Modulating glutamine metabolism to control viral immuno-inflammatory lesions.

Infection of the cornea with HSV results in an immune-inflammatory reaction orchestrated by proinflammatory T cells that is a major cause of human vision impairment. The severity of lesions can be reduced if the representation of inflammatory T cells is changed to increase the presence of T cells with regulatory function. This report shows that inhibiting glutamine metabolism using 6-Diazo-5-oxo-l-norleucine (DON) administered via intraperitoneal (IP) starting 6 days after ocular infection and continued until day 15 significantly reduced the severity of herpetic stromal keratitis lesions. The therapy resulted in reduced neutrophils, macrophages as well proinflammatory CD4 Th1 and Th17 T cells in the cornea, but had no effect on levels of regulatory T cells. A similar change in the representation of inflammatory and regulatory T cells occurred in the trigeminal ganglion (TG) the site where HSV infection establishes latency. Glutamine metabolism was shown to be required for the in-vitro optimal induction of both Th1 and Th17 T cells but not for the induction of Treg that were increased when glutamine metabolism was inhibited. Inhibiting glutamine metabolism also changed the ability of latently infected TG cells from animals previously infected with HSV to reactivate and produce infectious virus.

Gilteritinib Inhibits Glutamine Uptake and Utilization in FLT3-ITD-Positive AML.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib's antileukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. In addition, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Finally, we found that the glutaminase inhibitor CB-839 enhanced antileukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD-positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.

Dose-Dependent Carbon-Dot-Induced ROS Promote Uveal Melanoma Cell Tumorigenicity via Activation of mTOR Signaling and Glutamine Metabolism.

Uveal melanoma (UM) is the most common intraocular malignant tumor in adults and has a low survival rate following metastasis; it is derived from melanocytes susceptible to reactive oxygen species (ROS). Carbon dot (Cdot) nanoparticles are a promising tool in cancer detection and therapy due to their unique photophysical properties, low cytotoxicity, and efficient ROS productivity. However, the effects of Cdots on tumor metabolism and growth are not well characterized. Here, the effects of Cdots on UM cell metabolomics, growth, invasiveness, and tumorigenicity are investigated in vitro and in vivo zebrafish and nude mouse xenograft model. Cdots dose-dependently increase ROS levels in UM cells. At Cdots concentrations below 100 µg mL-1, Cdot-induced ROS promote UM cell growth, invasiveness, and tumorigenicity; at 200 µg mL-1, UM cells undergo apoptosis. The addition of antioxidants reverses the protumorigenic effects of Cdots. Cdots at 25-100 µg mL-1 activate Akt/mammalian target of rapamycin (mTOR) signaling and enhance glutamine metabolism, generating a cascade that promotes UM cell growth. These results demonstrate that moderate, subapoptotic doses of Cdots can promote UM cell tumorigenicity. This study lays the foundation for the rational application of ROS-producing nanoparticles in tumor imaging and therapy.

Association of Age, Antipsychotic Medication, and Symptom Severity in Schizophrenia With Proton Magnetic Resonance Spectroscopy Brain Glutamate Level: A Mega-analysis of Individual Participant-Level Data.

Importance: Proton magnetic resonance spectroscopy (1H-MRS) studies indicate that altered brain glutamatergic function may be associated with the pathophysiology of schizophrenia and the response to antipsychotic treatment. However, the association of altered glutamatergic function with clinical and demographic factors is unclear. Objective: To assess the associations of age, symptom severity, level of functioning, and antipsychotic treatment with brain glutamatergic metabolites. Data Sources: The MEDLINE database was searched to identify journal articles published between January 1, 1980, and June 3, 2020, using the following search terms: MRS or magnetic resonance spectroscopy and (1) schizophrenia or (2) psychosis or (3) UHR or (4) ARMS or (5) ultra-high risk or (6) clinical high risk or (7) genetic high risk or (8) prodrome* or (9) schizoaffective. Authors of 114 1H-MRS studies measuring glutamate (Glu) levels in patients with schizophrenia were contacted between January 2014 and June 2020 and asked to provide individual participant data. Study Selection: In total, 45 1H-MRS studies contributed data. Data Extraction and Synthesis: Associations of Glu, Glu plus glutamine (Glx), or total creatine plus phosphocreatine levels with age, antipsychotic medication dose, symptom severity, and functioning were assessed using linear mixed models, with study as a random factor. Main Outcomes and Measures: Glu, Glx, and Cr values in the medial frontal cortex (MFC) and medial temporal lobe (MTL). Results: In total, 42 studies were included, with data for 1251 patients with schizophrenia (mean [SD] age, 30.3 [10.4] years) and 1197 healthy volunteers (mean [SD] age, 27.5 [8.8] years). The MFC Glu (F1,1211.9 = 4.311, P = .04) and Glx (F1,1079.2 = 5.287, P = .02) levels were lower in patients than in healthy volunteers, and although creatine levels appeared lower in patients, the difference was not significant (F1,1395.9 = 3.622, P = .06). In both patients and volunteers, the MFC Glu level was negatively associated with age (Glu to Cr ratio, F1,1522.4 = 47.533, P < .001; cerebrospinal fluid-corrected Glu, F1,1216.7 = 5.610, P = .02), showing a 0.2-unit reduction per decade. In patients, antipsychotic dose (in chlorpromazine equivalents) was negatively associated with MFC Glu (estimate, 0.10 reduction per 100 mg; SE, 0.03) and MFC Glx (estimate, -0.11; SE, 0.04) levels. The MFC Glu to Cr ratio was positively associated with total symptom severity (estimate, 0.01 per 10 points; SE, 0.005) and positive symptom severity (estimate, 0.04; SE, 0.02) and was negatively associated with level of global functioning (estimate, 0.04; SE, 0.01). In the MTL, the Glx to Cr ratio was positively associated with total symptom severity (estimate, 0.06; SE, 0.03), negative symptoms (estimate, 0.2; SE, 0.07), and worse Clinical Global Impression score (estimate, 0.2 per point; SE, 0.06). The MFC creatine level increased with age (estimate, 0.2; SE, 0.05) but was not associated with either symptom severity or antipsychotic medication dose. Conclusions and Relevance: Findings from this mega-analysis suggest that lower brain Glu levels in patients with schizophrenia may be associated with antipsychotic medication exposure rather than with greater age-related decline. Higher brain Glu levels may act as a biomarker of illness severity in schizophrenia.

Changes in Brain Glutamate on Switching to Clozapine in Treatment-Resistant Schizophrenia.

It has been suggested that the antipsychotic clozapine may modulate brain glutamate, and that this effect could contribute to its efficacy in treatment-resistant schizophrenia (TRS). The aim of this study was to examine the effects of clozapine on brain glutamate in TRS longitudinally. This study examined individuals with TRS before and 12 weeks after switching from a non-clozapine antipsychotic to treatment with clozapine as part of their normal clinical care. Proton magnetic resonance spectroscopy (1H-MRS) measured concentrations, corrected for voxel tissue content, of glutamate (Glucorr), and glutamate plus glutamine (Glxcorr) in the anterior cingulate cortex (ACC) and right caudate nucleus. Symptoms were monitored using the Positive and Negative Syndrome Scale (PANSS). Of 37 recruited patients (27 men, 39.30 years old, 84% clozapine naïve), 25 completed 1H-MRS at both timepoints. 12 weeks of clozapine was associated with a longitudinal reduction in Glucorr in the caudate (n = 23, F = 7.61 P = .01) but not in the ACC (n = 24, F = 0.02, P = .59). Percentage reduction in caudate Glucorr was positively correlated with percentage improvement in symptoms (total PANSS score, n = 23, r = .42, P = .04). These findings indicate that reductions in glutamate in the caudate nucleus may contribute to symptomatic improvement during the first months of clozapine treatment.

N-acetylcysteine reduces cocaine-seeking behavior and anterior cingulate glutamate/glutamine levels among cocaine-dependent individuals.

N-acetylcysteine (NAC) is a cystine prodrug shown to reduce cocaine- and cue-primed reinstatement of cocaine-seeking behavior in preclinical studies. In this inpatient study, the effects of NAC maintenance versus placebo on cocaine-seeking behavior were examined during cocaine-primed and unprimed self-administration sessions among non-treatment-seeking, cocaine-dependent individuals. Twelve participants completed this double-blind, placebo-controlled, within-subject crossover study. Each participant was maintained for 1 week (Sat-Fri) on NAC (1200-mg TID; 3600 mg/day total) and 1 week on placebo (0-mg TID); medication order was randomized. A subset of participants underwent proton magnetic resonance spectroscopy scans (n = 8) on the third day of medication (Mon) to assess neurochemistry in the rostral anterior cingulate (rACC; voxel = 4.5 cm3 ). In four randomized sessions (Tue-Fri) each week, each participant could earn unit amounts of cocaine (10 mg, fixed) versus money ($0.50 vs. $1.50) on a choice, progressive ratio schedule after insufflating active versus placebo cocaine-priming doses (110 mg vs. 4 mg). Relative to the placebo priming dose, the active cocaine priming dose (110 mg) increased cocaine-seeking behavior (p = .003). NAC reduced cocaine-primed cocaine-seeking behavior compared with placebo levels (p = .044) but did not alter placebo-primed cocaine-seeking behavior. The larger money alternative ($1.50) suppressed cocaine-seeking behavior relative to the smaller money alternative ($0.50; p = .011). Compared with placebo levels, NAC significantly decreased rACC glutamate + glutamine levels (p = .035) and numerically decreased rACC glutamate levels (p = .085). These preliminary findings indicate that NAC suppresses cocaine-seeking behavior in some, but not all, experimental scenarios. Further, our findings suggest NAC may exert its therapeutic effects by modulating excitatory tone in the rACC.

Glutaminase 1 Inhibition Reduces Glycolysis and Ameliorates Lupus-like Disease in MRL/lpr Mice and Experimental Autoimmune Encephalomyelitis.

OBJECTIVE: Glutaminase 1 (Gls1) is the first enzyme in glutaminolysis. The selective Gls1 inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) suppresses Th17 development and ameliorates experimental autoimmune encephalomyelitis (EAE). The present study was undertaken to investigate whether inhibition of glutaminolysis is beneficial for the treatment of systemic lupus erythematosus (SLE), and the involved mechanisms. METHODS: MRL/lpr mice were treated with BPTES or vehicle control, and disease activity was examined. Then naive CD4+ T cells from patients with SLE were cultured under Th17-polarizing conditions with BPTES or vehicle. Furthermore, using newly generated Gls1 conditional-knockout mice, in vitro Th17 differentiation was examined, and EAE was induced in the mice. Glutaminolysis and glycolysis were measured with an extracellular flux analyzer. The expression of hypoxia-inducible factor 1α (HIF-1α) was examined by Western blotting. RESULTS: Treatment of MRL/lpr mice with BPTES improved autoimmune pathology in a Th17-dependent manner. T cells from patients with SLE treated with BPTES displayed decreased Th17 differentiation (P < 0.05). Using the conditional-knockout mice, we demonstrated that both in vitro Th17 differentiation (P < 0.05) and the development of EAE were dependent on Gls1. Gls1 inhibition reduced glycolysis and the expression of HIF-1α protein, which induces glycolysis. CONCLUSION: We demonstrated that inhibition of glutaminolysis represents a potential new treatment strategy for patients with SLE and Th17-related autoimmune diseases. Mechanistically, we have shown that inhibition of glutaminolysis affects the glycolysis pathway by reducing HIF-1α protein in Th17 cells.

Brain neurochemistry in unmedicated obsessive-compulsive disorder patients and effects of 12-week escitalopram treatment: 1 H-magnetic resonance spectroscopy study.

AIM: The purpose of this study was to examine treatment-related neurochemical changes in 28 unmedicated obsessive-compulsive disorder (OCD) patients using 1 H-magnetic resonance spectroscopy (1 H-MRS). METHODS: We included subjects diagnosed with OCD (n = 28), each with a total duration of illness of less than 5 years, as a study group and age- and sex-matched healthy controls (n = 26). The inclusion criteria for the OCD group were right-handed individuals aged 18 years or older who had not been on any specific treatment for OCD for the last at least 8 weeks and who had no other psychiatric comorbidity. A pre-post and case-control design was employed in which OCD patients underwent 1 H-MRS at baseline and 12 weeks after treatment with escitalopram (n = 21). Clinical assessment was carried out using a semi-structured pro forma Yale-Brown Obsessive Compulsive Scale and the World Health Organization Disability Assessment Scale 2.0 before and after treatment. Volume-localized 1 H-MRS was carried out with a 3-Tesla Philips MR scanner. RESULTS: Our data suggested higher levels of myoinositol (mI), total choline (tCho), and glutamate+glutamine (Glx) in the medial thalamus at pre-assessment in OCD subjects as compared to healthy controls and a significant reduction in tCho and Glx after treatment in OCD subjects. The mI levels in the caudate nucleus and Glx levels in the anterior cingulate cortex were significantly correlated with disease severity on the Yale-Brown Obsessive Compulsive Scale. CONCLUSION: Our study supports the hypothesis of a hyper-glutaminergic state (as suggested by increased Glx levels) and neurodegeneration (as suggested by increased tCho and mI in the thalamus) in cortico-striato-thalamocortical circuitry in OCD patients as suggested by previous studies using MRS as well as other functional imaging studies.

Anxiolytic properties of compounds that counteract oxidative stress, neuroinflammation, and glutamatergic dysfunction: a review

Objective: Anxiety disorders are highly prevalent and the efficacy of the available anxiolytic drugs is less than desired. Adverse effects also compromise patient quality of life and adherence to treatment. Accumulating evidence shows that the pathophysiology of anxiety and related disorders is multifactorial, involving oxidative stress, neuroinflammation, and glutamatergic dysfunction. The aim of this review was to evaluate data from animal studies and clinical trials showing the anxiolytic effects of agents whose mechanisms of action target these multiple domains. Methods: The PubMed database was searched for multitarget agents that had been evaluated in animal models of anxiety, as well as randomized double-blind placebo-controlled clinical trials of anxiety and/or anxiety related disorders. Results: The main multitarget agents that have shown consistent anxiolytic effects in various animal models of anxiety, as well in clinical trials, are agomelatine, N-acetylcysteine (NAC), and omega-3 fatty acids. Data from clinical trials are preliminary at best, but reveal good safety profiles and tolerance to adverse effects. Conclusion: Agomelatine, NAC and omega-3 fatty acids show beneficial effects in clinical conditions where mainstream treatments are ineffective. These three multitarget agents are considered promising candidates for innovative, effective, and better-tolerated anxiolytics.

Discovery and development of small molecule modulators targeting glutamine metabolism.

Cancer cells have been confirmed diverge significantly from normal cells on the metabolic properties. Energy production in cancer cells is abnormally dependent on the glycolysis pathway and other atypical metabolic characteristics such as increased fatty acid synthesis and increased rates of glutamine metabolism. Among these metabolic features of cancers, glutamine metabolism has been reported to be the main energy supply for the growth and viability of a potentially large subset of malignant tumors. In addition, the significance of glutamine metabolism in cancer cells derives from the ability of donate its nitrogen and carbon atoms for the synthesis of important biologically substances. During recent years, emerging evidences have proved the inhibitors targeting glutamine metabolism pathway could be efficient anticancer drugs. Therefore, in this review, we would briefly introduce the regulation of glutamine metabolism, and then summarize the recent advances of small molecule modulators targeting various nodes in glutamine signaling pathway. The current potential obstacles and future therapeutic perspectives in glutamine metabolism are also put forward in order to provide reference for the drug discovery of novel and potent glutamine metabolism modulators.

Anxiolytic properties of compounds that counteract oxidative stress, neuroinflammation, and glutamatergic dysfunction: a review.

OBJECTIVE: Anxiety disorders are highly prevalent and the efficacy of the available anxiolytic drugs is less than desired. Adverse effects also compromise patient quality of life and adherence to treatment. Accumulating evidence shows that the pathophysiology of anxiety and related disorders is multifactorial, involving oxidative stress, neuroinflammation, and glutamatergic dysfunction. The aim of this review was to evaluate data from animal studies and clinical trials showing the anxiolytic effects of agents whose mechanisms of action target these multiple domains. METHODS: The PubMed database was searched for multitarget agents that had been evaluated in animal models of anxiety, as well as randomized double-blind placebo-controlled clinical trials of anxiety and/or anxiety related disorders. RESULTS: The main multitarget agents that have shown consistent anxiolytic effects in various animal models of anxiety, as well in clinical trials, are agomelatine, N-acetylcysteine (NAC), and omega-3 fatty acids. Data from clinical trials are preliminary at best, but reveal good safety profiles and tolerance to adverse effects. CONCLUSION: Agomelatine, NAC and omega-3 fatty acids show beneficial effects in clinical conditions where mainstream treatments are ineffective. These three multitarget agents are considered promising candidates for innovative, effective, and better-tolerated anxiolytics.

Glutamine-dependent lysosome homeostatic changes induced by starvation and lysosome inhibition.

Lysosomes are a major organelle for degrading macromolecules. When deprived of nutrients, cells activate the autophagy and lysosome biogenesis pathways to recycle cytoplasmic materials and to increase lysosomal degradation capacity for survival, respectively. We have identified a condition in which cells accumulated enlarged lysosomes upon starvation and lysosome inhibition. Selective autophagy and inhibition of the mechanistic target of rapamycin (mTOR) in combination with lysosome inhibition were not able to induce this phenomenon. Conversely, knocking out autophagy genes, ATG5 or ATG7, had no effects on the enlarged lysosome formation. This suggests that the enlarged lysosome formation is an autophagy independent process. Remarkably, adding glutamine to the treatment can prevent formation of the enlarged lysosomes and dissipate the pre-existing ones. Furthermore, the nucleus/cytoplasm translocation of the transcription factor EB (TFEB), but not mTOR activity, correlates with the formation/dissipation of enlarged lysosomes. Knockdown of TFEB, however, suggests that TFEB-mediated lysosome biogenesis is not directly involved in the process. These results indicate that there is a novel mechanism by which lysosome homeostasis can be regulated under certain stress conditions.

Cerebral dopaminergic and glutamatergic transmission relate to different subjective responses of acute alcohol intake: an in vivo multimodal imaging study.

Converging preclinical evidence links extrastriatal dopamine release and glutamatergic transmission via the metabotropic glutamate receptor 5 (mGluR5) to the rewarding properties of alcohol. To date, human evidence is lacking on how and where in the brain these processes occur. Mesocorticolimbic dopamine release upon intravenous alcohol administration and mGluR5 availability were measured in 11 moderate social drinkers by single-session [18 F]fallypride and [18 F]FPEB positron emission tomography, respectively. Additionally, baseline and postalcohol glutamate and glutamine levels in the anterior cingulate cortex (ACC) were measured by using proton-magnetic resonance spectroscopy. To investigate differences in reward domains linked to both neurotransmitters, regional imaging data were related to subjective alcohol responses. Alcohol induced significant [18 F]fallypride displacement in the prefrontal cortex (PFC), temporal and parietal cortices and thalamus (P < 0.05, corrected for multiple comparisons). Dopamine release in the ACC and orbitofrontal and ventromedial PFCs were correlated with subjective 'liking' and 'wanting' effects (P < 0.05). In contrast, baseline mGluR5 availability was positively correlated with the 'high' effect of alcohol in dorsolateral, ventrolateral and ventromedial PFCs and in the medial temporal lobe, thalamus and caudate nucleus (P < 0.05). Although neither proton-magnetic resonance spectroscopy glutamate nor glutamine levels were affected by alcohol, baseline ACC glutamate levels were negatively associated with the alcohol 'liking' effect (P < 0.003). These data reveal new mechanistic understanding and differential neurobiological underpinnings of the effects of acute alcohol consumption on human behavior. Specifically, prefrontal dopamine release may encode alcohol 'liking' and 'wanting' effects in specific areas underlying value processing and motivation, whereas mGluR5 availability in distinct prefrontal-temporal-subcortical regions is more related to the alcohol 'high' effect.

Modeling human methamphetamine use patterns in mice: chronic and binge methamphetamine exposure, reward function and neurochemistry.

Different methamphetamine use patterns in human subjects may contribute to inconsistent findings regarding the effects of methamphetamine abuse on brain and behavior. The present study investigated whether human-derived chronic and binge methamphetamine use patterns have differential effects on reward and neurochemistry in mice. Brain reward function in mice was evaluated during acute/prolonged withdrawal, and in response to methamphetamine challenge using the intracranial self-stimulation procedure. Brain dopaminergic, serotonergic and glutamatergic neurochemistry was determined with high-performance liquid chromatography. Chronic and binge regimens induced withdrawal-related decreases in reward function that were more severe during the binge regimen during cycles 1-2. Despite large differences in methamphetamine dose, both regimens induced similar reward deficits during cycles 3-4. Neither methamphetamine regimen led to persistent alterations in the sensitivity to the reward-enhancing effects of acute methamphetamine challenge. The binge regimen severely depleted striatal dopamine levels and increased brain glutamine levels. The chronic regimen had milder effects on striatal dopamine levels and altered cortical dopamine and serotonin levels. This work highlights that the magnitude of acute/prolonged withdrawal may not reflect amount or frequency of methamphetamine intake. In contrast, the array of underlying neurochemical alterations was methamphetamine regimen dependent. Thus, stratifying methamphetamine-dependent individuals based on use pattern may help to cater therapeutic interventions more appropriately by targeting use pattern-specific neurotransmitter systems.

Safety and efficacy of glycerol phenylbutyrate for management of urea cycle disorders in patients aged 2months to 2years.

INTRODUCTION: Glycerol phenylbutyrate (GPB) is approved in the US for the management of patients 2months of age and older with urea cycle disorders (UCDs) that cannot be managed with protein restriction and/or amino acid supplementation alone. Limited data exist on the use of nitrogen conjugation agents in very young patients. METHODS: Seventeen patients (15 previously on other nitrogen scavengers) with all types of UCDs aged 2months to 2years were switched to, or started, GPB. Retrospective data up to 12months pre-switch and prospective data during initiation of therapy were used as baseline measures. The primary efficacy endpoint of the integrated analysis was the successful transition to GPB with controlled ammonia (<100µmol/L and no clinical symptoms). Secondary endpoints included glutamine and levels of other amino acids. Safety endpoints included adverse events, hyperammonemic crises (HACs), and growth and development. RESULTS: 82% and 53% of patients completed 3 and 6months of therapy, respectively (mean 8.85months, range 6days-18.4months). Patients transitioned to GPB maintained excellent control of ammonia and glutamine levels. There were 36 HACs in 11 patients before GPB and 11 in 7 patients while on GPB, with a reduction from 2.98 to 0.88 episodes per year. Adverse events occurring in at least 10% of patients while on GPB were neutropenia, vomiting, diarrhea, pyrexia, hypophagia, cough, nasal congestion, rhinorrhea, rash/papule. CONCLUSION: GPB was safe and effective in UCD patients aged 2months to 2years. GPB use was associated with good short- and long-term control of ammonia and glutamine levels, and the annualized frequency of hyperammonemic crises was lower during the study than before the study. There was no evidence for any previously unknown toxicity of GPB.

[Effect of ASCT2 gene knock-down by shRNA on biological behaviors of colorectal cancer cells].

OBJECTIVE: To investigate the effect of ASCT2 gene (glutamine transporter) knock-down by shRNA on biological behaviors of colorectal cancer cells. METHODS: shRNA was transfected into colorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine. RESULTS: ASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells(P<0.01). MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells (A490) and decrease the number of invasive Lovo and SW480 cells from the membrane (both P<0.01). The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells (both P<0.01). CONCLUSIONS: ASCT2 plays an oncogenic role in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.

Temporal Dynamics of Antidepressant Ketamine Effects on Glutamine Cycling Follow Regional Fingerprints of AMPA and NMDA Receptor Densities.

The anterior cingulate cortex (ACC) has shown decreased glutamate levels in patients with major depressive disorder. Subanesthetic doses of ketamine were repeatedly shown to improve depressive symptoms within 24 h after infusion and this antidepressant effect was attributed to increased α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) throughput. To elucidate ketamine's mechanism of action, we tested whether the clinical time course of the improvement is mirrored by the change of glutamine/glutamate ratio and if such effects show a regional and temporal specificity in two distinct subdivisions of ACC with different AMPA/N-methyl-D-aspartate receptor profiles. In a double-blind, placebo-controlled intravenous infusion study of ketamine, we measured glutamate and glutamine in the pregenual ACC (pgACC) and the anterior midcingulate cortex at 1 and 24 h post infusion with magnetic resonance spectroscopy at 7 T. A significant interaction of time, region, and treatment was found for the glutamine/glutamate ratios (placebo, n=14; ketamine, n=12). Post-hoc analyses revealed that the glutamine/glutamate ratio increased significantly in the ketamine group, compared with placebo, specifically in the pgACC after 24 h. The glutamine/glutamate increase in the pgACC caused by ketamine at 24 h post infusion was reproduced in an enlarged sample (placebo, n=24; ketamine, n=20). Our results support a significant temporal and regional response in glutamine/glutamate ratios to a single subanesthetic dose of ketamine, which mirrors the time course of the antidepressant response and reversal of the molecular deficits in patients and which may be associated with the histoarchitectonical receptor fingerprints of the ACC subregions.

Selenite inhibits glutamine metabolism and induces apoptosis by regulating GLS1 protein degradation via APC/C-CDH1 pathway in colorectal cancer cells.

Glutaminolysis is important for metabolism and biosynthesis of cancer cells, and GLS is essential in the process. Selenite is widely regarded as a chemopreventive agent against cancer risk. Emerging evidence suggests that it also has chemotherapeutic potential in various cancer types, but the mechanism remains elusive. We demonstrate for the first time that supranutritional dose of selenite suppresses glutaminolysis by promoting GLS1 protein degradation and apoptosis. Mechanistically, selenite promotes association of APC/C-CDH1 with GLS1 and leads to GLS1 degradation by ubiquitination, this process is related to induction of PTEN expression. In addition, GLS1 expression is increased in human colorectal cancer tissues compared with normal mucosae. Our data provide a novel mechanistic explanation for the anti-cancer effect of selenite from a perspective of cell metabolism. Moreover, our results indicate that glutaminolysis especially GLS1 could be an attractive therapeutic target in colorectal cancer.

Kinetic properties of Streptomyces canarius L- Glutaminase and its anticancer efficiency

Abstract L-glutaminase was produced by Streptomyces canarius FR (KC460654) with an apparent molecular mass of 44 kDa. It has 17.9 purification fold with a final specific activity 132.2 U/mg proteins and 28% yield recovery. The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8.0 at 40 °C for 30 min. It maintained its stability at wide range of pH from 5.0 11.0 and thermal stable up to 60 °C with Tm value 57.5 °C. It has high affinity and catalytic activity for L-glutamine (Km 0.129 mM, Vmax 2.02 U/mg/min), followed by L-asparagine and L-aspartic acid. In vivo, L-glutaminase showed no observed changes in liver; kidney functions; hematological parameters and slight effect on RBCs and level of platelets after 10 days of rabbit's injection. The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell (IC50, 6.8 μg/mL) and HeLa cells (IC50, 8.3 μg/mL), while the growth of MCF-7 cells was not affected. L-glutaminase has a moderate cytotoxic effect against HCT-116 cell (IC50, 64.7 μg/mL) and RAW 264.7 cell (IC50, 59.3 μg/mL).

How to understand the results of studies of glutamine supplementation.

The lack of understanding of the mechanisms behind possible beneficial and possible harmful effects of glutamine supplementation makes the design of interventional studies of glutamine supplementations difficult, perhaps even hazardous. What is the interventional target, and how might it relate to outcomes? Taking one step further and aggregating results from interventional studies into meta-analyses does not diminish the difficulties. Therefore, conducting basic research seems to be a better idea than groping in the dark and exposing patients to potential harm in this darkness.