Glutamine Pool Research Articles

Orientia tsutsugamushi infection reduces host gluconeogenic but not glycolytic substrates.

Orientia tsutsugamushi a causal agent of scrub typhus, is an obligate intracellular bacterium that, akin to other rickettsiae, is dependent on host cell-derived nutrients for survival and thus pathogenesis. Based on limited experimental evidence and genome-based in silico predictions, O. tsutsugamushi is hypothesized to parasitize host central carbon metabolism (CCM). Here, we (re-)evaluated O. tsutsugamushi dependency on host cell CCM as initiated by glucose and glutamine. Orientia infection had no effect on host glucose and glutamine consumption or lactate accumulation, indicating no change in overall flux through CCM. However, host cell mitochondrial activity and ATP levels were reduced during infection and correspond with lower intracellular glutamine and glutamate pools. To further probe the essentiality of host CCM in O. tsutsugamushi proliferation, we developed a minimal medium for host cell cultivation and paired it with chemical inhibitors to restrict the intermediates and processes related to glucose and glutamine metabolism. These conditions failed to negatively impact O. tsutsugamushi intracellular growth, suggesting the bacterium is adept at scavenging from host CCM. Accordingly, untargeted metabolomics was utilized to evaluate minor changes in host CCM metabolic intermediates across O. tsutsugamushi infection and revealed that pathogen proliferation corresponds with reductions in critical CCM building blocks, including amino acids and TCA cycle intermediates, as well as increases in lipid catabolism. This study directly correlates O. tsutsugamushi proliferation to alterations in host CCM and identifies metabolic intermediates that are likely critical for pathogen fitness.IMPORTANCEObligate intracellular bacterial pathogens have evolved strategies to reside and proliferate within the eukaryotic intracellular environment. At the crux of this parasitism is the balance between host and pathogen metabolic requirements. The physiological basis driving O. tsutsugamushi dependency on its mammalian host remains undefined. By evaluating alterations in host metabolism during O. tsutsugamushi proliferation, we discovered that bacterial growth is independent of the host's nutritional environment but appears dependent on host gluconeogenic substrates, including amino acids. Given that O. tsutsugamushi replication is essential for its virulence, this study provides experimental evidence for the first time in the post-genomic era of metabolic intermediates potentially parasitized by a scrub typhus agent.

Metabolic profiling of glioblastoma stem cells reveals pyruvate carboxylase as a critical survival factor and potential therapeutic target.

Glioblastoma (GBM) is a highly aggressive tumor with unmet therapeutic needs, which can be explained by extensive intra-tumoral heterogeneity and plasticity. In this study, we aimed to investigate the specific metabolic features of Glioblastoma stem cells (GSC), a rare tumor subpopulation involved in tumor growth and therapy resistance. We conducted comprehensive analyses of primary patient-derived GBM cultures and GSC-enriched cultures of human GBM cell lines using state-of-the-art molecular, metabolic, and phenotypic studies. We showed that GSC-enriched cultures display distinct glycolytic profiles compared with differentiated tumor cells. Further analysis revealed that GSC relies on pyruvate carboxylase (PC) activity for survival and self-renewal capacity. Interestingly, inhibition of PC led to GSC death, particularly when the glutamine pool was low, and increased differentiation. Finally, while GSC displayed resistance to the chemotherapy drug etoposide, genetic or pharmacological inhibition of PC restored etoposide sensitivity in GSC, both in vitro and in orthotopic murine models. Our findings demonstrate the critical role of PC in GSC metabolism, survival, and escape to etoposide. They also highlight PC as a therapeutic target to overcome therapy resistance in GBM.

Aminoacylase efficiently hydrolyses fatty acid amino acid conjugates of Helicoverpa armigera potentially to increase the pool of glutamine

One of the most prevalent bioactive molecules present in the oral secretion (OS) of lepidopteran insects is fatty acid amino acid conjugates (FACs). Insect dietary components have influence on the synthesis and retaining the pool of FACs in the OS. We noted differential and diet-specific accumulation of FACs in the OS of Helicoverpa armigera by using Liquid Chromatography-Quadrupole Time of Flight Mass Spectrometry. Interestingly, we identified FACs hydrolyzing enzyme aminoacylase (HaACY) in the OS of H. armigera through proteomic analysis. Next, we have cloned, expressed, and purified active recombinant HaACY in the bacterial system. Recombinant HaACY hydrolyzes all the six identified FACs in the OS of H. armigera larvae fed on host and non-host plants and releases respective fatty acid and glutamine. In these six FACs, fatty acid moieties vary while amino acid glutamine was common. Glutamine obtained upon hydrolysis of FACs by HaACY might serve as an amino acid pool for insect growth and development. To understand the substrate choices of HaACY, we chemically synthesized, purified, and characterized all the six FACs. Interestingly, rHaACY also shows hydrolysis of synthetic FACs into respective fatty acid and glutamine. Our results underline the importance of diet on accumulation of FACs and role of aminoacylase(s) in regulating the level of FACs and glutamine.

Evaluation of (2S,4S)-4-[18F]FEBGln as a Positron Emission Tomography Tracer for Tumor Imaging.

Glutamine metabolism-related tracers have the potential to visualize numerous tumors because glutamine is the second largest source of energy for tumors. (2S,4S)-4-[18F]FEBGln was designed by introducing [18F]fluoroethoxy benzyl on carbon-4 of glutamine. The aim of this study was to investigate the pharmacokinetic properties and tumor positron emission tomography (PET) imaging characteristics of (2S,4S)-4-[18F]FEBGln in detail. The biodistribution results of nude mice bearing MCF-7 tumor showed that (2S,4S)-4-[18F]FEBGln had high initial tumor uptake, and a fast clearance rate, resulting in a high tumor-to-muscle ratio at 30 min postinjection. There was no obvious defluorination in vivo. The micro-PET-CT imaging results of (2S,4S)-4-[18F]FEBGln orthotopic MCF-7 tumor-bearing nude mice were consistent with the biological distribution results. Compared with (2S,4R)-4-[18F]FGln, (2S,4S)-4-[18F]FEBGln showed poor tumor retention, but its clearance in normal tissues was also fast, so it had better PET image contrast than the former. Unlike poor retention in MCF-7-bearing nude mice, (2S,4S)-4-[18F]FEBGln has good retention in NCI-h1975 and 22Rv1 tumor models. Since (2S,4S)-4-[18F]FEBGln has low uptake in normal lungs and high uptake in the bladder, it is expected to be used in the accurate diagnosis of lung cancer but cannot accurately determine prostate cancer. Consistent with the advantages of radiolabeled amino acids in the application of brain tumors, (2S,4S)-4-[18F]FEBGln accurately diagnoses U87MG glioma with higher contrast than [18F]FET and [18F]FDG, and there is a correlation between (2S,4S)-4-[18F]FEBGln uptake and tumor growth cycle. Further kinetic model analysis showed that (2S,4S)-4-[18F]FEBGln was similar to (2S,4R)-4-[18F]FGln, conforming to the one-compartment model and the Logan graphical model, and was expected to assess the size of the glutamine pool of the tumor. Therefore, (2S,4S)-4-[18F]FEBGln is expected to provide a strong imaging basis for the diagnosis, formulation of personalized plans, and efficacy evaluation of glioma, lung cancer, and breast cancer.

18F-Fluciclovine PET Imaging of Glutaminase Inhibition in Breast Cancer Models.

Aggressive cancers such as triple-negative breast cancer (TNBC) avidly metabolize glutamine as a feature of their malignant phenotype. The conversion of glutamine to glutamate by the glutaminase enzyme represents the first and rate-limiting step of this pathway and a target for drug development. Indeed, a novel glutaminase inhibitor (GLSi) has been developed and tested in clinical trials but with limited success, suggesting the potential for a biomarker to select patients who could benefit from this novel therapy. Here, we studied a nonmetabolized amino acid analog, 18F-fluciclovine, as a PET imaging biomarker for detecting the pharmacodynamic response to GLSi. Methods: Uptake of 18F-fluciclovine into human breast cancer cells was studied in the presence and absence of inhibitors of glutamine transporters and GLSi. To allow 18F-fluciclovine PET to be performed on mice, citrate in the tracer formulation is replaced by phosphate-buffered saline. Mice bearing triple-negative breast cancer (TNBC) xenografts (HCC38, HCC1806, and MBA-MD-231) and estrogen receptor-positive breast cancer xenografts (MCF-7) were imaged with dynamic PET at baseline and after a 2-d treatment of GLSi (CB839) or vehicle. Kinetic analysis suggested reversible uptake of the tracer, and the distribution volume (VD) of 18F-fluciclovine was estimated by Logan plot analysis. Results: Our data showed that cellular uptake of 18F-fluciclovine is mediated by glutamine transporters. A significant increase in VD was observed after CB839 treatment in TNBC models exhibiting high glutaminase activity (HCC38 and HCC1806) but not in TNBC or MCF-7 exhibiting low glutaminase. Changes in VD were corroborated with changes in GLS activity measured in tumors treated with CB839 versus vehicle, as well as with changes in VD of 18F-(2S,R4)-fluoroglutamine, which we previously validated as a measure of cellular glutamine pool size. A moderate, albeit significant, decrease in 18F-FDG PET signal was observed in HCC1806 tumors after CB839 treatment. Conclusion: 18F-fluciclovine PET has potential to serve as a clinically translatable pharmacodynamic biomarker of GLSi.

P851: TARGETING GENE DEPENDENCIES IN MYC OVEREXPRESSING MULTIPLE MYELOMA

Background: Multiple myeloma (MM) is an incurable hematological malignancy characterized by a proliferation of clonal plasma cells in bone marrow. MM progresses from precursor stages, named monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM), to the symptomatic myeloma, MM. MYC abnormalities play a critical role in the disease progression. However, MYC is not therapeutically targetable due to its nuclear localization and the short half-life of the protein. Aims: To overcome this, we hypothesized that the proliferative advantage promoted by MYC overexpression induces differential genomic dependencies on other signaling pathways thus creates vulnerabilities that can be exploited therapeutically. Methods: We searched for genetic vulnerabilities associated with MYC overexpression by leveraging genome-scale pooled short hairpin RNA screening data for 236 cancer cell lines from Project Achilles. We generated an isogenic model of MYC overexpression (OE) in U266 cell line using EF1A-C-MYC lentiviral vector. Then, we performed RNA-seq, quantitative proteomics by Tandem Mass Tag mass spectrometry (TMT-MS) and a drug screening with ~2000 compounds. For validation, we performed pharmacological inhibition of glutamine catabolism as well as shRNA-mediated GLS1 knockdown. To determine the functional mechanisms, we used capillary electrophoresis-mass spectrometry (CE-MS) and Agilent Seahorse XF analyzer. Results: Achilles analysis revealed main dependencies associated with MYC overexpression on glutamine metabolism and specifically GLS1 (glutaminase) and SLC1A1 (glutamine transporter). Using a screening of 2000 small molecules, we further observed that inhibitors of NAD synthesis and mTORC1, which rely on the intracellular glutamine pool, had preferential effect on the proliferation of U266/MYC. Both RNA-seq and quantitative proteomics showed no significant upregulation of glutaminolysis related genes, suggesting a non-oncogenic dependency. Our validation tests confirmed this metabolic dependency, MYC OE cell lines failed to proliferate in the context of glutamine starvation and showed higher sensitivity to CB-839 and V-9302 inhibiting GLS1 and SLC1A5, respectively. Using the Seahorse analyzer, we measured the oxygen consumption rate (OCR) induced by glutamine. U266/MYC cells possess the ability to oxidize glutamine at a higher rate compared to U266/Ctrl. Additionally, higher sensitivity of U266/MYC to CB-839 was observed on both baseline and FCCP-induced OCR highlighting the role of glutamine in controlling mitochondrial OXPHOS in MYC OE cells. To understand the differential metabolic rewiring in MYC OE cells, we performed metabolomic analysis and observed higher GLS1 activity in U266/MYC presented by elevated glutamine to glutamate flux. We also identified the enriched metabolic pathways under GLS1 inhibition. Notably, CB-839 in U266/MYC results in more pronounced changes in TCA cycle and energy debt. This effect was blunted by the co-incubation with a-ketoglutarate, in a rescue experiment. Interestingly GLS1 inhibition was not limited to this effect, but extended to redox balance. CB839 significantly decreased glutathione level in U266/MYC. Furthermore, the intracellular concentrations of the glutamate-dependent amino acids were more depleted under GLS1 inhibition in U266/MYC compared to U266/Ctrl. Summary/Conclusion: Combining these observations, we were able to identify vulnerabilities to glutamine metabolism in MYC overexpressing cells. These results may lead to developing new therapeutic strategies to target MYC in clinical practice.

Kinetic Modeling of 18F-(2S,4R)4-Fluoroglutamine in Mouse Models of Breast Cancer to Estimate Glutamine Pool Size as an Indicator of Tumor Glutamine Metabolism.

The PET radiotracer 18F-(2S,4R)4-fluoroglutamine (18F-Gln) reflects glutamine transport and can be used to infer glutamine metabolism. Mouse xenograft studies have demonstrated that 18F-Gln uptake correlates directly with glutamine pool size and is inversely related to glutamine metabolism through the glutaminase enzyme. To provide a framework for the analysis of 18F-Gln-PET, we have examined 18F-Gln uptake kinetics in mouse models of breast cancer at baseline and after inhibition of glutaminase. We describe results of the preclinical analysis and computer simulations with the goal of model validation and performance assessment in anticipation of human breast cancer patient studies. Methods: Triple-negative breast cancer and receptor-positive xenografts were implanted in athymic mice. PET mouse imaging was performed at baseline and after treatment with a glutaminase inhibitor or a vehicle solution for 4 mouse groups. Dynamic PET images were obtained for 1 h beginning at the time of intravenous injection of 18F-Gln. Kinetic analysis and computer simulations were performed on representative time-activity curves, testing 1- and 2-compartment models to describe kinetics. Results: Dynamic imaging for 1 h captured blood and tumor time-activity curves indicative of largely reversible uptake of 18F-Gln in tumors. Consistent with this observation, a 2-compartment model indicated a relatively low estimate of the rate constant of tracer trapping, suggesting that the 1-compartment model is preferable. Logan plot graphical analysis demonstrated late linearity, supporting reversible kinetics and modeling with a single compartment. Analysis of the mouse data and simulations suggests that estimates of glutamine pool size, specifically the distribution volume (VD) for 18F-Gln, were more reliable using the 1-compartment reversible model than the 2-compartment irreversible model. Tumor-to-blood ratios, a more practical potential proxy of VD, correlated well with volume of distribution from single-compartment models and Logan analyses. Conclusion: Kinetic analysis of dynamic 18F-Gln-PET images demonstrated the ability to measure VD to estimate glutamine pool size, a key indicator of cellular glutamine metabolism, by both a 1-compartment model and Logan analysis. Changes in VD with glutaminase inhibition support the ability to assess response to glutamine metabolism-targeted therapy. Concordance of kinetic measures with tumor-to-blood ratios provides a clinically feasible approach to human imaging.

The Molecular Determinants of Thermoadaptation:Methanococcalesas a Case Study

Previous reports have shown that environmental temperature impacts proteome evolution in Bacteria and Archaea. However, it is unknown whether thermoadaptation mainly occurs via the sequential accumulation of substitutions, massive horizontal gene transfers, or both. Measuring the real contribution of amino acid substitution to thermoadaptation is challenging, because of confounding environmental and genetic factors (e.g., pH, salinity, genomic G + C content) that also affect proteome evolution. Here, using Methanococcales, a major archaeal lineage, as a study model, we show that optimal growth temperature is the major factor affecting variations in amino acid frequencies of proteomes. By combining phylogenomic and ancestral sequence reconstruction approaches, we disclose a sequential substitutional scheme in which lysine plays a central role by fine tuning the pool of arginine, serine, threonine, glutamine, and asparagine, whose frequencies are strongly correlated with optimal growth temperature. Finally, we show that colonization to new thermal niches is not associated with high amounts of horizontal gene transfers. Altogether, although the acquisition of a few key proteins through horizontal gene transfer may have favored thermoadaptation in Methanococcales, our findings support sequential amino acid substitutions as the main factor driving thermoadaptation.

Crosstalk Between Skeletal Muscle and Immune System: Which Roles Do IL-6 and Glutamine Play?

The skeletal muscle was always seen from biomechanical and biochemical views. It is well-established that an active muscle brings many benefits for different body organs and tissues, including the immune system. Since the 1970s, many studies have shown the importance of regular exercise and physical activity in increasing the body’s ability to fight opportunist infections, as well as a strategy to fight established diseases. This interaction was mainly attributed to the glutamine, a non-essential amino acid produced by the active skeletal muscle and primarily consumed by rapidly dividing cells, including lymphocytes and monocytes/macrophages, as their main source of energy. Therefore, these cells’ function would be significantly improved by the presence of a bigger glutamine pool, facilitating phagocytosis, antigen-presentation, proliferative capacity, cytokine synthesis and release, among other functions. Despite its importance, glutamine is not the only molecule to connect these two tissues. The presence of cytokines is crucial for a proper immune system function. Many of them have well-established pro-inflammatory properties, while others are known for their anti-inflammatory role. Interleukin-6 (IL-6), however, has been in the center of many scientific discussions since it can act as pro- and anti-inflammatory cytokine depending on the tissue that releases it. Skeletal muscle is an essential source of IL-6 with anti-inflammatory properties, regulating the function of the immune cells after tissue injury and the healing process. Therefore, this review aims to discuss further the role of these four components (glutamine, and interleukin-6, and its interface with monocytes/macrophages, and lymphocytes) on the communication between the skeletal muscle and the immune system.

Efeito de imunodulador glutamina e probiótico pool de Lactobacillus na atenuação dos sintomas e crises de asma em crianças

Introduction: Immunomodulator glutamine and probiotic Lactobacillus at certain doses have beneficial effects by modulation of the immune system and may assist in the integrity of therespiratory system. However, studies about the effects of glutamine or Lactobacillus pool on the prevention and control of asthma in children are still scarce. The aim of study was to evaluate the effect of the use of glutamine and Lactobacillus pool on the attenuation of clinical asthma symptoms in children. Methods: Longitudinal study, 45 children with medical diagnosis of asthma distributed in three groups: control group (C), glutamine group (G), Lactobacillus group (L). Group G was supplemented with L-glutamine powder (0.3 g/kg/day). Group L used a pool of Lactoba- cillus (Lactobacillus casei, paracasei, rhamnosus, acidophilus and Bifidobacterium lactis) (2 g/day). Group C received no glutamine or Lactobacillus pool. Clinical characteristics and symptoms of respiratory diseases were assessed by study-specific anamnesis and ISSAC Questionnaire (to obtain diagnostic scores and asthma symptoms) for 4 months. To test for differences between groups, the ANOVA test with Tukey post-hoc test was used. It was considered significant p <0.05. Results: Initial ISSAC score was (C=8.67±1.77, L=7.80±1.52 and G=8.00 ± 1.46, p=0.31). At the first follow-up, the ISSAC score indicated that group G had improvement in the clinical characteristics of asthma (C=6.47±2.29, L = 5.07±2.28, G = 4.00±1.73, p <0.05), as also occurred in the following months until the last follow-up (C=5.93±2.28, L=5.13±2.13, G=4.00±1.96, p<0.05). After supplementation, group G presented lower mean duration of asthma attack (p=0.01), lower number of asthma attack (p<0.05), lower prevalence of typical asthma symptoms, as cough and wheezing (p<0.05). Conclusion: Glutamine supplementation attenuated the typical asthma symptoms, while the use of Lactobacillus pool did not attenuate the symptoms. Glutamine may be a new strategy for prevention and control of asthma in children.

Abstract PD4-11: [18F]Fluciclovine PET tracks cellular glutamine pool size in breast cancer and changes in response to metabolic inhibition

Abstract Background: Some forms of triple negative breast cancer (TNBC) rely on glutamine (Gln) metabolism for survival and growth (1), therefore, targeting this metabolic pathway provides a viable strategy for managing TNBC. Drugs that inhibit glutaminase (GLS), a key enzyme of glutaminolysis, are being developed (1,2). [18F]Fluciclovine is a PET imaging agent that enters/exits cells via glutamine transporters and undergoes minimal metabolism. Therefore, we hypothesize, that akin to our prior work with [18F]fluoroglutamine (3), the distribution volume (VT) of fluciclovine obtained from dynamic PET can be used to estimate the cellular glutamine level (pool size) and to mark the effect of pharmacological inhibitors of tumor glutaminase (GLS). We tested this hypothesis in human TNBC and ER+ breast cancer xenograft exhibiting a high and low GLS activity, respectively. Methods: To make [18F]fluciclovine preparation suitable for mouse imaging, citrate in the formulation was removed and replaced with PBS by eluting through a column (Bio-Rad). Cellular uptake was performed in the presence and absence of Gln transporter inhibitors and GLS inhibitor. In vivo dynamic PET imaging were performed on mice bearing HCC1806 (TNBC) and MCF-7 (ER+ BC) xenografts. Dynamic PET images were analyzed by Logan Plot (PMOD) to estimate VT. Results: Cellular uptake of [18F]fluciclovine in HCC1806 and MCF-7 cells were sensitively inhibited by cold glutamine (Gln) and GPNA (a pharmacologic inhibitor of ASCT-2), confirming that the uptake is mediated by Gln transporters. The peak uptake in MCF-7 cells was 5-fold higher than HCC1806. In mouse models, VT from in vivo [18F]Fluciclovine PET in MCF-7 tumor is 1.4-fold of HCC1806. These data are consistent with a higher cellular Gln pool size in MCF-7 as the result of its lower GLS activity. After inhibition of tumor GLS activity, VT of [18F]fluciclovine in HCC-1806 tumors was increased by 56% from baseline values (n=2), whereas VT in MCF-7 tumors decreased 1% after treatment (n=2). Only a small change of FDG PET signal (5% decrease, n=5) was detected in TNBC tumors after GLS inhibitor treatment. Discussions: These data suggest that VT obtained from [18F]fluciclovine PET is sensitive to changes of the Gln pool size induced by GLS inhibition whereas FDG PET is not. Since the Gln pool size is inversely related to the GLS activity, increased VT is consistent with the increased intracellular Gln level when metabolic conversion of Gln to glutamate by GLS is inhibited. Our results suggest that [18F]fluciclovine, an imaging agent approved for prostate cancer imaging, may be useful for assessing glutamine pool size in breast cancer and changes in response to GLS inhibition Support: R21CA198563, R01CA211337, and Komen SAC130060. We thank Blue Earth Diagnostics for supplies of [18F]fluciclovine. 1.Gross MI, Demo SD, Dennison JB, et al. Mol Cancer Ther 2014;13(4):890-901. 2.Le A, Lane AN, Hamaker M, et al. Cell Metab 2012;15(1):110-21. 3.Zhou R, Pantel AR, Li S, et al. Cancer Res 2017;77(6):1476-84. Citation Format: Cao J, Choi H, Pantel A, Kranseler D, Lee H, Mankoff D, Zhou R. [18F]Fluciclovine PET tracks cellular glutamine pool size in breast cancer and changes in response to metabolic inhibition [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD4-11.

High-fat diet induces a neurometabolic state characterized by changes in glutamate and N-acetylaspartate pools associated with early glucose intolerance: An in vivo multimodal MRI study.

Type-2 diabetes mellitus (T2DM) is a metabolic disorder with a broad range of complications in the brain that depend on the conditions that precede its onset, such as obesity and metabolic syndromes. It has been suggested that neurotransmitter and metabolic perturbations may emerge even before the early stages of T2DM and that high-caloric intake could adversely influence the brain in such states. Notwithstanding, evidence for neurochemical and structural alterations in these conditions are still sparse and controversial. To evaluate the influence of high-fat diet in the neurochemical profile and structural integrity of the rodent brain. Prospective. Wistar rats (n = 12/group). A PRESS, ISIS, RARE, and EPI sequences were performed at 9.4T. Neurochemical and structural parameters were assessed by magnetic resonance spectroscopy, voxel-based morphometry, volumetry, and diffusion tensor imaging. Measurements were compared through Student and Mann-Whitney tests. Pearson correlation was used to assess relationships between parameters. Animals submitted to high-caloric intake gained weight (P = 0.003) and developed glucose intolerance (P < 0.001) but not hyperglycemia. In the hippocampus, the diet induced perturbations in glutamatergic metabolites reflected by increased levels of glutamine (P = 0.016) and glutamatergic pool (Glx) (P = 0.036), which were negatively correlated with glucose intolerance (glutamine, r = -0.804, P = 0.029), suggesting a link with neurometabolic dysregulation. At caudate-putamen, high-fat diet led to a surprising increase in the pool of N-acetylaspartate (P = 0.028). A relation with metabolic changes was again suggested by the negative correlation between glucose intolerance and levels of glutamatergic metabolites in this region (glutamate, r = -0.845, P = 0.014; Glx, r = -0.834, P = 0.020). Neither changes in phosphate compounds nor major structural alterations were observed for both regions. We found evidence that high-fat diet-induced obesity leads to distinct early and region-specific metabolic/neurochemical imbalances in the presence of early glucose intolerance even when structural alterations or T2DM are absent. 1 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2018.

GlnA Truncation in Salmonella enterica Results in a Small Colony Variant Phenotype, Attenuated Host Cell Entry, and Reduced Expression of Flagellin and SPI-1-Associated Effector Genes.

Many pathogenic bacteria use sophisticated survival strategies to overcome harsh environmental conditions. One strategy is the formation of slow-growing subpopulations termed small colony variants (SCVs). Here we characterize an SCV that spontaneously emerged from an axenic Salmonella enterica serovar Typhimurium water culture. We found that the SCV harbored a frameshift mutation in the glutamine synthetase gene glnA, leading to an ∼90% truncation of the corresponding protein. Glutamine synthetase, a central enzyme in nitrogen assimilation, converts glutamate and ammonia to glutamine. Glutamine is an important nitrogen donor that is required for the synthesis of cellular compounds. The internal glutamine pool serves as an indicator of nitrogen availability in Salmonella In our study, the SCV and a constructed glnA knockout mutant showed reduced growth rates, compared to the wild type. Moreover, the SCV and the glnA mutant displayed attenuated entry into host cells and severely reduced levels of exoproteins, including flagellin and several Salmonella pathogenicity island 1 (SPI-1)-dependent secreted virulence factors. We found that these proteins were also depleted in cell lysates, indicating their diminished synthesis. Accordingly, the SCV and the glnA mutant had severely decreased expression of flagellin genes, several SPI-1 effector genes, and a class 2 motility gene (flgB). However, the expression of a class 1 motility gene (flhD) was not affected. Supplementation with glutamine or genetic reversion of the glnA truncation restored growth, cell entry, gene expression, and protein abundance. In summary, our data show that glnA is essential for the growth of S. enterica and controls important motility- and virulence-related traits in response to glutamine availability.IMPORTANCESalmonella enterica serovar Typhimurium is a significant pathogen causing foodborne infections. Here we describe an S Typhimurium small colony variant (SCV) that spontaneously emerged from a long-term starvation experiment in water. It is important to study SCVs because (i) SCVs may arise spontaneously upon exposure to stresses, including environmental and host defense stresses, (ii) SCVs are slow growing and difficult to eradicate, and (iii) only a few descriptions of S. enterica SCVs are available. We clarify the genetic basis of the SCV described here as a frameshift mutation in the glutamine synthetase gene glnA, leading to glutamine auxotrophy. In Salmonella, internal glutamine limitation serves as a sign of external nitrogen deficiency and is thought to regulate cell growth. In addition to exhibiting impaired growth, the SCV showed reduced host cell entry and reduced expression of SPI-1 virulence and flagellin genes.

Abstract 2851: Cellular glutamine pool size change in response to glutaminase inhibition detected by kinetic analysis of [18F](2S,4R)4-fluoroglutamine PET

Abstract INTRODUCTION Oncogene-dependent reliance on glutamine is a cancer vulnerability that can be exploited for therapeutic gain. Inhibitors of glutaminase, the enzyme that catalyzes the conversion of glutamine to glutamate, have been developed and have shown antitumor effects in several tumor models. The specific and potent glutaminase inhibitor CB-839 demonstrated marked antitumor efficacy in a triple-negative breast cancer (TNBC) cell line with inherently high glutaminase activity (HCC-1806), but not in an estrogen receptor-positive (ER+) cell line with inherently low glutaminase activity. An early phase 1 clinical trial of CB-839 in combination with paclitaxel has shown encouraging results in TNBC patients. Cell-line specific efficacy of glutaminase inhibitors supports the need for clinical biomarkers that can predict and evaluate therapeutic efficacy. As a minimally metabolized glutamine analog with similar transport properties, [18F](2S,4R)4-Fluoroglutamine ([18F]4F-Gln) is an ideal radiotracer to infer glutamine pool size through estimates of tracer distribution volume (DV). In this study, we demonstrate differences in DV of [18F]4F-Gln in two xenografts with different levels of glutaminolysis (TNBC vs. ER+) using dynamic PET imaging, as well as show the effect of anti-glutaminase therapy on [18F]4F-Gln DV. METHODS TNBC (HCC-1806) and ER+ (MCF-7) xenografts were established in athymic nu/nu mice. Mice were scanned in a dedicated animal PET scanner at baseline and after CB-839 administration. Dynamic imaging was obtained for one hour upon injection of [18F]4F-Gln (300-350 μCi) via the tail vein. Kinetic analysis was performed with PMOD. RESULTS [18F]4F-Gln uptake was largely reversible with Logan plots demonstrating late linearity and k3 in a two-compartment (trapping) model was low (less than 0.01/min in most cases). Strong correlation was seen in DV estimates by Logan plot and a single-compartmental model (R2&amp;gt;0.9), but not with estimates from a two-compartment model. MCF-7 xenografts demonstrated greater than 60% larger DV at baseline than TNBC xenografts indicative of increased cellular glutamine concentrations in MCF-7 xenografts. An increase in DV was detected in TNBC xenografts post-glutaminase inhibition (&amp;gt;30% mean change), but not in MCF-7 xenografts. CONCLUSION Estimates of [18F]4F-Gln DV offer the ability to non-invasively infer tumor glutaminolysis. Low [18F]4F-Gln uptake in highly glutaminolytic tumors and an increase in [18F]4F-Gln uptake with glutaminase inhibition is concordant with changes in cellular glutamine concentration as measured by MR spectroscopy of tumor extracts. Our studies indicate promise for the use of [18F]4F-Gln as a predictive and pharmacodynamic marker for glutaminase-targeted therapy. Supported by: Komen SAC130060, DE-SE0012476, R21-CA198563 We thank Calithera Inc. for providing CB-839 and Dr. Susan Demo for discussions. Citation Format: Austin R. Pantel, Hsiaoju Lee, Shihong Li, Robert K. Doot, Robert H. Mach, David A. Mankoff, Rong Zhou. Cellular glutamine pool size change in response to glutaminase inhibition detected by kinetic analysis of [18F](2S,4R)4-fluoroglutamine PET [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2851. doi:10.1158/1538-7445.AM2017-2851

L-glutamine Enhances Plasma Glutamine And Maintains Concentrations Of Alanine And Arginine Following High Intensity Cycling

Glutamine is a naturally occurring nonessential, gluconeogenic amino acid. Prolonged exercise is associated with a decrease in intramuscular and plasma concentrations of glutamine, which may be linked to performance decrements. Thus, we hypothesized that exogenous glutamine supplementation would preserve the plasma glutamine pool following high intensity cycling, as well as improve performance in a repeated bout of cycling. Two solutions [61 g glucose polymer (PLC); 61 g glucose polymer and 0.3 g-kg-1-bodyweight L-glutamine (GLN)] were tested using a double-blind, randomized, cross-over design. During each trial, ten cyclists ingested one liter of test solution immediately following an initial exercise bout (30 min at 70% VO2 max, 6 x 1 min sprints at 140% VO2 max, 45 min at 70% VO2max) and recovered for two hours in a seated position. Immediately following the recovery period, subjects completed a time to exhaustion (cycle at 80% VO2 max until no longer maintaining 100 RPM) test. Blood was collected immediately following the initial exercise bout, after 2 h recovery, and immediately following the time to exhaustion test. Glutamine concentration was increased (p < 0.05) by approximately twofold (676±126 to 1410±636 μmol/L) from baseline to the completion of recovery for GLN, while no difference was detected in the PLC trial. At the end of recovery, GLN maintained alanine and arginine concentrations, while these amino acids significantly decreased in the PLC trial. However, the increased concentration of plasma glutamine and maintained concentrations of alanine and arginine did not influence performance (GLN: 20.98±10.35; PLC: 21.5±8.53 min).

18F](2S,4R)4-Fluoroglutamine PET Detects Glutamine Pool Size Changes in Triple-Negative Breast Cancer in Response to Glutaminase Inhibition.

Glutaminolysis is a metabolic pathway adapted by many aggressive cancers, including triple-negative breast cancers (TNBC), to utilize glutamine for survival and growth. In this study, we examined the utility of [18F](2S,4R)4-fluoroglutamine ([18F]4F-Gln) PET to measure tumor cellular glutamine pool size, whose change might reveal the pharmacodynamic (PD) effect of drugs targeting this cancer-specific metabolic pathway. High glutaminase (GLS) activity in TNBC tumors resulted in low cellular glutamine pool size assayed via high-resolution 1H magnetic resonance spectroscopy (MRS). GLS inhibition significantly increased glutamine pool size in TNBC tumors. MCF-7 tumors, with inherently low GLS activity compared with TNBC, displayed a larger baseline glutamine pool size that did not change as much in response to GLS inhibition. The tumor-to-blood-activity ratios (T/B) obtained from [18F]4F-Gln PET images matched the distinct glutamine pool sizes of both tumor models at baseline. After a short course of GLS inhibitor treatment, the T/B values increased significantly in TNBC, but did not change in MCF-7 tumors. Across both tumor types and after GLS inhibitor or vehicle treatment, we observed a strong positive correlation between T/B values and tumor glutamine pool size measured using MRS (r2 = 0.71). In conclusion, [18F]4F-Gln PET tracked cellular glutamine pool size in breast cancers with differential GLS activity and detected increases in cellular glutamine pool size induced by GLS inhibitors. This study accomplished the first necessary step toward validating [18F]4F-Gln PET as a PD marker for GLS-targeting drugs. Cancer Res; 77(6); 1476-84. ©2017 AACR.

Glutamine in critically ill patients: is it a fundamental nutritional supplement?

Glutamine is the most abundant free amino acid in the human body and is necessary to modulate the inflammatory and oxidative stress responses in patients.(1,2) Systemic glutamine availability is determined by the balance of endogenous glutamine production (mainly in muscular tissue) and its use by glutamine consuming organs (gut, kidney, liver and the immune system). Several studies show that in catabolic intensive care unit (ICU) patients, the endogenous production of muscular glutamine is increased while the plasma levels of glutamine are decreased, indicating elevated glutamine needs.(3,4) We know also that low plasma glutamine values (< 420μmol/L) upon admission are related to increased mortality.(5) These findings are the rationale for the use of glutamine supplementation in the ICU population in order to replenish the muscle pool of glutamine, attenuate the efflux of this amino acid, and provide exogenous glutamine required to meet the elevated organ needs for improvement in protein synthesis, modulation of the immune system, reduction of oxidative stress, and preservation of the gut barrier. However, recent observations challenge this hypothesis. The plasma levels of glutamine are extremely variable in the ICU population(6) and are not always associated with increased mortality.(7) The glutamine supplementation does not stop the glutamine efflux from muscle because the endogenous muscular production and plasma levels of glutamine are related to the severity of the illness.(8) Intensive care unit patients are considered to be immunosuppressed as evidenced by reduced levels of and the presence of dysfunctional T lymphocytes, altered neutrophil activity and an imbalance in the production of cytokines.(9-13) The demonstration of the benefit of glutamine in increasing the number and functionality of effector cells of the immune response is evident in some studies,(14-19) while in others that answer is not so obvious.(20,21) We found (personal communication)(22) that in the ICU population, glutamine supplementation (0.40g/Kg/day) via parenteral nutrition improved cellular immune function with significant increases in CD14 and CD14 HLA-DR monocytes and significant decreases in T regulatory cells with a significant reduction in the appearance of nosocomial infection. Over the years, many studies examining glutamine in ICU populations have presented controversial results. We can differentiate between the small monocentric studies of the early years showing a significant reduction in Paulo Martins1

The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7

Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5'UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus [Formula: see text] assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.

Membrane transporters for the special amino acid glutamine: structure/function relationships and relevance to human health.

Glutamine together with glucose is essential for body's homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7, and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5, and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologs. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.

Abstract C154: Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase).

Abstract Glutamine is an essential nutrient for cancer cells and increased glutaminolysis is widely recognised as a key feature of the metabolic reprogramming that occurs during malignant transformation. As a consequence of the role glutamine plays in cancer biology, targeting glutamine metabolism is of considerable interest with inhibition of the mitochondrial enzyme glutaminase showing promise in experimental models. An alternative or adjunct to the strategy of inhibiting glutamine metabolism intracellularly is to deplete extracellular pools of glutamine. L-asparaginases are known to possess glutaminase activity and the purpose of this study is to characterise the kinetics of the glutaminase activity of two clinically approved L-asparaginases, Kidrolase (derived from E. coli) and Erwinase (derived from Erwinia chrysanthemi). Glutaminase activity was measured using a two step reaction: initial reduction of glutamine to glutamate by Kidrolase/Erwinase followed by the NAD dependent conversion of glutamate to ammonia by L-glutamate dehydrogenase (GDH). Each reaction consisted of NAD (2mM), K2PO4 (50mM), GDH (3U), Erwinase or Kidrolase (10U), L-glutamine (concentrations ranged from 5mM to 1.25 μM) in a final volume of 1 ml Tris Acetate buffer (50mM, pH8.6). The reaction was started by the addition of Erwinase/Kidrolase and the formation of NADH monitored for 30 seconds at 340nm using a Cary UV/Vis spectrophotometer and a NADH molar extinction coefficient of 6,220 M-1cm-1. The reaction was linear between glutamine concentrations 100 and 1.25 μM. Using 10U in each reaction, Hanes Woolf kinetic plots generated Vmax parameters of 46.29 and 7.57 μM/min for Erwinase and Kidrolase respectively. Km values were 96.05 and 33.06 μM for Erwinase and Kidrolase respectively. Preliminary studies demonstrate that glutamine depletion induced a dose dependent reduction in cell proliferation in HCT116 p53+/+, HCT116 p53-/-, MDA-MD-231, MDA-MB-453, MCF7, TK10, A549 and T47D cell lines in vitro. In conclusion, these studies demonstrate that Erwinase has significant glutaminase activity and is superior to Kidrolase in terms of glutaminase activity. Erwinase is a good candidate for exploring the efficacy of glutamine depletion strategies and further studies are required to determine whether Erwinase can potentiate the activity of other approaches aimed at restricting the glutamine dependence of tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C154. Citation Format: Roger M. Phillips, Mohammed U. Saleem, Adrian Williams, Jon Morgan. Kinetic analysis of the glutaminase activity of L-asparaginases derived from Erwinia chrysanthemi (Erwinase®) and Eshericia coli (Kidrolase). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C154.