Exogenous Gln Research Articles

The Role of Glutamine Homeostasis in Emotional and Cognitive Functions.

Glutamine (Gln), a non-essential amino acid, is synthesized de novo by glutamine synthetase (GS) in various organs. In the brain, GS is exclusively expressed in astrocytes under normal physiological conditions, producing Gln that takes part in glutamatergic neurotransmission through the glutamate (Glu)-Gln cycle. Because the Glu-Gln cycle and glutamatergic neurotransmission play a pivotal role in normal brain activity, maintaining Gln homeostasis in the brain is crucial. Recent findings indicated that a neuronal Gln deficiency in the medial prefrontal cortex in rodents led to depressive behaviors and mild cognitive impairment along with lower glutamatergic neurotransmission. In addition, exogenous Gln supplementation has been tested for its ability to overcome neuronal Gln deficiency and reverse abnormal behaviors induced by chronic immobilization stress (CIS). Although evidence is accumulating as to how Gln supplementation contributes to normalizing glutamatergic neurotransmission and the Glu-Gln cycle, there are few reviews on this. In this review, we summarize recent evidence demonstrating that Gln supplementation ameliorates CIS-induced deleterious changes, including an imbalance of the Glu-Gln cycle, suggesting that Gln homeostasis is important for emotional and cognitive functions. This is the first review of detailed mechanistic studies on the effects of Gln supplementation on emotional and cognitive functions.

Unveiling Putative Pathway Enrichment Following Crisantaspase Treatment in AML through RNA Sequencing Analysis

Background: Tumor development is intricately influenced by metabolic reprogramming, resulting in the emergence of exploitable vulnerabilities that can be targeted. Glutamine (Gln) depletion has emerged as a promising therapeutic strategy for malignancies, including AML, that heavily rely on Gln for their survival. Erwinia-derived asparaginase, also called crisantaspase, has exhibited anti-AML activity in both preclinical and clinical models by disrupting Gln supply, albeit with the persistence of residual leukemia burden. To identify cellular pathways modulated after crisantaspase-induced Gln depletion, here we report our comprehensive results using a whole transcriptome profiling approach (RNA-Seq) after crisantaspase treatment. Methods: Whole-transcriptomic profiling using RNA-Seq was performed to profile gene expression of MOLM-14 AML cells following crisantaspase treatment. RNA was isolated from each sample and total RNA was utilized for this study. The samples were sequenced at IGS using the Illumina HiSeq sequencing platform. Raw sequencing reads generated for each sample were analyzed using the CAVERN analysis pipeline. Read quality was assessed using the FastQC toolkit to ensure good-quality reads for downstream analyses. P values were generated using a Wald test implemented in DESeq2 and then corrected for multiple hypothesis testing using the Benjamini-Hochberg correction method. Results: We utilized Rylaze, an FDA-approved Erwinia crisantaspase, to deplete exogenous Gln. MOLM-14 cells were treated with Rylaze for 16h, followed by whole-transcriptome/gene expression profiling (GEP) using RNA-seq. The transcriptomic analysis provided information on the regulation of gene expression between vehicle control and Rylaze treatment in AML, as well as on the potential association with several putative pathways including serine family amino acids biosynthetic process (GO: 0009070, p-value = 2.18E-08), regulation of programmed cell death (GO:0043067, p-value = 7.96E-06), and amino acid transmembrane transport (GO:0003333, p-value = 5.10E-05). Furthermore, Figure 1 illustrates the gene ontology terms enriched by the DEGs, offering insights into the underlying molecular mechanisms involved in the response to Rylaze-induced Gln depletion in MOLM-14 cells. For example, the upregulation of genes associated with the serine family amino acid biosynthetic process may suggest a compensatory mechanism to counteract the depletion of Gln induced by Rylaze. By employing generalized linear regression models, we observed significant changes in gene expression following Rylaze (141 genes) compared to the vehicle control. This finding is further illustrated by a heatmap generated from transcriptome data analysis (Figure 2). We identified 141 significantly differentially expressed genes (DEGs); among the Rylaze-treated samples, consisting of 114 up-regulated and 27 down-regulated genes. Specifically, we focused on five genes that exhibited significant modulation in response to the treatment with Rylaze. These genes included CHAC1, ADM2, S100P, CTH and SPOCK2. These genes might play crucial roles in the cellular response to Gln depletion and could potentially serve as biomarkers or therapeutic targets in AML treatment. Validation of these observations was performed using immunoblotting techniques and quantitative real-time polymerase chain reaction (qRT-PCR), which confirmed the significant increase in CHAC1, ADM2, S100P, and CTH, as well as the significant decrease in SPOCK2 (p<0.0001) following Rylaze-induced Gln depletion. These findings contribute to a better understanding of the drug's mode of action and the impact of Gln depletion on cellular processes in AML. Conclusion: This study represents a significant contribution towards advancing our understanding of amino acid-regulated metabolic pathways in AML, with potential implications for other Gln metabolism-dependent cancers as well.

Glutamine Metabolism, Sensing andSignaling inPlants.

Glutamine (Gln) is the first amino acid synthesized in nitrogen (N) assimilation in plants. Gln synthetase (GS), converting glutamate (Glu) and NH4+ into Gln at the expense of ATP, is one of the oldest enzymes in all life domains. Plants have multiple GS isoenzymes that work individually or cooperatively to ensure that the Gln supply is sufficient for plant growth and development under various conditions. Gln is a building block for protein synthesis and an N-donor for the biosynthesis of amino acids, nucleic acids, amino sugars and vitamin B coenzymes. Most reactions using Gln as an N-donor are catalyzed by Gln amidotransferase (GAT) that hydrolyzes Gln to Glu and transfers the amido group of Gln to an acceptor substrate. Several GAT domain-containing proteins of unknown function in the reference plant Arabidopsis thaliana suggest that some metabolic fates of Gln have yet to be identified in plants. In addition to metabolism, Gln signaling has emerged in recent years. The N regulatory protein PII senses Gln to regulate arginine biosynthesis in plants. Gln promotes somatic embryogenesis and shoot organogenesis with unknown mechanisms. Exogenous Gln has been implicated in activating stress and defense responses in plants. Likely, Gln signaling is responsible for some of the new Gln functions in plants.

Transcriptome Analysis Reveals Critical Genes and Pathways in Carbon Metabolism and Ribosome Biogenesis in Poplar Fertilized with Glutamine.

Exogenous Gln as a single N source has been shown to exert similar roles to the inorganic N in poplar ‘Nanlin895′ in terms of growth performance, yet the underlying molecular mechanism remains unclear. Herein, transcriptome analyses of both shoots (L) and roots (R) of poplar ‘Nanlin895’ fertilized with Gln (G) or the inorganic N (control, C) were performed. Compared with the control, 3109 differentially expressed genes (DEGs) and 5071 DEGs were detected in the GL and GR libraries, respectively. In the shoots, Gln treatment resulted in downregulation of a large number of ribosomal genes but significant induction of many starch and sucrose metabolism genes, demonstrating that poplars tend to distribute more energy to sugar metabolism rather than ribosome biosynthesis when fertilized with Gln-N. By contrast, in the roots, most of the DEGs were annotated to carbon metabolism, glycolysis/gluconeogenesis and phenylpropanoid biosynthesis, suggesting that apart from N metabolism, exogenous Gln has an important role in regulating the redistribution of carbon resources and secondary metabolites. Therefore, it can be proposed that the promotion impact of Gln on poplar growth and photosynthesis may result from the improvement of both carbon and N allocation, accompanied by an efficient energy switch for growth and stress responses.

Endogenous glutamine is rate-limiting for anti-CD3 and anti-CD28 induced CD4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1–4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.

AMPK-mediated glutaminolysis maintains coelomocytes redox homeostasis in Vibrio splendidus-challenged Apostichopus japonicus

Glutaminolysis has been proved to play an irreplaceable role in vertebrate immunity, including effects on cytokine production, bacterial killing, and redox homeostasis maintenance. Our previous metabolomics analysis indicated that glutaminolysis metabolic substrates glutamine (Gln) and metabolites glutamate (Glu) were significantly lower in Skin ulceration syndrome (SUS)-diseased Apostichopus japonicus. To further delineate the role of glutaminolysis, we assayed the levels of Gln and Glu. We found that their contents in coelomocytes were decreased, accompanied by an increase in glutathione (GSH) in pathogen-challenged Apostichopus japonicus. Consistently, the mRNA transcripts of three key genes in glutaminolysis (AjASCT2, AjGOT, and AjGCS) were significantly induced. Moreover, the increased MDA and NADPH/NADP + levels in response to pathogen infection indicated that oxidative stress occurs during the immune response. The metabolic regulator AMPKβ could regulate glutaminolysis in vertebrates by inducing cells to take up extracellular Gln. To explore the underlying regulatory mechanism behind glutaminolysis that occurred in coelomocytes, the full-length cDNA of AMPKβ was identified from A. japonicus (designated as AjAMPKβ). AjAMPKβ expression was significantly induced in the coelomocytes after pathogen challenge, which was consistent with the expression of key genes of glutaminolysis. A functional assay indicated that AjAMPKβ silencing by siRNA transfection could increase the levels of Gln and Glu and depress the production of GSH. Moreover, the expression of glutaminolysis-related genes was significantly inhibited, and the reduction of redox homeostasis indexes (MDA and NADPH/NADP+) was also observed. Contrastingly, AjAMPKβ overexpression promoted redox homeostasis balance. Intracellular ROS is mostly responsible for breaking redox homeostasis and leading to oxidative stress, contributing to cell fate changes in immune cells. Exogenous Gln and GSH treatments could significantly reduce ROS level while the AjAMPKβ silencing induced the level of ROS and accelerated the necrosis rate. All these results collectively revealed that AjAMPKβ could modulate cellular redox homeostasis by affecting the glutaminolysis in A. japonicus.

Effects of exogenous L-Glutamine as a sole nitrogen source on physiological characteristics and nitrogen use efficiency of poplar

L-Glutamine (Gln) is a proteinogenic amino acid, N transporter and NH3 carrier, engaging in diversified pathways for synthesizing many important molecules. However, the effects of exogenous Gln on plant growth and development remain largely unknown. In this study, different concentrations of Gln were supplemented in the poplar hybrid ‘Nanlin895’ culture medium as a sole N source. Their effects on poplar growth, photosynthesis, N metabolism-related enzymes and metabolites were elucidated. Strikingly, 0.5 mM Gln-fed poplars showed no considerable growth compromise compared to the inorganic N control (CK-N), even though their N supply level was only half that of the CK-N control. What's more, their NUE was enhanced. In addition, 0.5 mM Gln treatment significantly increased the contents of amino acids in coordination with soluble sugars in the roots, while marginal effects in the leaves were observed compared to CK-N. By contrast, applying a high level of Gln (>0.5 mM) resulted in larger accumulation of amino acids and starch, but lower level of soluble sugars, particularly in the roots, followed by adverse effects on poplar biomass, photosynthesis, enzyme activities and NUE; consequently, poplar growth was inhibited. Collectively, these findings allow us to deduce that poplar plants are competent to take up and utilize Gln as a sole N source. When applied at an appropriate level, Gln could promote a dynamic equilibrium of N and C, conferring sound growth performance and additional benefit for the environment as indicated by higher NUE, lower N input and higher biocompatible nature than the inorganic N.

Glutamine metabolism in the proliferation of GS-expression pituitary tumor cells.

ObjectiveMany cancer cells cannot survive without exogenous glutamine (Gln); however, cancer cells expressing glutamine synthetase (GS) do not have this restriction. Previous metabolomics studies have indicated that glutamine metabolism is altered during pituitary tumorigenesis. However, the main role of Gln in pituitary adenoma (PA) pathophysiology remains unknown. The aim of this study was to evaluate the expression of GS and the main role of Gln in human PAs.MethodsWe used cell proliferation assay and flow cytometry to assess the effect of Gln depletion on three different pituitary cell lines and human primary PA cells. We then investigated the expression level of Gln synthetase (GS) in 24 human PA samples. At last, we used LC-MS/MS to identify the differences in metabolites of PA cells after the blockage of both endogenous and exogenous Gln.ResultsPA cell lines showed different sensitivities to Gln starvation, and the sensitivity is correlated with GS expression level. GS expressed in 21 out of the 24 human PA samples. Furthermore, a positive p53 and ki-67 index was correlated with a higher GS expression level (P < 0.05). Removal of both endogenous and exogenous Gln from GS-expressing PA cells resulted in blockage of nucleotide metabolism and cell cycle arrest.ConclusionsOur data indicate that GS is needed for PA cells to undergo proliferation during Gln deprivation, and most human PA cells express GS and might have a negative response to exogenous Gln depletion. Moreover, Gln is mainly responsible for nucleotide metabolism in the proliferation of GS-expressing pituitary tumor cells.

The glutamine-glutamate cycle regulates synaptic glutamate release in the ventrolateral ventromedial nucleus of the hypothalamus of perinatal female rats.

The astrocytic glutamine (Gln)-glutamate (Glu) cycle (GGC) supplies Gln for the regulation of glutamatergic synaptic transmission (GST) in the adult hippocampus. Increased synaptic Glu release in the perinatal ventrolateral ventromedial nucleus of the hypothalamus (vlVMH) modulates sexual differentiation, however, whether GGC regulates GST in the perinatal vlVMH has not been determined. Sex differences in oestradiol (E2 ) levels exist in the neonatal hypothalamus, and E2 increases levels of glutamine synthetase and glutaminase, two key enzymes involved in the GGC. Thus, it is hypothesised that sexually dimorphic phenotypes may exist in glutamatergic synapses associated with the GGC in the vlVMH in perinatal rats. Whole-cell voltage-clamp recordings in vlVMH neurones in brain slices from male and female pups revealed that pharmacological disruption of the GGC by α-(methylamino) isobutyric acid (5 mmol L-1 ), which blocks neuronal Gln uptake; or by l-methionine sulphoximine (1.5 mmol L-1 ), which inhibits astrocytic Gln synthesis, decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in female but not male pups. By contrast, GGC interruptions decreased evoked (e)EPSC amplitudes in both sexes following increased synaptic activity produced by a period of stimulation. In male pups, the decreased eEPSCs were attributable to reduced Glu release, as assessed by paired-pulse stimulations, whereas, in female pups, they were attributable to decreased Glu content in the synaptic vesicles, as measured by strontium-evoked mEPSCs. The l-methionine sulphoximine-mediated decrease in eEPSCs was rapidly rescued by exogenous Gln in female but not male pups. The reductions in mEPSCs and eEPSCs in female pups were accompanied by enhanced blocking effects of the low-affinity Glu AMPA receptor antagonist, γ-d-glutamylglycine, consistent with diminished Glu release. In conclusion, female, but not male pups, rely on constitutive astrocytic Gln for sustained synaptic Glu release in the vlVMH. This glutamatergic synaptic phenotype may be associated with brain and behaviour feminisation and/or defeminisation in rats.

Glutamine has antidepressive effects through increments of glutamate and glutamine levels and glutamatergic activity in the medial prefrontal cortex

Emerging evidence has shown the low levels of glutamate (Glu) and glutamine (Gln) and the hypoactivity in the cortex of patients with depression. The hypoactivity is closely related with low frequency of glutamatergic signaling that is affected by the levels of Glu and Gln. Thus, we hypothesized that there might be a causality among low levels of Glu and Gln, hypoactive glutamatergic neurotransmissions, and depressive behaviors. Here, we found low Glu and Gln levels and low frequency of spontaneous excitatory postsynaptic current (sEPSC) of glutamatergic neurons in the medial prefrontal cortex (mPFC) of chronic immobilization stress (CIS)-induced depressed mice. The depressed mice also showed hypoactive Gln synthetase (GS). Inhibition of GS by methionine sulfoximine (MSO) decreased Glu and Gln levels and increased depressive behaviors with low frequency of sEPSC in the mPFC, indicating that Glu and Gln decrements cause hypoactive glutamatergic neurotransmissions and depressive behaviors. Both Glu and Gln could increase sEPSC of glutamatergic neurons in the mPFC on slice patch, but only Gln overcame MSO to increase sEPSC, suggesting that exogenous Gln would recover CIS-induced low frequency of sEPSC caused by hypoactive GS and act as an antidepressant. Expectedly, Gln supplementation showed antidepressant effects against CIS; it increased glutamatergic neurotransmissions with Glu and Gln increment in the mPFC and attenuated depressive behaviors. Moreover, selective glutamatergic activation in the mPFC by optogenetics decreased depressive behavior. In conclusion, depressive behaviors evoked by chronic stress were due to hypoactive glutamatergic neurons in the mPFC caused by low levels of Glu and Gln, and exogenous Gln can be used as an alternative antidepressant to increase glutamatergic neurotransmission.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

Interactive effects of glutamine and gamma-aminobutyric acid on growth performance and skeletal muscle amino acid metabolism of 22-42-day-old broilers exposed to hot environment.

The present experiment was conducted to investigate the interactive effects between dietary glutamine (Gln, 0 and 5g/kg) and gamma-aminobutyric acid (GABA, 0 and 100mg/kg) on growth performance and amino acid (AA) metabolism of broilers under hot environment. A total of 360 22-day-old Arbor Acres male chickens were randomly assigned to five treatment groups under thermoneutral chamber (PC, 23°C) and cyclic heat stress (HS, 30-34°C cycling) conditions. Compared with the PC group, cyclic HS decreased (P < 0.05) daily weight gain (DWG), daily feed consumption (DFC), the concentrations of Gln, glutamate (Glu), and GABA, and the activities of glutaminase and glutamic acid decarboxylase (GAD) in breast muscle at 28, 35, and 42days, while it increased (P < 0.05) the activities of glutamine synthetase (GS) and gamma-aminobutyric acid transaminase (GABA-T) at 28, 35, and 42days. Dietary Gln and GABA improved (P < 0.05) DWG and DFC of broilers under cyclic HS during 28-42days. In breast muscle, the Gln supplementation increased (P < 0.05) the concentrations of Gln (28, 35, and 42days), Glu (28, 35, and 42days), and GABA (42days) and the activities of glutaminase (28, 35, and 42days) and GAD (28, 35, and 42days) but decreased (P < 0.05) GS activities at 28, 35, and 42days and GABA-T activities at 28days. The addition of GABA increased (P < 0.05) the concentrations of Gln and Glu and activities of glutaminase and GAD, while it decreased (P < 0.05) GABA-T activities at 28, 35, and 42days. Significant interactions (P < 0.05) between Gln and GABA were found on breast skeletal muscle Gln concentrations, glutaminase activities, GS activities at 28 and 35days, and DWG, GABA concentrations, and GABA-T activities at 28, 35, and 42days in broilers under cyclic HS. In conclusion, the present results indicated that the interactions of exogenous Gln and GABA could offer a potential nutritional strategy to prevent HS-related depression in skeletal muscle Gln and GABA metabolism of broilers.

Abstract P2-09-03: Antitumor activity of the glutaminase inhibitor, CB-839, in triple-negative breast cancer

Abstract Glutamine (Gln) serves as an important source of energy and building blocks for many tumor cells. The first step in Gln utilization is its conversion to glutamate (Glu) by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 inhibits recombinant GAC with an IC50 of &amp;lt;15 nM. Treatment of the triple-negative breast cancer (TNBC) cell line MDA-MB-231 in vitro with CB-839 reduced cell viability and suppressed cellular Glu pools with comparable potency (IC50 ∼15 nM). TNBC breast cancer cell lines have been reported to have a heightened dependence on exogenous Gln for growth relative to estrogen receptor (ER) positive cells (Kung et al, 2011). Consistent with this observation, an analysis of 58 breast cancer cell lines in the Cancer Cell Line Encyclopedia revealed that low ER and/or Her2 expression is correlated with high glutaminase expression (particularly the GAC isoform) and low expression of glutamine synthetase (GLUL), an enzyme that converts Glu to Gln. This reciprocal pattern of GAC and GLUL expression suggests that TNBC may be particularly sensitive to glutaminase inhibition. To test this hypothesis, the anti-tumor activity of CB-839 was evaluated on a panel of breast tumor cell lines (n&amp;gt;25) that included a mixture of TNBC and ER/Her2-positive subtypes. The TNBC subtype displayed the greatest sensitivity to CB-839 treatment (IC50s ranging from 5-100 nM) and this sensitivity was correlated with: i) dependence on extracellular Gln for growth (r = 0.83, p&amp;lt;0.0001); ii) GAC (but not KGA) expression (r = -0.76, p&amp;lt;0.0001); iii) glutaminase activity in cell lysates (r = -0.75, p = 0.0002); and iv) the ratio of cellular Glu to Gln (r = 0.53, p = 0.014) indicative of efficient Gln to Glu conversion. CB-839 also displayed significant anti-tumor activity in two TNBC xenograft models: 1) as a single agent in a patient-derived tumor chosen based on Gln utilization biomarkers and 2) in a cell line model (JIMT1) both as a single agent and in combination with paclitaxel. To determine if potential markers of Gln utilization and CB-839 sensitivity are reflected in primary tumors, an analysis of large mRNA expression and metabolomic datasets was undertaken. Consistent with the observations in cell lines, there was significantly elevated expression of GAC and reduced expression of GLUL in TNBC tumors relative to the ER+ population in The Cancer Genome Atlas mRNA expression dataset (n = 756). Likewise, analysis of the metabolite levels in 262 primary breast tumors showed that the Glu to Gln ratio in the ER- populations was significantly elevated relative to the ER+ population (fold increase = 3.2, p = 8.0E-09). This elevated metabolite ratio in the ER- population was also significantly correlated with the mRNA expression level of GLUL (r = -0.27, p = 0.001). These observations suggest that biomarkers of Gln utilization and CB-839 sensitivity extend to primary TNBC tumors. Together with the in vitro and in vivo preclinical anti-tumor activity of CB-839 in TNBC models, this primary tumor biomarker evaluation provides a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in TNBC patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-03.

Antitumor Activity Of The Glutaminase Inhibitor CB-839 In Hematological Malignances

Many tumor cells are dependent on glutamine (Gln) for growth and survival. Gln is the most abundant amino acid in plasma and tumor cells can utilize Gln for energy production and generation of building blocks for the synthesis of macromolecules. A key step in Gln utilization is its conversion to glutamate (Glu) by the mitochondrial enzyme glutaminase (GLS). Suppressing GLS activity via either siRNA knockdown or small molecule inhibitor treatment has anti-proliferative activity in several tumor types. GLS mRNA levels are elevated in several hematological malignances, particularly those derived from the B-cell lineage including multiple myeloma (MM), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and several types of non-Hodgkin's lymphoma (NHL). Interestingly, these B-cell derived tumors express low levels of glutamine synthetase (GLUL), an enzyme that reverses the action of GLS by converting Glu to Gln. A high GLS/low GLUL mRNA expression pattern may be indicative of tumor-dependence on extracellular Gln for growth. This expression pattern is observed in triple negative breast cancer (TNBC), a tumor type that we and others have shown to be highly dependent on exogenous Gln for cell proliferation and sensitive to GLS inhibitor treatment. CB-839 is a novel, potent, orally bioavailable GLS inhibitor that inhibits recombinant human GLS with an IC50 of 15 nM. The anti-proliferative activity of CB-839 on a panel of breast cell lines is correlated with a high ratio of GLS to GLUL expression, a pattern associated with the TNBC subtype. Given the similarity of the GLS/GLUL expression pattern observed in TNBC with some hematologic malignancies, we evaluated the anti-tumor activity of CB-839 in cell culture and xenograft models of hematological malignancies predicted to be dependent on Gln for growth. Across a panel of 18 cell lines derived from MM, ALL and diffuse large B-cell lymphoma (DLBCL), Gln deprivation resulted in a minimum of 70% reduction in growth for 16 cell lines and induced cell death in 5 cell lines. Across the same panel of cell lines, the majority were sensitive to CB-839 treatment with 16 cell lines exhibiting an antiproliferative IC50 of<100nM (range 2-72 nM). Importantly, there was a strong correlation between sensitivity to Gln withdrawal and CB-839 sensitivity (r=0.63, p=0.005), suggesting that the dominant pathway by which Gln supports tumor cell growth and survival is through its conversation to Glu by GLS. Consistent with this notion, CB-839 treatment caused a decrease in the intracellular pool of Glu and an increase in the intracellular pool of Gln. The extent of decrease in Glu was highly correlated with the effect of CB-839 on cell proliferation (r=-0.64, p=0.006). The in vivo antitumor activity of CB-839 was also evaluated in a xenograft model using the multiple myeloma RPMI-8226 model. Oral, twice daily dosing of CB-839 resulted in a significant decrease in tumor volume (70% tumor growth inhibition). As observed in vitro, CB-839 dosing caused an increase in tumor Gln levels and a decrease in the level of tumor Glu. In a dose dependent fashion, there was a correlation between tumor Gln accumulation, tumor GLS inhibition and antitumor activity. Efficacious doses of CB-839 were well tolerated with no effect on hematological cell counts or body weight. These data suggest that GLS inhibition with CB-839 may provide therapeutic benefit in hematological malignances and motivate the evaluation of this compound in clinical trials that include patients with B-cell malignancies. Disclosures:Parlati:Calithera BIosciences: Employment. Bromley-Dulfano:Calithera Biosciences: Employment. Demo:Calithera Biosciences: Employment. Janes:Calithera Biosciences: Employment. Gross:Calithera Biosciences: Employment. Lewis:Calithera Biosciences: Employment. MacKinnon:Calithera Biosciences: Employment. Rodriguez:Calithera Biosciences: Employment. Yang:Calithera Biosciences: Employment. Zhao:Calithera Biosciences: Employment. Bennett:Calithera Biosciences: Employment.

Proinflammatory Cytokine TNF‐α Inhibits Na+‐glutamine Cotransport in Intestinal Epithelial Cell line (IEC‐6)

Mucosal Na+-glutamine (Gln) cotransport is critical in absorbing exogenous Gln that the body needs during extreme stress (e.g. infections and trauma). Although stress has been shown to inhibit Gln synthesis, it is not known whether stress also inhibits Gln absorption. Thus, the aim of this study was to determine whether TNF-α, the major proinflammatory cytokine is produced during infection would inhibit Gln cotransport in rat intestinal epithelial (IEC-6) cells. [3H]-Gln uptake was measured in 10-day post-confluent normal and TNF-α (25 ng/ml) treated IEC-6 cells, grown in 6-well plates. Na+/K+-ATPase activity, and B0AT1 specific mRNA abundance and protein expression were measured by qRT-PCR and western blot analyses, respectively. Na+-Gln cotransport is present in IEC-6 cells. TNF-α exposure (24 hrs) inhibited Na+-Gln cotransport by 75% (1.9 ± 0.1 vs 0.5 ± 0.01 nmol/mg protein • 2 min). Kinetic studies revealed that TNF-α inhibited Na+-Gln cotransport by reducing the affinity for Gln (Km: normal vs TNF-α = 3.5 ± 0.3 vs 8.5 ± 1.0 mM). TNF-α did not alter either B0AT1 specific mRNA abundance and protein expression or Na+/K+-ATPase activity. We concluded from these results that, in addition to inhibiting Gln synthesis, extreme stress also inhibits exogenous Gln absorption. We speculate that TNF-α might regulate Na+-Gln cotransport mediated via B0AT1 by second messenger mediated signal transduction process(es).

Glutamine inhibits ammonia-induced accumulation of cGMP in rat striatum limiting arginine supply for NO synthesis

Brain l-glutamine (Gln) accumulation and increased activity of the NO/cGMP pathway are immediate consequences of acute exposure to ammonia. This study tested whether excess Gln may influence NO and/or cGMP synthesis. Intrastriatal administration of the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine or the system A-specific Gln uptake inhibitor methylaminoisobutyrate increased microdialysate Gln concentration and reduced basal and ammonia-induced NO and cGMP accumulation. Gln applied in vivo ( via microdialysis) or in vitro (to rat brain cortical slices) reduced NO and cGMP accumulation in the presence and/or absence of ammonia, but not cGMP synthesis induced by the NO donor sodium nitroprusside. Attenuation of cGMP synthesis by Gln was prevented by administration of l-arginine (Arg). The l-arginine co-substrates of y +LAT2 transport system, l-leucine and cyclo-leucine, mimicked the effect of exogenous Gln, suggesting that Gln limits Arg supply for NO synthesis by interfering with y+LAT2-mediated Arg uptake across the cell membrane.

Exogenous ammonium inhibits petal pigmentation and expansion in Gerbera hybrida

Petal pigmentation is the most important aspect in natural flower coloration. In the present study, the inhibition of petal pigmentation by exogenous ammonium was investigated. Ray floret petals detached from inflorescences of Gerbera hybrida (Shenzhen No. 5) were cultured in vitro on media supplied with different forms of nitrogen and its assimilated compounds. The expression of a set of genes involved in anthocyanin biosynthesis and regulation was determined by Northern blotting assay. It was found that ammonium (NH4+), not nitrate (NO3-), in millimolar concentrations inhibited anthocyanin accumulation. The expressions of Gerbera chalcone synthase 1 (GCHS1), Gerbera chalcone synthase 2 (GCHS2) and Gerbera dihydroflavonol-4-reductase (GDFR) decreased, while six other related genes showed no significant changes after NH4+ treatment. Further studies on NH4+ function indicated that glutamine (Gln) acted as a downstream factor of NH4+ to suppress petal pigmentation. Both exogenous Gln and NH4+ were found to inhibit anthocyanin accumulation in the petals, and the application of Gln was also found to inhibit the expressions of GCHS1, GCHS2 and GDFR. The application of NH4+ also resulted in an increase in the activity of Gerbera glutamine synthetase (EC 6.3.1.2) along with a rapid increase of Gln content. When methionine sulfoximine, an inhibitor of glutamine synthetase (GS), was added, it was found to block the NH4+-induced inhibition of pigmentation. From these experiments, we conclude that the NH4+-induced suppression of petal pigmentation is not because of NH4+ toxicity, and the inhibition of pigmentation caused by the addition of exogenous NH4+ is the result of its assimilation into Gln.

L-Glutamine Administration Reduces Oxidized Glutathione and MAP Kinase Signaling in Dystrophic Muscle of mdx Mice

To determine whether glutamine (Gln) reduces the ratio of oxidized to total glutathione (GSSG/GSH) and extracellular signal-regulated kinase (ERK1/2) activation in dystrophic muscle. Four-week old mdx mice, an animal model for Duchenne muscular dystrophy and control (C57BL/10) received daily intraperitoneal injections of l-Gln (500 mg/kg/d) or 0.9% NaCl for 3 d. GSH and GSSG concentrations in gastrocnemius were measured using a standard enzymatic recycling procedure. Free amino acid concentrations in gastrocnemius were determined by ion exchange chromatography. Phosphorylated protein levels of ERK1/2 in quadriceps were examined using Western Blot. l-Gln decreased GSSG and GSSG/GSH (an indicator of oxidative stress). This was associated with decreased ERK1/2 phosphorylation. Muscle free Gln, glutamate (Glu), and the sum (Gln + Glu) were higher in mdx versus C57BL/10, at the basal level. Exogenous Gln decreased muscle free Glu and Gln + Glu in mdx only, whereas Gln was not affected. In conclusion, exogenous Gln reduces GSSG/GSH and ERK1/2 activation in dystrophic skeletal muscle of young mdx mice, which is associated with decreased muscle free Glu and Gln + Glu. This antioxidant protective mechanism provides a molecular basis for Gln's antiproteolytic effect in Duchenne muscular dystrophy children.

Substitutes for glutamine in proliferation of rat intestinal epithelial cells

Objectives Glutamine (Gln) is important for intestinal epithelial proliferation. The purpose of this study was to determine whether glutamate (Glu), a mixture of nucleotide monophosphates, arginine, or glucosamine could support proliferation of rat intestinal crypt cells (IEC-6) in the absence of Gln. Methods Glu with added ammonia acetate, glucosamine, arginine, and nucleotide monophosphates were tested at concentrations that were isonitrogenous with respect to Gln. To determine whether de novo synthesis of Gln was affected by these nutrients, a duplicate set of treatment groups was also tested with 1.0 mM/L of methionine sulfoximine, an inhibitor of Gln synthetase. Results Gln + methionine sulfoximine-treated cells showed suboptimal proliferation below 0.6 mM/L but normal proliferation between 0.6 and 4.0 mM/L of Gln. In the absence of exogenous Gln, isonitrogenous concentrations of Glu, glucosamine, arginine, or nucleotide monophosphates yielded similar proliferation as Gln. Cells treated with Glu, glucosamine, arginine, or nucleotide monophosphate mixture showed a decrease in proliferation compared with cells treated with Gln across all treatment doses ( P < 0.03). Conclusions The importance of these results is that, in the presence of active Gln synthetase, these nutrients can maintain intestinal epithelial proliferation similar to that observed with Gln.

Effect of parenteral l-glutamine on muscle in the very severely Ill

Glutamine (Gln)-supplemented perioperative total parenteral nutrition (TPN) has been reported to reduce the loss of intramuscular glutamine following routine surgery. This study investigates whether glutamine-supplemented TPN can alter muscle biochemistry acutely in the very severely ill patient. Thirty-eight patients (age 19–77 yr; mean 55 yr), critically ill (APACHE II range 8–31; median 17) admitted to the intensive care unit (ICU) were recruited to receive either conventional TPN (CTPN) or an isonitrogenous, isoenergetic feed supplemented with 25 g crystalline l-glutamine per 24 h (GTPN) in a prospective, double blind, block-randomized study. In a representative sample of these patients, relatives consented to a paired muscle biopsy taken before feeding (10 GTPN/9 CTPN patients; ICU Day 2–4) and repeated 5 days later (16 patients; ICU Day 7–9). Muscle biopsies and matching plasma samples were analyzed using a coupled glutaminase-glutamate dehydrogenase enzymatic assay. A correction was made using sodium to account for the massive changes in extracellular fluid volume. The average muscle Gln content before feeding was very low. Between biopsies no consistent pattern of change was seen with or without exogenous Gln. It also proved difficult in these very sick patients to correct a low plasma Gln with l-Gln-TPN during the initial phase of the severe illness. TPN supplementation with 25 g/24 h, l-glutamine appears inadequate in the acute period to counteract the muscle and plasma biochemical changes seen in these patients. It is unknown whether any larger dose could alter this state.