Role Of Glutamine Research Articles

Glutamine and type 1 diabetes mellitus

Recent literature suggests dietary glutamine supplementation may lower blood glucose in patients with type 1 diabetes (T1D), who have no residual insulin secretion. The mechanisms and potential relevance to the care of T1D remain unclear. Glutamine is involved in multiple pathways including gluconeogenesis, lipolysis, antioxidant defense, the production of nitric oxide, the secretion of peptides (e.g., glucagon-like peptide 1, GLP-1), or neuromediators (e.g., [Latin Small Letter Gamma]-aminobutyric acid), all processes that may impact insulin sensitivity and/or glucose homeostasis. The article reviews potential mechanisms and literature evidence suggesting a role in improving glucose tolerance in patients with illness associated with insulin resistance, as well as the preliminary evidence for the increased incidence of postexercise hypoglycemia in T1D after oral glutamine. Further studies are warranted to determine whether the lowering effect of glutamine on blood glucose is sustained over time. If so, long-term randomized trials would be warranted to determine whether there is a role for glutamine as an adjunct dietary supplement to improve glucose control in patients with T1D.

Abstract IA06: Molecular mechanisms of lung cancer development: Between metabolic reprogramming and genomic instability in the field of cancerization

Abstract Airway epithelial cells are prone to assault by risk factors and considered to be the primary cell type involved in the field of cancerization. To investigate risk-associated changes in the bronchial epithelium proteome that may offer new insights into molecular pathogenesis of lung cancer, proteins were identified in the airway epithelial cells of bronchial brushings specimens from risk-stratified individuals by shotgun proteomics. Differential expression of selected proteins was validated by parallel reaction monitoring mass spectrometry in an independent set of individual bronchial brushings. We identified over 300 proteins with a significant trend in expression. Pathway analysis revealed enrichment of carbohydrate metabolic enzymes in high-risk individuals. Glucose consumption and lactate production were increased in human bronchial epithelial BEAS2B cells treated with cigarette smoke condensate for seven months. Increased lipid biosynthetic capacity and net reductive carboxylation were revealed by metabolic flux analyses of [U-13C5] glutamine in this in vitro model, suggesting profound metabolic reprogramming in the airway epithelium of high-risk individuals. These results also pointed towards a fundamental role of glutamine and cysteine metabolism in lung cancer progression. Two amino acid transporters, specifically SLC1A5 and SLC7A11, captured our attention for their high level of expression in lung cancer. These transporters are associated with worse survival in lung cancer. A small-molecular inhibitor of SLC1A5 prevents tumor progression in nude mice, and a glutaminase inhibitor CB-839 exhibits radiosensitization to lung cancer cells, an effect that may be mediated by decreased GSH production. Taken together, these studies provide a rationale for new chemopreventive strategies, selection of patients for surveillance programs, and determining how metabolic reprogramming may contribute genomic instability a requirement to cancer development. Further investigation into how glutamine and cystine metabolism contribute to lung cancer development and progression may lead to novel early-interception strategies in lung cancer. This work is supported by NCI CA186145, CA152662, and DoD W81XWH-11-2-0161. Citation Format: Jamshed Rahman, Xiangming Ji, Chirayu Patel, Jamey Young, Pierre P. Massion. Molecular mechanisms of lung cancer development: Between metabolic reprogramming and genomic instability in the field of cancerization [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA06.

Role of glutamine in the mediation of E-cadherin, p120-catenin and inflammation in ventilator-induced lung injury.

Background:Ventilator-induced lung injury (VILI) is commonly associated with barrier dysfunction and inflammation reaction. Glutamine could ameliorate VILI, but its role has not been fully elucidated. This study examined the relationship between inflammatory cytokines (interleukin [IL]-6, tumor necrosis factor [TNF]-α, and IL-10) and adherens junctions (E-cadherin, p120-catenin), which were ameliorated by glutamine in VILI, both in vitro and in vivo.Methods:For the in vivo study, 30 healthy C57BL/6 mice weighing 25–30 g were randomly divided into five groups with random number table (n = 6 in each group): control (Group C); low tidal volume (Group L); low tidal volume + glutamine (Group L + G); high tidal volume (Group H); and high tidal volume + glutamine (Group H + G). Mice in all groups, except Group C, underwent mechanical ventilation for 4 h. For the in vitro study, mouse lung epithelial 12 (MLE-12) cells pretreated with glutamine underwent cyclic stretching at 20% for 4 h. Cell lysate and lung tissue were obtained to detect the junction proteins, inflammatory cytokines, and lung pathological changes by the Western blotting, cytokine assay, hematoxylin and eosin staining, and immunofluorescence.Results:In vivo, compared with Group C, total cell counts (t = −28.182, P < 0.01), the percentage of neutrophils (t = −28.095, P < 0.01), IL-6 (t = −28.296, P < 0.01), and TNF-α (t = −19.812, P < 0.01) in bronchoalveolar lavage (BAL) fluid, lung injury scores (t = −6.708, P < 0.01), and the wet-to-dry ratio (t = −15.595, P < 0.01) were increased in Group H; IL-10 in BAL fluid (t = 9.093, P < 0.01) and the expression of E-cadherin (t = 10.044, P < 0.01) and p120-catenin (t = 13.218, P < 0.01) were decreased in Group H. Compared with Group H, total cell counts (t = 14.844, P < 0.01), the percentage of neutrophils (t = 18.077, P < 0.01), IL-6 (t = 18.007, P < 0.01), and TNF-α (t = 10.171, P < 0.01) in BAL fluid were decreased in Group H + G; IL-10 in BAL fluid (t = −7.531, P < 0.01) and the expression of E-cadherin (t = −14.814, P < 0.01) and p120-catenin (t = −9.114, P < 0.01) were increased in Group H + G. In vitro, compared with the nonstretching group, the levels of IL-6 (t = −21.111, P < 0.01) and TNF-α (t = −15.270, P < 0.01) were increased in the 20% cyclic stretching group; the levels of IL-10 (t = 5.450, P < 0.01) and the expression of E-cadherin (t = 17.736, P < 0.01) and p120-catenin (t = 16.136, P < 0.01) were decreased in the 20% cyclic stretching group. Compared with the stretching group, the levels of IL-6 (t = 11.818, P < 0.01) and TNF-α (t = 8.631, P < 0.01) decreased in the glutamine group; the levels of IL-10 (t = −3.203, P < 0.05) and the expression of E-cadherin (t = −13.567, P < 0.01) and p120-catenin (t = −10.013, P < 0.01) were increased in the glutamine group.Conclusions:High tidal volume mechanical ventilation and 20% cyclic stretching could cause VILI. Glutamine regulates VILI by improving cytokines and increasing the adherens junctions, protein E-cadherin and p120-catenin, to enhance the epithelial barrier function.

“When is Glutamine Supplementation Beneficial?”

The role of glutamine in metabolic processes has been quite extensively researched and described, though no consensus exists as to its role in treatment. It is considered significant mainly for ICU patients; though no clear criteria for including glutamine in treatment have been defined. It is known that glutamine should be administered as a complement to nutritional treatment, and not independently. The purpose of the paper was to identify practical criteria for determining the clinical benefits of glutamine supplementation. The study was performed in the years 2007-2015 at the 1st Department of General and Transplantation Surgery and Nutritional Therapy of the Lublin Medical University in Lublin, Poland. It included patients scheduled for surgery due to a gastrointestinal cancer. The final study group included 105 patients, 48 female and 57 male. We found that low blood concentration of glutamine was correlated with a higher incidence of postoperative complications. ROC analysis allowed for identification of glutamine concentration below which there is a very high risk of complications. The threshold glutamine value identified was 205.15 nmol/ml. Low total lymphocyte count and serum albumin concentration can help identify patients in whom glutamine supplementation can decrease postoperative complication incidence, especially in the case of malnourished patients. Glutamine supplementation before a scheduled surgical procedure may benefit patients with a preoperative glutamine concentration below 205.15 nmol/ml. Glutamine supplementation can benefit malnourished patients.

The Roles of Glutamine in the Intestine and Its Implication in Intestinal Diseases.

Glutamine, the most abundant free amino acid in the human body, is a major substrate utilized by intestinal cells. The roles of glutamine in intestinal physiology and management of multiple intestinal diseases have been reported. In gut physiology, glutamine promotes enterocyte proliferation, regulates tight junction proteins, suppresses pro-inflammatory signaling pathways, and protects cells against apoptosis and cellular stresses during normal and pathologic conditions. As glutamine stores are depleted during severe metabolic stress including trauma, sepsis, and inflammatory bowel diseases, glutamine supplementation has been examined in patients to improve their clinical outcomes. In this review, we discuss the physiological roles of glutamine for intestinal health and its underlying mechanisms. In addition, we discuss the current evidence for the efficacy of glutamine supplementation in intestinal diseases.

Short communication: Glutamine modulates inflammatory responses to lipopolysaccharide in ex vivo bovine endometrium

Bacteria infect the endometrium lining the uterus of cattle after parturition, and clearance of these microbes depends on a robust innate immune response to bacterial molecules, such as the endotoxin lipopolysaccharide (LPS). Endometrial inflammation is characterized by secretion of the cytokines IL-1β and IL-6 and the chemokine IL-8. However, animals often fail to clear invading bacteria and develop uterine disease if they are in negative energy balance, with reduced abundance of glucose and glutamine, which are substrates for energy in tissues. Depletion of glucose blunts inflammatory responses in the endometrium, but the role of glutamine is not clear. The present study tested the hypothesis that depletion of glutamine compromises inflammatory responses to LPS in endometrial tissue. Ex vivo organ cultures of endometrium were challenged with LPS, and culture supernatants accumulated IL-1β, IL-6, and IL-8, as expected. However, reducing the availability of glutamine in culture medium containing glucose reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%. Surprisingly, in the absence of glucose, supplying increasing amounts of glutamine was not sufficient to augment inflammatory responses to LPS, whereas, in the absence of glutamine, supplying more glucose increased inflammation. Furthermore, inhibiting glycolysis reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%, even when glutamine and glucose were abundant. In conclusion, depletion of glutamine reduces inflammatory responses to LPS in the endometrium, and the activity of glutamine depends on glucose and glycolysis. These data provide mechanistic insights into how negative energy balance may be linked to postpartum uterine disease.

The role of glutamine in Pseudomonas mediterranea in biotechnological processes

In this work, in order to study the effect of glutamine as co-feeder on growth kinetics, biomass and PHA production in Pseudomonas mediterranea, different co-metabolic strategies were employed. Unrelated (glycerol and glucose) and related (sodium octanoate) carbon sources both in presence and absence of glutamine have been tested. For each cultural condition, we (i) evaluated growth kinetics and measured the cell metabolic activity by MTT assay, (ii) monitored PHA production and (iii) estimated the expression of phaC1 and phaC2 genes through RT-PCR. Our results show that the use of glutamine as co-feeder in P. mediterranea led to an improvement of the specific growth rate and cell metabolic activity and enhanced the uptake of all the carbon sources assayed. Moreover, the use of glutamine reduced significantly the time required for PHA production and increased biopolymer yield, as consequence of an early activation of phaC1 and phaC2.

Role of Glutamine in Metabolic and Epigenetic Reprogramming in AML

Glutamine (Gln) was shown to play a role in generation of oncometabolite 2-hydroxyglutarate (2-HG) in tumors with high GLS (glutaminase) expression, whereby IDH2 (Isocitrate Dehydrogenase 2) enzyme catalyzes carboxylating reduction of glutamine-derived α-ketoglutarate (α-KG) to isocitrate and noncarboxylating reduction to 2-HG (Wise PNAS 2011). 2-HG in turn is known to inhibit α-KG dependent dioxygenases that mediate epigenetic events, including DNA and histone demethylation (Licht Cancer Cell 2010). A recent report demonstrated that hypoxia induces production of the L-enantiomer of 2-HG (L-2HG), through enzymatic reduction of α-KG by lactate dehydrogenase A (LDHA) (Intlekofer Cell Metabolism 2015). We have previously demonstrated that leukemic bone marrow microenvironment is highly hypoxic (Benito PLoS One 2011). Further, our unpublished data indicate upregulation of GLS protein and increase production of total 2-HG in AML (acute myeloid leukemia) cells cultured under hypoxia. We therefore propose a link between hypoxia, Gln metabolism, and epigenetic regulation in AML. Since increased methylation (and decreased hydroxymethylation) is seen in AML, we hypothesize that GLS inhibition can abrogate these changes via reduction of 2-HG levels.First, we examined effects of hypoxia and selective GLS inhibitor CB-839 (Calithera Biosciences) on cellular growth of AML cells with wild type IDH (OCI-AML3 and HL-60), cultured alone or co-cultured with bone marrow derived stromal cells (MSC). The culture of untreated OCI-AML3 alone in normoxic and hypoxic conditions caused a decrease in viability from 96 ± 2.5% to 84 ± 4.1% respectively, while the treatment with CB-839 (1 mM) for 6 days decreased viability in OCI-AML3 cells from 94 ± 0.23% to 71 ± 2.3% respectively (P=0.015). While MSC co-cultures improved survival of floating AML cells, the attached cells that were in direct contact with MSC were more affected under hypoxic conditions, having a viability of 64 ± 8.7% at the end of the experiment. These data indicate that GLS inhibitor is more effective under hypoxic conditions mimicking leukemic BM microenvironment.Hypoxia selectively induced the production of L-2HG (measured by liquid chromatography-tandem mass spectrometry) under hypoxic conditions (>40 fold) in OCI-AML3 cells, both with and without MSC co-culture. This increase in L-2HG was partially inhibited by co-treating OCI-AML3 cells with GLS inhibitor CB-839 (reduction of 1.7-fold in media only and 1.3-fold in MSC co-culture). Determination of hydroxymethylation (hmc) levels using HELP-GT assay demonstrated a significant increase in hmc in cells treated with CB-839. Of importance, genes that were differentially hydroxymethylated after CB-839 treatment belonged to important functional categories with cancer being the dominant pathway affected by these changes.Under hypoxia, glucose metabolism is known to be directed towards anaerobic glycolysis, with increased pyruvate-lactate enzymatic conversion by LDHA. To characterize the role of Gln and GLS on these processes within leukemia microenvironment, we performed nuclear magnetic resonance imaging with hyperpolarized pyruvate in NSG (NOD scid gamma) mice engrafted with GFP/luc-labeled OCI-AML3 cells. Inhibition of GLS in vivo following exposure of mice with 200 mg/kg dose of CB-839 showed a decrease in lactate conversion rate within leukemic bone marrow (femur area) (0.31 + 0.03 (pre) to 0.20 + 0.04 (post) P < 0.05), possibly due to the reduction of the level of NADH from decreased flux of Gln in the TCA cycle.In summary, our results indicate that Gln and GLS contribute towards hypoxia-induced production of L-2HG and critical epigenetic changes in AML; as well as playing a role in enhanced production of lactate from pyruvate. These findings suggest a major importance of Gln in metabolic and epigenetic reprogramming of microenvironment. DisclosuresOff Label Use: CB-839 is a potent, selective, reversible and orally bioavailable glutaminase (mitochondrial enzyme able to convert glutamine in glutamate) inhibitor that has shown to reduce cell growth and/or induce cell death in solid tumors and hematological malignancies.. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Reducing the serine availability complements the inhibition of the glutamine metabolism to block leukemia cell growth

Leukemia cells are described as a prototype of glucose-consuming cells with a high turnover rate. The role of glutamine in fueling the tricarboxylic acid cycle of leukemia cells was however recently identified confirming its status of major anaplerotic precursor in solid tumors. Here we examined whether glutamine metabolism could represent a therapeutic target in leukemia cells and whether resistance to this strategy could arise. We found that glutamine deprivation inhibited leukemia cell growth but also led to a glucose-independent adaptation maintaining cell survival. A proteomic study revealed that glutamine withdrawal induced the upregulation of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT), two enzymes of the serine pathway. We further documented that both exogenous and endogenous serine were critical for leukemia cell growth and contributed to cell regrowth following glutamine deprivation. Increase in oxidative stress upon inhibition of glutamine metabolism was identified as the trigger of the upregulation of PHGDH. Finally, we showed that PHGDH silencing in vitro and the use of serine-free diet in vivo inhibited leukemia cell growth, an effect further increased when glutamine metabolism was blocked. In conclusion, this study identified serine as a key pro-survival actor that needs to be handled to sensitize leukemia cells to glutamine-targeting modalities.

Enteral/Oral Glutamine Supplementation in Patients Following Abdominal Surgery

The objective of this investigation was to study the effect of enteral/oral glutamine supplementation in patients following abdominal surgery on plasma glutamine levels, rate of infection and Length Of Hospitalization (LOS). A randomized control trial was used and the patients were randomly divided into two groups namely experimental and control with 15 participants each. Glutamine supplement (0.5g/kg) was administered (oral and enteral) to the experimental group for a period of 5 days postsurgery immediately after the feeding began. In both the feeding groups the plasma glutamine levels were analyzed spectrophotometrically using a glutamine determination kit on day 1 following surgery and day 6 (post 5 days of feeding). For statistical analysis t test and Chi square test were used. The study was carried out in Topiwala National Medical College and BYL Nair Hospital, Mumbai, India. There was a significant difference in the baseline plasma glutamine levels in both the groups (p<0.05). And on day 6 the plasma glutamine analysis revealed significant higher levels in the experimental group as compared to the control group. The incidence of infection in the control group was found to be almost twice that in the experimental group considering the role of glutamine in combating infection. Also the length of hospitalization was found to be slightly higher in the control group as compared to the experimental group. The present study has provided evidence that the supplementation of enteral glutamine in post-operative patients decreases the incidence of post-surgical infection, shortening of hospital stay and reduction in the overall hospital costs.

Glutamine Supplementation in Intensive Care Patients: A Meta-Analysis of Randomized Clinical Trials.

The role of glutamine (GLN) supplementation in critically ill patients is controversial. Our aim was to analyze its potential effect in patients admitted to intensive care unit (ICU).We performed a systematic literature review through Medline, Embase, Pubmed, Scopus, Ovid, ISI Web of Science, and the Cochrane-Controlled Trials Register searching for randomized clinical trials (RCTs) published from 1983 to 2014 and comparing GLN supplementation to no supplementation in patients admitted to ICU. A random-effect meta-analysis for each outcome (hospital and ICU mortality and rate of infections) of interest was carried out. The effect size was estimated by the risk ratio (RR).Thirty RCTs were analyzed with a total of 3696 patients, 1825 (49.4%) receiving GLN and 1859 (50.6%) no GLN (control groups). Hospital mortality rate was 27.6% in the GLN patients and 28.6% in controls with an RR of 0.93 (95% CI = 0.81–1.07; P = 0.325, I2 = 10.7%). ICU mortality was 18.0 % in the patients receiving GLN and 17.6% in controls with an RR of 1.01 (95% CI = 0.86–1.19; P = 0.932, I2 = 0%). The incidence of infections was 39.7% in GLN group versus 41.7% in controls. The effect of GLN was not significant (RR = 0.88; 95% CI = 0.76–1.03; P = 0.108, I2 = 56.1%).These results do not allow to recommend GLN supplementation in a generic population of critically ills. Further RCTs are needed to explore the effect of GLN in more specific cohort of patients.

The protective role of glutamine against acute induced toxicity in rats

Context: Deltamethrin (DLM) is an insecticide commonly used to protect agricultural crops against pests. QT prolongation with malignant ventricular arrhythmias are amongst the most common cardiovascular complications. DLM intoxication cause decreased level of antioxidant enzymes. Glutamine is the precursor of glutathione which is an antioxidant and has been demonstrated to improve outcome after several critical illnesses.Objective: We hypothesized that glutamine, by means of antioxidant characteristics, may antagonize the cardiotoxic effects of DLM.Materials and methods: All experiments were performed on 8-week-old male Wistar albino rats. The rats were divided into following groups (n = 10); Group I: control, Group II: l-glutamine, Group III: DLM, Group IV: DLM and after 4 h l-glutamine. Total antioxidant status (TAS), total oxidant status (TOS) and parameter analyses were performed in cardiac tissue.Results: We found that TAS was higher and TOS lower in DLM group. We also found that interstitial edema and inflammatory cell infiltration was significantly more frequent in DLM group and QT and QTc of DLM group were higher than others.Discussion: Recent studies have shown that several special amino acids, such as glutamine, glycine, arginine and taurine, exhibit cytoprotective effect on the cardiocyte, and have established the cardioprotective properties of glutamine.Conclusion: In this study, we showed the protective role of glutamine against cardiotoxic effects of DLM in rats. This protective effect was confirmed by showing both tissue level improvement in oxidative stress markers and improvement in prolonged QT interval.

Probing treatment response of glutaminolytic prostate cancer cells to natural drugs with hyperpolarized [5-(13) C]glutamine.

The correlation between glutamine metabolism and oncogene expression in cancers has led to a renewed interest in the role of glutamine in cancer cell survival. Hyperpolarized [5-(13) C]glutamine is evaluated as a potential biomarker for noninvasive metabolic measurements of drug response in prostate cancer cells. Hyperpolarized [5-(13) C]glutamine is used to measure glutamine metabolism in two prostate cancer cell lines (PC3 and DU145) before and after treatment with the two natural anticancer drugs resveratrol and sulforaphane. An invasive biochemical assay simulating the hyperpolarized experiment is used to independently quantify glutamine metabolism. Glutamine metabolism is found to be 4 times higher in the more glutaminolytic DU145 cells compared with PC3 cells under proliferating growth conditions by using hyperpolarized [5-(13) C]glutamine as a noninvasive probe. A significant decrease in glutamine metabolism occurs upon apoptotic response to treatment with resveratrol and sulforaphane. Hyperpolarized NMR using [5-(13) C]glutamine as a probe permits the noninvasive observation of glutaminolysis in different cell lines and under different treatment conditions. Hyperpolarized [5-(13) C]glutamine metabolism thus is a promising biomarker for the noninvasive detection of tumor response to treatment, as it directly monitors one of the hallmarks in cancer metabolism - glutaminolysis - in living cells.

The Pasteur’s Dictum: Nitrogen Promotes Growth and Oxygen Reduces the Need for Sugar

In our previous article (1), we presented a unifying principle in the hypotheses of Pasteur, Warburg, and Crabtree that “an inverse relation exists between glucose uptake and respiration.” Warburg’s hypothesis laid emphasis on glucose metabolism and damaged respiration (mitochondria) for growth of cells in cancer (2). Pasteur recognized that yeast can grow only if ammonium tartrate (nitrogen source) is available, whether oxygen is available or not (3) and ammonium is transformed into a “complex albuminoid” (protein) material. Growth is faster in the presence of oxygen and for producing one unit of mass, yeast requires only 1/15th of glucose when compared to that in the absence of oxygen (4–10 parts as against 60–80 parts). Since the ratio of ATP produced per one glucose molecule in glycolysis/oxidative phosphorylation (OXPHOS) is 1:15, the energy consumed per unit of growth of yeast remains same (60X1 and 4X15 ATP). We call this: “the Pasteur’s dictum,” which is different from the “Pasteur effect” introduced by Warburg. Lagunas et al. (4) in early 80s reported that Pasteur effect is observed only in resting cells in the absence of nitrogen, but not in growing cells. Warburg’s hypothesis relies on energetics of glucose metabolism and damaged respiration but ignores Pasteur’s demonstration of nitrogen as the primary requirement for triggering growth. It also ignores the anabolic functions of mitochondria (5, 6) and the role of glutamine, another key nutrient avidly consumed by actively proliferating cells (7, 8). We present in this article, a hypothesis that activation of complexes of mechanistic target of rapamycin (mTORC1 and C2) by amino acids (nitrogen source) is the molecular explanation of the “Pasteur’s dictum.” Amino acids activate both mTORC1 and C2 independent of insulin or growth factor signaling (IIS). Amino acids are also required for activation of mTORC1 in IIS dependent pathway.

Abstract P3-01-01: Targeting glutamine metabolism in breast cancer for therapy

Abstract Metabolic reprogramming of cancer cells is observed in different types of tumors including breast. Oncogenic signals aid changes in metabolism that provide selective advantage to the cancer cells to meet their energy requirements to accomplish rapid proliferation. The increased dependence on the glycolytic pathway for energy called the “Warburg effect” was reported by Otto Warburg several decades ago. Our recent understanding of cancer metabolism has thrown light on alternative energy sources, especially glutamine and other branched chain amino acids. The role of glutamine in breast cancer cell growth has not yet been studied extensively. In this study we found that a number of breast cancer cell lines, especially those negative for ER, PR, HER2, display a high dependence on glutamine for their survival and growth. Interestingly, most of these glutamine-dependent cell lines express high levels of c-myc protein. Consistent with their growth dependency on glutamine, transaminases responsible for entry of glutamine into the tricarboxylic acid cycle are transcriptionally up regulated under low glutamine conditions. Consequently, growth of these cancer cell lines was found to be specifically inhibited by the transaminase inhibitor, amino oxyacetate (AOA). Moreover, the AOA mediated cytotoxic effect was partially c-myc dependent. Through 1H-NMR studies of AOA-treated cells we show that in addition to glutamine, AOA treatment decreases the aspartate and alanine content in the cells. In line with these findings, exogenous supplementation with aspartate partially rescued the cells from the growth inhibitory effects of AOA. Flow cytometry analysis showed that AOA causes cell cycle arrest in the S phase. AOA also had significant inhibitory effect on in vivo growth of rapidly growing SUM 149 and SUM159 xenografts in immunodeficient mice. When combined with chemotherapeutic agents, doxorubicin and carboplatin, AOA inhibited growth of MDA-MB-231 xenograft tumors more effectively than AOA alone. Lastly we present evidence that the cytotoxic effect of AOA is mediated through activation of the ER stress pathway, combined with depletion of key amino acids and likely, a reduction in the nucleoside pool in the cells. Our preclinical studies, both in vitro and in vivo, combined with development of intermediate markers of response, provide a strong rationale for testing AOA for therapy in Phase 0 clinical trials. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-01.

Cerebral microdialysis reflects the neuroprotective effect of fractionated plasma separation and adsorption in acute liver failure better and earlier than intracranial pressure: a controlled study in pigs

BackgroundCerebral edema is a well-recognized and potentially fatal complication of acute liver failure (ALF). The effectiveness of treatments that address intracranial hypertension is generally assessed by measuring intracranial pressure (ICP). The aim of this study was to determine the role of cerebral microdialysis in monitoring the efficacy of fractionated plasma separation and adsorption (FPSA) treatment for ALF. We hypothesized that in ALF cerebral microdialysis reflects the benefits of FPSA treatment on cerebral edema before ICP.MethodsA surgical resection model of ALF was used in 21 pigs. We measured plasma ammonia concentration, brain concentrations of glucose, lactate, pyruvate, glutamate and glutamine, and ICP. Animals were randomized into three groups: in one group eight animals received 6 hours of FPSA treatment 2 hours after induction of ALF; in another group 10 animals received supportive treatment for ALF only; and in the final group three underwent sham surgery.ResultsThe ICP was significantly higher in the ALF group than in the FPSA group 9 hours after surgery. The lactate/pyruvate (L/P) ratio was significantly lower in the FPSA group than the ALF group 5 hours after surgery, before any significant difference in ICP was detected. Indeed, significant changes in the L/P ratio could be observed within 1 hour of treatment. Glutamine levels were significantly lower in the FPSA group than the ALF group between 6 hours and 10 hours after surgery.ConclusionsBrain lactate/pyruvate ratio and concentration of glutamine measured by cerebral microdialysis reflected the beneficial effects of FPSA treatment on cerebral metabolism more precisely and rapidly than ICP in pigs with fulminant ALF. The role of glutamine as a marker of the efficacy of FPSA treatment for ALF appears promising, but needs further evaluation.

The B55α Subunit of PP2A Drives a p53-Dependent Metabolic Adaptation to Glutamine Deprivation

Glutamine is an essential nutrient for cancer cell survival and proliferation, yet the signaling pathways that sense glutamine levels remain uncharacterized. Here, we report that the protein phosphatase 2A (PP2A)-associated protein, α4, plays a conserved role in glutamine sensing. α4 promotes assembly of an adaptive PP2A complex containing the B55α regulatory subunit via providing the catalytic subunit upon glutamine deprivation. Moreover, B55α is specifically induced upon glutamine deprivation in a ROS-dependent manner to activate p53 and promote cell survival. B55α activates p53 through direct interaction and dephosphorylation of EDD, a negative regulator of p53. Importantly, the B55α-EDD-p53 pathway is essential for cancer cell survival and tumor growth under low glutamine conditions in vitro and in vivo. This study delineates a previously unidentified signaling pathway that senses glutamine levels as well as provides important evidence that protein phosphatase complexes are actively involved in signal transduction.

Glutaminolysis Activates Rag-mTORC1 Signaling

Amino acids control cell growth via activation of the highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.

Abstract 5161: Role of glutamine in cancer growth, invasion and progression: Therapeutic modulation of glutamine pathway

Abstract Glutamine, an alternative carbon source to glucose, has recently been shown to be important for the cancer cell survival, growth and progression. The link between glycolysis, glutaminolysis and fatty acid oxidation is not known. Thus, there is a strong motivation to study metabolic alternations caused by glutamine and uncover links between various fuel sources for highly and less invasive cancer cells. Here, we postulate that glutamine will increase glutamine uptake through up-regulating ASCT2 expression, while decreasing the glucose uptake by down-regulating GLUT1 expression for aggressive cancer cells. However, for non-aggressive cancer cells, glutamine will have no effect on glutamine and glucose uptake. Our findings indicate that NAD+/NADH ratio regulates the glutamine's direct effect on the lactate production irrespective of the bioenergetic substrate requirement for different kinds of cancer cells. Our findings using quantification of metabolites pertaining to central carbon and nitrogen metabolism provided clues as to which pathways relevant to glycolysis, the pentose phosphate pathway, the TCA and urea cycles, lipid cycle, and amino acid and nucleotide metabolisms, may be altered by glutamine in cancer cells, resulting in identification of dominant factors which control cancer metabolism. Through this study, we clearly demonstrate the exact of glutamine's effect on cancer metabolism. Our results set the stage for the development of glutaminolysis inhibitors based therapeutics for reprogramming of cancer metabolism. We believe that development of our oncosecretomics based metabolic strategy provides an unprecedented opportunity for the development of nutritional supplementation that can deregulate the adaptations and alterations induced by various nutrients in cancer metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5161. doi:1538-7445.AM2012-5161

Abstract PL08-01: Clinical achievements reached by interfering with the IGF1R-PI3K-PTEN-AKT-mTOR axis.

Abstract PL08-01: Clinical achievements reached by interfering with the IGF1R-PI3K-PTEN-AKT-mTOR axis.