeutic approach for CML. Activation of metabolism signaling pathway has been connected with BCR::ABL1-independent TKIs resistance in CML cells. However, the specific mechanism by which metabolism signaling mediates this drug resistance remains unclear. Here, we identified one relationship between glutamine synthetase (GS) and BCR::ABL1-independent Imatinib resistance in CML cells.MethodsGS and PXN-AS1 in bone marrow samples of CML patients with Imatinib resistance (IR) were screened and detected by whole transcriptome sequencing. GS expression was upregulated using LVs and blocked using shRNAs respectively, then GS expression, Gln content, and cell cycle progression were respectively tested. The CML IR mice model were established by tail vein injection, prognosis of CML IR mice model were evaluated by Kaplan–Meier analysis, the ratio of spleen/body weight, HE staining, and IHC. PXN-AS1 level was blocked using shRNAs, and the effects of PXN-AS1 on CML IR cells in vitro and in vivo were tested the same as GS. Several RNA-RNA tools were used to predict the potential target microRNAs binding to both GS and PXN-AS1. RNA mimics and RNA inhibitors were used to explore the mechanism through which PXN-AS1 regulates miR-635 or miR-635 regulates GS.ResultsGS was highly expressed in the bone marrow samples of CML patients with Imatinib resistance. In addition, the lncRNA PXN-AS1 was found to mediate GS expression and disorder cell cycle in CML IR cells via mTOR signaling pathway. PXN-AS1 regulated GS expression by binding to miR-635. Additionally, knockdown of PXN-AS1 attenuated BCR::ABL1-independent Imatinib resistance in CML cells via PXN-AS1/miR-635/GS/Gln/mTOR signaling pathway.ConclusionsThus, PXN-AS1 promotes GS-mediated BCR::ABL1-independent Imatinib resistance in CML cells via cell cycle signaling pathway.Graphic

Role of ammonia and glutamine in the pathogenesis and progression of metabolic dysfunction-associated steatotic liver disease: A systematic review.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects over 30% of the global population, with a significant risk of advancing to liver cirrhosis and hepatocellular carcinoma. The roles of ammonia and glutamine in MASLD's pathogenesis are increasingly recognized, prompting this systematic review. This systematic review was conducted through a meticulous search of literature on December 21, 2023, across five major databases, focusing on studies that addressed the relationship between ammonia or glutamine and MASLD. The quality of the included studies was evaluated using CASP checklists. This study is officially registered in the PROSPERO database (CRD42023495619) and was conducted without external funding or sponsorship. Following PRISMA guidelines, 13 studies were included in this review. The studies were conducted globally, with varying sample sizes and study designs. The appraisal indicated a mainly low bias, confirming the reliability of the evidence. Glutamine's involvement in MASLD emerged as multifaceted, with its metabolic role being critical for liver function and disease progression. Variable expressions of glutamine synthetase and glutaminase enzymes highlight metabolic complexity whereas ammonia's impact through urea cycle dysfunction suggests avenues for therapeutic intervention. However, human clinical trials are lacking. This review emphasizes the necessity of glutamine and ammonia in understanding MASLD and identifies potential therapeutic targets. The current evidence, while robust, points to the need for human studies to corroborate preclinical findings. A personalized approach to treatment, informed by metabolic differences in MASLD patients, is advocated, alongside future large-scale clinical trials for a deeper exploration into these metabolic pathways.

Antisense RNA regulates glutamine synthetase in a heterocyst-forming cyanobacterium.

Glutamine synthetase (GS) is a key enzyme involved in nitrogen assimilation and the maintenance of C/N balance, and it is strictly regulated in all bacteria. In cyanobacteria, glutamine synthetase expression is controlled by nitrogen control A (NtcA) transcription factor, which operates global nitrogen regulation in these photosynthetic organisms. Furthermore, post-translational regulation of GS is operated by protein-protein interaction with GS inactivating factors (IFs). Here, we describe an additional regulatory mechanism involving an antisense RNA. In Nostoc sp. PCC 7120, the GS inactivating factor A (gifA) gene (encoding GS inactivating factor IF7) is transcribed downstream of the glutamine synthetase (glnA) gene, from the opposite strand, and the gifA mRNA extends into the glnA coding sequence in antisense orientation. Therefore, the dual RNA transcript that encodes gifA constitutes two functional regions: a 5' protein-coding region, encoding IF7, and a 3' untranslated region that acts as an antisense to glnA. By increasing the levels of such antisense RNA either in cis or in trans, we demonstrate that the amount of GS activity can be modulated by the presence of the antisense RNA. The tail-to-tail disposition of the glnA and gifA genes observed in many cyanobacterial strains from the Nostocales clade suggests the prevalence of such antisense RNA-mediated regulation of GS in this group of cyanobacteria.

A case study of a liver transplant-treated patient with glycogen storage disease type Ia presenting with multiple inflammatory hepatic adenomas: an analysis of clinicopathologic and genetic data

BackgroundGlycogen storage disease (GSD) is a disease caused by excessive deposition of glycogen in tissues due to genetic disorders in glycogen metabolism. Glycogen storage disease type I (GSD-I) is also known as VonGeirk disease and glucose-6-phosphatase deficiency. This disease is inherited in an autosomal recessive manner, and both sexes can be affected. The main symptoms include hypoglycaemia, hepatomegaly, acidosis, hyperlipidaemia, hyperuricaemia, hyperlactataemia, coagulopathy and developmental delay.Case presentationHere, we present the case of a 13-year-old female patient with GSD Ia complicated with multiple inflammatory hepatic adenomas. She presented to the hospital with hepatomegaly, hypoglycaemia, and epistaxis. By clinical manifestations and imaging and laboratory examinations, we suspected that the patient suffered from GSD I. Finally, the diagnosis was confirmed by liver pathology and whole-exome sequencing (WES).WES revealed a synonymous mutation, c.648 G > T (p.L216 = , NM_000151.4), in exon 5 and a frameshift mutation, c.262delG (p.Val88Phefs*14, NM_000151.4), in exon 2 of the G6PC gene. According to the pedigree analysis results of first-generation sequencing, heterozygous mutations of c.648 G > T and c.262delG were obtained from the patient's father and mother.Liver pathology revealed that the solid nodules were hepatocellular hyperplastic lesions, and immunohistochemical (IHC) results revealed positive expression of CD34 (incomplete vascularization), liver fatty acid binding protein (L-FABP) and C-reactive protein (CRP) in nodule hepatocytes and negative expression of β-catenin and glutamine synthetase (GS). These findings suggest multiple inflammatory hepatocellular adenomas. PAS-stained peripheral hepatocytes that were mostly digested by PAS-D were strongly positive.This patient was finally diagnosed with GSD-Ia complicated with multiple inflammatory hepatic adenomas, briefly treated with nutritional therapy after diagnosis and then underwent living-donor liver allotransplantation.After 14 months of follow-up, the patient recovered well, liver function and blood glucose levels remained normal, and no complications occurred.ConclusionThe patient was diagnosed with GSD-Ia combined with multiple inflammatory hepatic adenomas and received liver transplant treatment. For childhood patients who present with hepatomegaly, growth retardation, and laboratory test abnormalities, including hypoglycaemia, hyperuricaemia, and hyperlipidaemia, a diagnosis of GSD should be considered. Gene sequencing and liver pathology play important roles in the diagnosis and typing of GSD.

The impact of sestrin2 on reactive oxygen species in diabetic retinopathy.

Diabetic retinopathy (DR) is a significant complication of diabetes that often leads to blindness, impacting Müller cells, the primary retinal macroglia involved in DR pathogenesis. Reactive oxygen species (ROS) play a crucial role in the development of DR. The objective of this study was to investigate the involvement of sestrin2 in DR using a high-glucose (HG)-induced Müller cell model and assessing cell proliferation with 5-ethynyl-2-deoxyuridine (EdU) labeling. Following this, sestrin2 was upregulated in Müller cells to investigate its effects on ROS, tube formation, and inflammation both in vitro and in vivo, as well as its interaction with the nuclear factor erythroid2-related factor 2 (Nrf2) signaling pathway. The findings demonstrated a gradual increase in the number of EdU-positive cells over time, with a subsequent decrease after 72 h of exposure to high glucose levels. Additionally, the expression of sestrin2 exhibited a progressive increase over time, followed by a decrease at 72 h. The rh-sestrin2 treatment suppressed the injury of Müller cells, decreased ROS level, and inhibited the tube formation. Rh-sestrin2 treatment enhanced the expression of sestrin2, Nrf2, heme oxygenase-1 (HO-1), and glutamine synthetase (GS); however, the ML385 treatment reversed the protective effect of rh-sestrin2. Finally, we evaluated the effect of sestrin2 in a DR rat model. Sestrin2 overexpression treatment improved the pathological injury of retina and attenuated the oxidative damage and inflammatory reaction. Our results highlighted the inhibitory effect of sestrin2 in the damage of retina, thus presenting a novel therapeutic sight for DR.

Adipocytes Are the Only Site of Glutamine Synthetase Expression Within the Lactating Mouse Mammary Gland

BackgroundGlutamine in milk is believed to play an important role in neonatal intestinal maturation and immune function. For lactating mothers, glutamine utilization is increased to meet the demands of the enlarged intestine and milk production. However, the source of such glutamine during lactation has not been studied. ObjectivesWe aimed to assess the effects of lactation on the expression of glutamine synthetase (GS) in the mammary gland and other tissues of lactating mice. MethodsMouse tissues were sampled at 4 time points: 8-wk-old (virgin, control), post-delivery day 5 (PD5, early lactation), PD15 (peak lactation), and involution (4 days after weaning at PD21). We examined the gene expression and protein concentrations of GS and the first 2 enzymes of branched-chain amino acid catabolism: branched-chain aminotransferase 2 (BCAT2) and branched-chain ketoacid dehydrogenase subunit E1α (BCKDHA). ResultsThe messenger RNA (mRNA) expression and protein concentrations of GS in mammary glands were significantly lower at PD5 and PD15 compared with the control but were restored at involution. Within the mammary gland, GS protein was only detected in adipocytes with no evidence of presence in mammary epithelial cells. Compared with the control, mRNA and protein concentrations of BCAT2 and BCKDHA in mammary glands significantly decreased during lactation and involution. No changes in GS protein concentrations during lactation were found in the liver, skeletal muscle, and lung. In non-mammary adipose tissue, GS protein abundance was higher during lactation compared with the virgin. ConclusionsThis work shows that, within the mouse mammary gland, GS is only expressed in adipocytes and that the relative GS abundance in mammary gland sections is lower during lactation. This suggests that mammary adipocytes may be a site of glutamine synthesis in the lactating mouse. Identifying the sources of glutamine production during lactation is important for optimizing milk glutamine concentration to enhance neonatal and maternal health.

Evaluation of the histologic and immunohistochemical (CD34, glutamine synthetase) findings in idiopathic non-cirrhotic portal hypertension (INCPH)

AimIdiopathic non-cirrhotic portal hypertension (INCPH) is a vascular disorder of uncertain origin. Diagnosis can be challenging on liver biopsy. Despite diverse histomorphologic findings documented in literature, studies on the frequency of these findings are lacking. This study aims to assess both the histomorphologic features and the immunoexpression patterns of CD34 and glutamine synthetase (GS) in liver biopsies and searched for their contribution to the pathologic diagnosis of INCPH.Materials and methodsHematoxylin–eosin, CD34, and GS-stained liver needle biopsy sections of 16 patients clinically diagnosed with INCPH were retrospectively analyzed. Histologic findings such as portal vein narrowing, obliteration, or loss were grouped as major findings, while portal vein herniation, hypervascularized portal tracts, and periportal abnormal vessels were grouped as minor findings, and their frequency were evaluated. Periportal endothelial CD34 stained areas were measured via ocular micrometer. The distribution of GS immunoexpression was evaluated. Eighteen healthy liver donor biopsies were evaluated as controls.ResultsIn INCPH cases, 58% of portal tracts showed major findings, compared to 15% in the control group (p < 0.001). Minor findings were observed in 16% of INCPH cases and 7% of controls (p = 0.014). The number of portal tracts with histologic findings is significantly higher in INCPH than in control liver biopsies. Abnormal portal tract distribution, like being close to each other, was seen in 75% of INCPH cases but not in controls (p < 0.001). Nodular regenerative hyperplasia (NRH) was present in 31% of cases. Periportal CD34 expression was higher in INCPH, and affected areas were larger than in controls (p < 0.001). Irregular GS staining, i.e. GS staining with patchy distribution in zone 3, and/or periportal and zone 2 hepatocytes, was found in 62% of INCPH cases, while controls showed the usual pattern (p < 0.001).ConclusionIn the biopsy diagnosis of INCPH, in addition to the presence of major histologic findings and the amount of portal tracts displaying these features, the expression of endothelial CD34 in periportal areas, and irregular hepatocellular GS expression can also be considered as supporting feature.

Post-Traumatic Expressions of Aromatase B, Glutamine Synthetase, and Cystathionine-Beta-Synthase in the Cerebellum of Juvenile Chum Salmon, Oncorhynchus keta.

In adult fish, neurogenesis occurs in many areas of the brain, including the cerebellum, with the ratio of newly formed cells relative to the total number of brain cells being several orders of magnitude greater than in mammals. Our study aimed to compare the expressions of aromatase B (AroB), glutamine synthetase (GS), and cystathionine-beta-synthase (CBS) in the cerebellum of intact juvenile chum salmon, Oncorhynchus keta. To identify the dynamics that determine the involvement of AroB, GS, and CBS in the cellular mechanisms of regeneration, we performed a comprehensive assessment of the expressions of these molecular markers during a long-term primary traumatic brain injury (TBI) and after a repeated acute TBI to the cerebellum of O. keta juveniles. As a result, in intact juveniles, weak or moderate expressions of AroB, GS, and CBS were detected in four cell types, including cells of the neuroepithelial type, migrating, and differentiated cells (graphic abstract, A). At 90 days post injury, local hypercellular areas were found in the molecular layer containing moderately labeled AroB+, GS+, and CBS+ cells of the neuroepithelial type and larger AroB+, GS+, and CBS+ cells (possibly analogous to the reactive glia of mammals); patterns of cells migration and neovascularization were also observed. A repeated TBI caused the number of AroB+, GS+, and CBS+ cells to further increase; an increased intensity of immunolabeling was recorded from all cell types (graphic abstract, C). Thus, the results of this study provide a better understanding of adult neurogenesis in teleost fishes, which is expected to clarify the issue of the reactivation of adult neurogenesis in mammalian species.

Glutamine Metabolism Promotes Renal Fibrosis through Regulation of Mitochondrial Energy Generation and Mitochondrial Fission.

Fibroblast activation and proliferation is an essential phase in the progression of renal fibrosis. Despite the recognized significance of glutamine metabolism in cellular growth and proliferation, its precise pathophysiological relevance in renal fibrosis remains uncertain. Therefore, this study aims to investigate the involvement of glutamine metabolism in fibroblast activation and its possible mechanism. Our findings highlight the importance of glutamine metabolism in fibroblast activation and reveal that patients with severe fibrosis exhibit elevated serum glutamine levels and increased expression of kidney glutamine synthetase. Furthermore, the deprivation of glutamine metabolism in vitro and in vivo could inhibit fibroblast activation, thereby ameliorating renal fibrosis. It was also detected that glutamine metabolism is crucial for maintaining mitochondrial function and morphology. These effects may partially depend on the metabolic intermediate α-ketoglutaric acid. Moreover, glutamine deprivation led to upregulated mitochondrial fission in fibroblasts and the activation of the mammalian target of rapamycin / mitochondrial fission process 1 / dynamin-related protein 1 pathway. Thus, these results provide compelling evidence that the modulation of glutamine metabolism initiates the regulation of mitochondrial function, thereby facilitating the progression of renal fibrosis. Consequently, targeting glutamine metabolism emerges as a novel and promising avenue for therapeutic intervention and prevention of renal fibrosis.

Ammonia scavenger and glutamine synthetase inhibitors cocktail in targeting mTOR/β-catenin and MMP-14 for nitrogen homeostasis and liver cancer.

The glutamine synthetase (GS) facilitates cancer cell growth by catalyzing de novo glutamine synthesis. This enzyme removes ammonia waste from the liver following the urea cycle. Since cancer development is associated with dysregulated urea cycles, there has been no investigation of GS's role in ammonia clearance. Here, we demonstrate that, although GS expression is increased in the setting of β-catenin oncogenic activation, it is insufficient to clear the ammonia waste burden due to the dysregulated urea cycle and may thus be unable to prevent cancer formation. In vivo study, a total of 165 male Swiss albino mice allocated in 11 groups were used, and liver cancer was induced by p-DAB. The activity of GS was evaluated along with the relative expression of mTOR, β-catenin, MMP-14, and GS genes in liver samples and HepG2 cells using qRT-PCR. Moreover, the cytotoxicity of the NH3 scavenger phenyl acetate (PA) and/or GS-inhibitor L-methionine sulfoximine (MSO) and the migratory potential of cells was assessed by MTT and wound healing assays, respectively. The Swiss target prediction algorithm was used to screen the mentioned compounds for probable targets. The treatment of the HepG2 cell line with PA plus MSO demonstrated strong cytotoxicity. The post-scratch remaining wound area (%) in the untreated HepG2 cells was 2.0%. In contrast, the remaining wound area (%) in the cells treated with PA, MSO, and PA + MSO for 48 h was 61.1, 55.8, and 78.5%, respectively. The combination of the two drugs had the greatest effect, resulting in the greatest decrease in the GS activity, β-catenin, and mTOR expression. MSO and PA are both capable of suppressing mTOR, a key player in the development of HCC, and MMP-14, a key player in the development of HCC. PA inhibited the MMP-14 enzyme more effectively than MSO, implying that PA might be a better way to target HCC as it inhibited MMP-14 more effectively than MSO. A large number of abnormal hepatocytes (5%) were found to be present in the HCC mice compared to mice in the control group as determined by the histopathological lesions scores. In contrast, PA, MSO, and PA + MSO showed a significant reduction in the hepatic lesions score either when protecting the liver or when treating the liver. The molecular docking study indicated that PA and MSO form a three-dimensional structure with NF-κB and COX-II, blocking their ability to promote cancer and cause gene mutations. PA and MSO could be used to manipulate GS activities to modulate ammonia levels, thus providing a potential treatment for ammonia homeostasis.

Acetylation of xenogeneic silencer H-NS regulates biofilm development through the nitrogen homeostasis regulator in Shewanella.

Adjusting intracellular metabolic pathways and adopting suitable live state such as biofilms, are crucial for bacteria to survive environmental changes. Although substantial progress has been made in understanding how the histone-like nucleoid-structuring (H-NS) protein modulates the expression of the genes involved in biofilm formation, the precise modification that the H-NS protein undergoes to alter its DNA binding activity is still largely uncharacterized. This study revealed that acetylation of H-NS at Lys19 inhibits biofilm development in Shewanella oneidensis MR-1 by downregulating the expression of glutamine synthetase, a critical enzyme in glutamine synthesis. We further found that nitrogen starvation, a likely condition in biofilm development, induces deacetylation of H-NS and the trimerization of nitrogen assimilation regulator GlnB. The acetylated H-NS strain exhibits significantly lower cellular glutamine concentration, emphasizing the requirement of H-NS deacetylation in Shewanella biofilm development. Moreover, we discovered in vivo that the activation of glutamine biosynthesis pathway and the concurrent suppression of the arginine synthesis pathway during both pellicle and attached biofilms development, further suggesting the importance of fine tune nitrogen assimilation by H-NS acetylation in Shewanella. In summary, posttranslational modification of H-NS endows Shewanella with the ability to respond to environmental needs by adjusting the intracellular metabolism pathways.

Butyrate Alleviates High-Fat-Induced Metabolic Disorders Partially through Increasing Systematic Glutamine.

Obesity has emerged as a worldwide epidemic. Both butyrate and glutamine counteract obesity-related metabolic disorders; however, whether and how they synergistically cooperate with each other remains a mystery. In the study, a high-fat diet (HFD, 60% calories from fat) was used to develop a model of obesity-related metabolic disorder and compared with administrated saline and sodium butyrate (SB, 300 mg/kg body weight) daily by gavage. Compared with HFD counterparts, oral administration of SB in mice exhibited significantly reduced body weight and fat mass and decreased hepatic triglyceride content. The targeted mass spectrum revealed that SB restored serum contents of glutamine, which were significantly decreased by HFD. Furthermore, SB significantly elevated the expression of glutamine synthetase (GS, encoded by GLUL) in the liver, accompanied by more enrichment of H3K27ac modifications within its promoter. In summary, the study verified the contribution of elevated glutamine to the beneficial effects of butyrate on metabolic disorders induced by a high-fat diet, providing a novel pathway for understanding how butyrate benefits metabolic homeostasis.

HIV-1 gp120 Induces Glutamate Excitotoxicity By Diminishing Glutamine Synthetase Expression in Astrocytes

Background： As the most abundant subtype of glial cells, astrocytes have an important role in maintaining homeostasis in the central nervous system(CNS) by regulating neurotransmitters. Glutamate is an excitatory neurotransmitter released from neurons, while astrocytes take up glutamate and mediate the conversion to glutamine, which is known as the glutamate-glutamine cycle between neurons and astrocytes. Glutamine synthetase (GS) is found primarily in astrocytes and is a key player in the glutamate-glutamine cycle. GS synthesizes glutamine using glutamate and contributes to the maintenance of glutamate homeostasis between neurons and astrocytes by facilitating glutamate clearance. In addition, glutamate excitotoxicity is a key pathological factor in neurodegenerative pathologies such as HIV-1-associated neurocognitive disorder (HAND) and Alzheimer's disease. Although the importance of GS in the glutamate-glutamine cycle has been demonstrated, the relationship between gp120 in regulating astrocyte glutamate metabolism and GS activity is unclear. Methods: To evaluate the glutamate clearance capacity, glutamate uptake assay was undertaken by using the glutamate colorimetric assay kit. Astrocytes (u251) were incubated for 24 hours with different concentrations of full-length HIV-1 gp120 and medium in each dish was replaced with serum-free HBSS that containing 2 mM glutamate. After 10, 30, 60, or 120 min in the incubator, 200 μL culture supernatants of the astrocytes were collected and mixed with the corresponding reagents provided in the kit as required by the protocol, then 100 μL of the mixed solution were transferred to each well of the 96-well culture plates. The absorbance of the mixed solution was measured at 340 nm using a microplate reader. The glutamate content of each sample was calculated according to the formula provided in the instruction manual. The expressions of GS in astrocytes which were incubated for 24 hours with different concentrations of full-length HIV-1 gp120 were detected by Western blot. Student's t-tests and ANOVA were applied to determine statistical significance. Results： The glutamate uptake assay reflects whether the glutamate uptake capacity of astrocytes is impaired by assessing the extracellular glutamate content. First, changes in extracellular glutamate content were detected by treating astrocytes with different concentrations of HIV-1 gp120 for 24h. As expected, the extracellular glutamate content increased relative to the blank control, indicating that HIV-1 gp120 impaired the glutamate uptake ability of astrocytes. The optimal experimental point to test the glutamate uptake capacity was 30 min after the addition of serum-free HBSS that containing 2 mM glutamate by time-point experiments. In addition, the results of western blot showed that GS expression in astrocytes was significantly decreased by HIV-1 gp120 treatment. The GS expression levels and glutamate uptake assay results followed the same trend. Conclusion: In conclusion, this study explored the specific mechanisms of glutamatergic excitotoxicity in the brains of HAND patients. HIV-1 gp120 could impair the glutamate uptake capacity of astrocytes by diminishing GS expression. ( Acknowledgements: National Natural Science Foundation of China, No. 82172259 to H.C.; Corresponding author: Hong Cao, gzhcao@smu.edu.cn )

Leat-associated seizures the possible role of EAAT2, pyruvate carboxylase and glutamine synthetase

BackgroundDrug-resistant epilepsy is a common condition in patients with brain neoplasms. The pathogenesis of tumor-associated seizures is poorly understood. Among the possible pathogenetic mechanisms, the increase in glutamate concentration has been proposed. Glutamate transporters, glutamine synthetase and pyruvate carboxylase are involved in maintaining the physiological concentration of glutamate in the intersynaptic spaces. In our previous research on angiocentric gliomas, we demonstrated that all tumors lacked the expression of the main glutamate transporter EAAT2, while the expression of glutamine synthetase and pyruvate carboxylase was mostly preserved. MethodsIn the present study, we evaluated the immunohistochemical expression of EAAT2, glutamine synthetase and pyruvate carboxylase in a heterogeneous series of 25 long-term epilepsy-associated tumors (10 dysembryoplastic neuroepithelial tumors, 7 gangliogliomas, 3 subependymal giant cell astrocytomas, 3 rosette forming glioneuronal tumors, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). In order to evaluate the incidence of variants in the SLC1A2 gene, encoding EAAT2, in a large number of central nervous system tumors we also queried the PedcBioPortal. ResultsEAAT2 protein expression was lost in 9 tumors (36 %: 3 dysembryoplastic neuroepithelial tumors, 1 ganglioglioma, 3 subependymal giant cell astrocytomas, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). Glutamine synthetase protein expression was completely lost in 2 tumors (8 %; 1 ganglioglioma and 1 diffuse astrocytoma MYB- or MYBL1-altered). All tumors of our series but rosette forming glioneuronal tumors (in which neurocytic cells were negative) were diffusely positive for pyruvate carboxylase. Consultation of the PedcBioPortal revealed that of 2307 pediatric brain tumors of different histotype and grade, 20 (< 1%) had variants in the SLC1A2 gene. Among the SLC1A2-mutated tumors, there were no angiocentric gliomas or other LEATs ConclusionsIn conclusion, unlike angiocentric gliomas where the EAAT2 loss is typical and constant, the current study shows the loss of EAAT2 expression only in a fraction of the LEATs. In these cases, we may hypothesize some possible epileptogenic role of the EAAT2 loss. The retained expression of pyruvate carboxylase may contribute to determining a pathological glutamate excess unopposed by glutamine synthetase that resulted expressed to a variable extent in the majority of the tumors. Furthermore, we can assume that the EAAT2 loss in brain tumors in general and in LEATs in particular is more conceivably epigenetic.

Glutamine synthetase and hepatocellular carcinoma

Glutamine synthetase (GS) is an enzyme that converts ammonia and glutamate to glutamine using adenosine triphosphate. GS is expressed in the brain, kidney, and liver tissues under normal physiological conditions. GS is involved in abnormal lipid metabolism of the liver by catalyzing de novo synthesis of glutamine, thereby inducing liver inflammation. Metabolic dysfunction-associated steatotic liver diseases (MASLD), such as Metabolic Associated Fatty Liver Disease and Metabolic Associated Steato Hepatitis, are considered risk factors for HCC. GS may also be involved in the development and progression of hepatocellular carcinoma (HCC) through other signaling pathways, including the rapamycin (mTOR) and Wnt/β-catenin signaling pathways. Furthermore, the correct combination of HSP70, GPC3, and GS can improve the accuracy and precision of HCC diagnosis. However, the prognostic value of GS in different HCC populations remains controversial. The expression of GS affects the sensitivity of HCC cells to radiotherapy and chemotherapy. In addition, immunotherapy has been approved for the treatment of advanced HCC. This article delves into the development and application of GS in HCC, laying a theoretical foundation for the subsequent exploration of GS as a potential target for treating HCC.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

Control of glnA (glutamine synthetase) expression by urea in non-pathogenic and uropathogenic Escherichia coli.

The bacteria that cause urinary tract infections often become resistant to antibiotic treatment, and genes expressed during an infection could suggest non-antibiotic targets. During growth in urine, glnA (specifying glutamine synthetase) expression is high, but our results show that urea induces glnA expression independent of the regulation that responds to nitrogen limitation. Although our results suggest that glnA is an unlikely target for therapy because of variation in urinary components between individuals, our analysis of glnA expression in urine-like environments has revealed previously undescribed layers of regulation. In other words, regulatory mechanisms that are discovered in a laboratory environment do not necessarily operate in the same way in nature.

Electroacupuncture exerts prolonged analgesic and neuroprotective effects in a persistent dental pain model induced by multiple dental pulp injuries: GABAergic interneurons-astrocytes interaction.

Pain within the trigeminal system, particularly dental pain, is poorly understood. This study aimed to determine whether single or multiple dental pulp injuries induce persistent pain, its association with trigeminal central nociceptive pathways and whether electroacupuncture (EA) provides prolonged analgesic and neuroprotective effects in a persistent dental pain model. Models of single dental pulp injury (SDPI) and multiple dental pulp injuries (MDPI) were used to induce trigeminal neuropathic pain. The signs of dental pain-related behavior were assessed using the mechanical head withdrawal threshold (HWT). Immunofluorescence and western blot protocols were used to monitor astrocyte activation, changes in apoptosis-related proteins, and GABAergic interneuron plasticity. SDPI mice exhibited an initial marked decrease in HWT from days one to 14, followed by progressive recovery from days 21 to 42. From days 49 to 70, the HWT increased and returned to the control values. In contrast, MDPI mice showed a persistent decrease in HWT from days one to 70. MDPI increased glial fibrillary acidic protein (GFAP) and decreased glutamine synthetase (GS) and glutamate transporter-1 (GLT1) expression in the Vi/Vc transition zone of the brainstem on day 70, whereas no changes in astrocytic markers were observed on day 70 after SDPI. Increased expression of cleaved cysteine-aspartic protease-3 (cleaved caspase-3) and Bcl-2-associated X protein (Bax), along with decreased B-cell lymphoma/leukemia 2 (Bcl-2), were observed at day 70 after MDPI but not after SDPI. The downregulation of glutamic acid decarboxylase (GAD65) expression was observed on day 70 only after MDPI. The effects of MDPI-induced lower HWT from days one to 70 were attenuated by 12 sessions of EA treatment (days one to 21 after MDPI). Changes in astrocytic GFAP, GS, and GLT-1, along with cleaved caspase-3, Bax, Bcl-2, and GAD65 expression observed 70 days after MDPI, were reversed by EA treatment. The results suggest that persistent dental pain in mice was induced by MDPI but not by SDPI. This effect was associated with trigeminal GABAergic interneuron plasticity along with morphological and functional changes in astrocytes. EA exerts prolonged analgesic and neuroprotective effects that might be associated with the modulation of neuron-glia crosstalk mechanisms.