Gln Pool Research Articles

Glutamate cycle changes in the putamen of patients with de novo Parkinson's disease using 1H MRS

IntroductionTo investigate glutamatergic metabolism changes in the putamen of patients with de novo Parkinson's Disease (PD) and test the hypothesis that glutamate (Glu) levels are abnormally elevated in the putamen contralateral to where the motor clinical signs predominate as expected from observations in animal models. Methods1H NMR spectra from 17 healthy control volunteers were compared with spectra from 17 de novo PD patients of who 14 were evaluated again after 2–3 years of disease progression. Statistical analysis used random-effects models. ResultsThe only significant difference between PD patients and controls was a higher glutamine (Gln) concentration in the putamen ipsilateral to the hemibody with predominant motor signs (Visit 1: 6.0 ± 0.4 mM vs. 5.2 ± 0.2 mM, p < 0.05; Visit 2: 6.2 ± 0.3 mM vs. 5.2 ± 0.2 mM, p < 0.05). At Visit 1, PD patients had higher Glu and Gln levels in the putamen ipsilateral versus contralateral to dominant clinical signs (Glu: 12.2 ± 0.6 mM vs. 10.4 ± 0.6 mM, p < 0.05; Gln: 6.0 ± 0.4 mM vs. 4.8 ± 0.4 mM, p < 0.05; Glu and Gln pool (Glx): 17.9 ± 0.8 mM vs. 14.7 ± 1.1 mM, p < 0.05). At Visit 2, the sum of the two metabolites remained significantly higher in the ipsilateral versus contralateral putamen (Glx: 18.3 ± 0.6 mM vs. 16.1 ± 0.9 mM, p < 0.05). ConclusionIn de novo PD patients, the putamen ipsilateral to the more affected hemibody showed elevated Gln versus controls and elevated Glu and Gln concentrations versus the contralateral side. Abnormalities in Glu metabolism therefore occur early in PD but unexpectedly in the putamen contralateral to the more damaged hemisphere, suggesting they are not dependent solely on dopamine loss.

Protein scaffold optimizes arrangement of constituent enzymes in indigoidine synthetic pathway to improve the pigment production.

Indigoidine is a dark-blue natural pigment with application prospect and synthesized from glutamine (Gln) by series of indigoidine synthetases (IndCs). Indigoidine production can be improved by enhancing Gln pool via supplementing Gln directly or converting metabolism glutamate (Glu) to Gln by glutamine synthetase (GlnA). But, Gln is expensive, and excess Gln inhibits indigoidine production of the recombinant strain. Supplementing Glu instead of Gln may improve the productive and economic efficiency of indigoidine, but the local activities and positions of the indigoidine pathway enzymes GlnA, Sc-IndC, and the helper protein of Sc-IndC (IndB) should be well arranged. We identified the Streptomyces chromofuscus ATCC 49982 derived IndC (Sc-IndC) as an more efficient IndC compared to other IndCs applied for constructing indigoidine-producting strains, and designed series of protein scaffold complexes with architectures of PDZ, SH3, and GBD domains (PxSyG1) to arrange the pathway enzymes. The strain recruiting GlnA, Sc-IndC, and IndB on the PDZ, SH3, and GBD domains of scaffold P1S2G1, respectively, was the most efficient. In the strain, the GlnA supplied sufficient local Gln for Sc-IndC from Glu, and the generated Gln was immediately consumed by Sc-IndC to relieve cell growth inhibition caused by Gln. The optimum Glu concentration (6g/L) for the strain was higher than those of the strains recruiting Sc-IndC on the GBD domain, which was away from the PDZ domain recruiting GlnA. The highest titer of indigoidine was 12g/L, which was two folds of the control without scaffold (5.8g/L). The titer is 5g/L higher than the control without Glu supplemented (6.9g/L), meaning that 97% of the supplemented Glu was transformed into indigoidine. The batch fermentation with the optimum strain in a 5-L reactor achieved an indigoidine titer of 14g/L in 60h. To our knowledge, this was the most efficient indigoidine productivity achieved so far. The optimization strategies by protein scaffold should be applicative to other pathways with complex substrate demands. KEY POINTS: •Protein scaffold systems were designed to arrange the indigoidine synthetic pathway. •The scaffold system improved supplement of Gln for indigoidine production from Glu. •The inhibition caused by excess Gln was relieved by proper designed scaffold. •The yield and titer of indigoidine was improved by arranging the pathway enzymes. Graphical abstract.

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.

Abstract 1250: Selection of rare and novel breast cancer cell variants based on their plasticity of glutamine metabolism

Abstract Breast cancer is a heterogeneous disease and only rare cancer cells succeed in metastasizing. The current approach of classifying subpopulations of cancer cells based on certain biomarkers derived from the bulk of primary tumor is unsatisfactory for identifying the rare cells that cause metastasis. One attribute of metastatic cells is their ability to stop proliferating and survive long-term under unfavorable conditions. While the majority of cells present in metastatic breast cancer cell lines are predicted to be dependent on Gln and thus will die upon Gln withdrawal, we hypothesized that some rare cells would survive and grow without Gln, thus suggesting a simple robust function-based selection method for metabolically plastic cancer cells. We analyzed several aggressive breast cancer cell lines including SUM149, SUM149-FP76 (variant cells cultured from a fat pad tumor in nude mice), SUM190, MDA-MB-231, MDA-MB-231-BSC60 (a metastatic clone), 168FARN, 4T07, and 4T1. Gln-independent variants were selected by growing cells in a medium without Gln. We evaluated protein level of glutaminase (GLS) that correlates with Gln addiction in cancer cells, Myc that drives Gln catabolism, and COX-2 that is involved in metastasis, by western blotting. We evaluated the sensitivity of cell lines to chemotherapeutic drugs, doxorubicin and paclitaxel, by a cell proliferation assay. We utilized the two-sample t-test for statistical analysis. We were able to select rare variants (one in 35,000 cancer cells) from the SUM149-Luc cell line that grow long-term in Gln-free medium. We found that Gln-independent variants produced a significantly lower level of GLS (1/5th) and a significantly higher level (5-fold) of COX-2 than did the parental cell line. The Gln-ind variants produced a high level of Myc protein similar to the Gln-addicted bulk population of cells. The Gln-ind phenotype in the rare variants is stable since exposure to Gln for multiple (&amp;gt; 10) passages did not lead to Gln addiction, although they were able to utilize Gln when available. Of interest, the Gln-ind variants were significantly more resistant to doxorubicin and paclitaxel than the parental cell lines, requiring approximately 10X drug concentrations for inhibiting the Gln-ind variants. The Gln-ind variants are unique in another significant manner- the variants, but not the parental SUM149 cell line, yielded colonies in a chemically defined medium that is devoid of serum. We were successful in isolating rare metabolically plastic Gln-ind variants from all the cell lines mentioned above. Our results revealed a link between metabolic plasticity (i.e., decreased GLS to maintain a sufficient Gln pool for cell growth) and metastatic ability (via increased COX-2 level) that could be exploited for selecting novel subpopulations of cancer cells and for targeted therapy development against these cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1250. doi:10.1158/1538-7445.AM2011-1250

Abstract P4-03-06: Selection of Rare and Novel Breast Cancer Cell Variants Based on Glutamine Metabolism

Abstract Background: Breast cancer is a heterogeneous disease composed of tumor cells that have different characteristics. Although there are several different ways to explain cellular heterogeneity, it is clear that only rare cancer cells out of the overall population succeed in metastasizing. The current approach of classifying subpopulations of cancer cells based on certain biomarkers derived from the bulk of primary tumor is unsatisfactory for identifying the rare cells that cause metastasis. The purpose of this study was to test a novel function-based, rather than biomarker-based, selection strategy using glutamine (Gln) metabolism (or Gln-dependence to be more specific) since it is a prominent feature of cancer cells. While the majority of cells present in metastatic breast cancer cell lines are predicted to be dependent on Gln and thus will die upon Gln withdrawal, we hypothesized that some rare cells would survive and grow without Gln as a nutrient, thus suggesting a simple robust selection method for “tumorigenic” cancer cells. Methods: We chose an aggressive inflammatory breast cancer cell line SUM149 and SUM149-FP76 cell line that was recently derived by “passaging” SUM149-Luc cells (luciferase transfected for imaging in mice) in nude mice in our laboratory, first selecting for growth subcutaneously and then in inguinal fat pad. Gln-independent variants were selected by growing cells in a medium without Gln, supplemented with dialyzed fetal bovine serum. We evaluated protein level of glutaminase (GLS), an enzyme that converts glutamine to glutamate and usually correlates with Gln addiction in cancer cells, by western blotting. Expression of COX-2, an enzyme associated with inflammation, metastasis, and with cellular hierarchy, was measured by western blotting and compared between parental and Gln-independent lines. Tests were repeated in triplicate and evaluated using paired T-test. Results: We were able to select rare variants (one in 35,000 cancer cells) from the SUM149 inflammatory breast cancer cell line that grow long-term in Gln-free medium. We found that Gln-independent variants produced a significantly lower level of GLS (1/5th) and a significantly higher level (5-fold) of COX-2 than did the parental cell line. To provide further support for a link between Gln metabolism and tumorigenesis, we were also able to select the rare Gln-independent variants from the SUM149-FP76 cell line. Further experiments involving siRNA-mediated COX-2 knockdown indicated that the high level of COX-2 protein present in Gln-independent variants was not a direct regulator of the reduced GLS level, but rather COX-2 may offer indirect selective advantage by influencing processes such as apoptosis and “developmental hierarchy”. Conclusions: Since COX-2 is very important in breast cancermetastasis, our results suggest the possibility of a strong link between a critical metabolic adaptation (decreased GLS to maintain a sufficient Gln pool) that may be essential for cell survival and growth, and metastatic ability (via increased COX-2) in cancer cells. Our study also revealed a deep connection between glutamine metabolism and metastasis that could be exploited for developing novel models of cancer cell function and for targeted therapy development against these cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-03-06.

Fasting up-regulates muscle glutamine synthetase while it down-regulates liver glutamine synthetase in male rats during aging

Summary Background & aims Muscle is an important site of Gln synthesis and release in the whole body, while liver can take up circulating Gln even if it has the capacity to both degrade and synthesize Gln. During fasting, skeletal muscle in rats exports large amounts of Gln but also increases Gln production by glutamine synthetase (GS) to maintain its Gln pool, whatever the age. The aim of this study was to determine whether fasting induced GS activity in muscle may have consequences on liver Gln homeostasis during aging (by changing GS and glutaminase (GA) activities). Methods Liver GS and GA activities were assessed in fed and 5 day fasted male Wistar rats during aging (2, 5, 15, 22 and 24 months). To compare potential cooperation between liver and muscle, GS activity was also measured in skeletal muscle. Results After 5 days fasting, both total GS and GA activities in liver were decreased irrespective of age. As expected, muscle GS activity was increased by a factor of 1.5–2 in 2 to 24 month rats. In addition, 24 month fed and fasted rats had the same GS response. In contrast, in the fed state, both age and nutritional state played a role in the response of GA activity. Conclusion Five day fasting induced an adaptation of Gln metabolism in male Wistar rats, in relation to aging. Muscle and liver synergistically played a role in this regulation. The specific role of Gln in satisfying increased requirements in very old rats was confirmed.

Glutamine-supplemented total parenteral nutrition attenuates plasma interleukin-6 in surgical patients with lower disease severity

Previous reports have shown that decrease in plasma glutamine (Gln) level following major surgery may contribute to the state of immunosuppression. Gln supplementation improves the depletion of body Gln pool, and may have indirect effect on reducing proinflammatory mediator release. This study evaluated whether the effect of Gln dipeptide-enriched total parenteral nutrition (TPN) on postoperative cytokine alteration depended on the disease severity of surgical patients. Forty-eight patients with major abdominal surgery were allocated to two groups to receive isonitrogenous (0.228 g nitrogen/kg per d) and isocaloric (30 kcal/kg per d) TPN for 6 d. Control group (Conv) using conventional TPN solution received 1.5 g amino acids/kg per day, whereas the test group received 0.972 g amino acids/kg per day and 0.417 g L-alanyl-L-glutamine (Ala-Gln)/kg per day. Blood samples were collected on d 1 and d 6 postoperatively for plasma interleukin (IL)-2, IL-6, IL-8, and interferon (IFN)-gamma analysis. Plasma IL-2 and IFN-gamma were not detectable. IL-6 concentrations were significantly lower on the 6(th) postoperative day in the Ala-Gln group than those in the Conv group in patients with APACHE II <=6, whereas no difference was noted in patients with APACHE II >6. There was no difference in IL-8 levels between the two groups. No difference in cumulative nitrogen balance was observed on d 2-5 after the operation between the two groups (Ala-Gln -3.2+/-1.6 g vs Conv -6.5+/-2.7 g). A significant inverse correlation was noted between plasma IL-6 levels and cumulative nitrogen balance postoperatively in the Ala-Gln group, whereas no such correlation was observed in the Conv group. TPN supplemented with Gln dipeptide had no effect on plasma IL-8 levels after surgery. However, Gln supplementation had a beneficial effect on decreasing systemic IL-6 production after surgery in patients with low admission illness severity, and lower plasma IL-6 may improve nitrogen balance in patients with abdominal surgery when Gln was administered.

The role of adrenal hormones in the response of glutamine synthetase to fasting in adult and old rats

Background & Aims: During fasting, skeletal muscle exports increased amounts of glutamine (Gln) while increasing the production of this amino acid by glutamine synthetase (GS) in order to maintain the intramuscular Gln pool. Glucocorticoid hormones are believed to be the principal mediators of GS induction during stress conditions. The aim of this study was to evaluate (1) the effect of fasting on GS activity and expression in skeletal muscle during aging and consequently, (2) the role of glucocorticoids in fasting-induced GS activity. Methods: Male Wistar rats (6-, 22-month old) were fasted for 5 days and both the activity and expression of GS were measured in tibialis anterior muscle. To better demonstrate the role of glucocorticoids in the response of GS to fasting, we suppressed their action by RU38486 administration (a potent glucocorticoid antagonist) and their production by adrenalectomy in fed and fasted rats. Results: An increase in fasting-induced GS activity was observed in skeletal muscles from both adult and aged rats. Adrenalectomy, but surprisingly not RU38486, suppressed the fasting-induced increase in GS activity and expression. Conclusion: The data clearly show that the GS responsiveness to fasting was not modified by aging in skeletal muscle.

The effect of glutamine-supplemented total parenteral nutrition on nitrogen economy depends on severity of diseases in surgical patients

Background: Gln is an important substrate for enterocyte and rapid proliferation cells. Studies have shown that parenteral supplementation of Gln maintains the intracellular Gln pool, improves nitrogen balance and shortens hospital stay. However, some studies showed Gln-supplemented TPN had no effect on restoring the Gln pool in critically ill patients. Objective: To evaluate the effect of glutamine (Gln) dipeptide supplementation of total parenteral nutrition (TPN) on postoperative nitrogen balance and immune response of patients undergoing surgery. Methods: This study is a prospective, randomized double-blind clinical trial. APACHE II score and TISS were used to evaluate the patients after admission. Forty-eight patients with major abdominal surgery were allocated to two groups to receive isonitrogenous (0.228 g nitrogen/kg/day) and isoenergetic (30 kcal/kg/day) TPN for 6 days. Two groups (Conv and Ala-Gln) were further divided to high (APACHE⩾6) and low (APACHE <6) groups. Control group (Conv) received 1.5 g amino acids/kg/day, whereas the Ala-Gln group received 0.972 g amino acids/kg/day and 0.417 g of L -alanyl- L -glutamine (Ala-Gln)/kg/day. Blood samples were collected on day 1 and day 6 after surgery for plasma amino acid and CD4, CD8 cell and T lymphocyte analysis. Cumulative nitrogen balance were also measured on day 2, 3, 4, 5 postoperatively. Results: Although there was a tendency to have better cumulative nitrogen balance on the postoperative days in the Ala-Gln group, no significant difference was observed between two groups. However, a better significant cumulative nitrogen balance was observed on the 2nd, 3rd and 5th postoperative day in the Ala-Gln group than in the Conv group in patients with APACHE II <6, whereas no significant difference was noted in patients with APACHE II ⩾ 6. No difference in urine 3-methylhistidine excretion were observed between the 2 groups. Patients in the Ala-Gln group had significant higher T lymphocyte and CD4 cells than did those in the Conv group.Conclusion: TPN supplemented with Gln dipeptide had beneficial effect on enhancing the immune response. However, the effect of Ala-Gln administration on improving nitrogen economy was only observed in patients with low APACHE II scores. These results may indicate that Gln required for reversing the catabolic condition may depend on the characteristics and severity of the diseases.

An in vivo N NMR study of agropine synthesis in transformed root cultures of Nicotiana tabacum

The biosynthesis of the opine agropine in transformed Nicotiana tabacum L. root cultures was studied using in vivo N nuclear magnetic resonance (NMR) spectroscopy. Roots were incubated with [ N]ammonium and/or [ N]nitrate, and the incorporation of the label into agropine, conjugated polyamines, Γ‐aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln) and nicotine was monitored by NMR. The largest labelled pool was agropine in cells grown on a N‐enriched medium and its synthesis was maintained, to the detriment of the Gln pool, under conditions of nitrogen (N) starvation. These observations indicate that the synthesis of agropine is not tightly regulated and that it represents a significant sink for carbon (C) and N in the plant. The addition of α‐naphthaleneacetic acid (NAA) and kinetin to the growth medium caused de‐differentiation of the root cultures and perturbation of secondary N metabolism. The amount of agropine relative to Gln increased but the total accumulation of agropine decreased, in part because of the increase in the synthesis of conjugated polyamines. These results show that agropine biosynthesis perturbs both primary and secondary N metabolism, and that the perturbation differs according to the culture conditions and the imposed stress.

Glutamine synthetase expression in muscle is regulated by transcriptional and posttranscriptional mechanisms.

Skeletal muscle exports glutamine (Gln) and increases the expression of the enzyme glutamine synthetase (GS) in response to physiological stress. Acute stress or direct glucocorticoid administration raises muscle GS mRNA levels dramatically without a parallel increase in GS protein levels. In the lung, this discrepancy is caused by feedback destabilization of the GS protein by its product Gln. It was hypothesized that muscle GS protein levels increase during stress only when the intracellular Gln pool has been depleted. Adult male rats were injected with the glucocorticoid hormone dexamethasone (DEX) to mimic the acute stress response and with the GS inhibitor methionine sulfoximine (MSO) to deplete muscle Gln stores. DEX increased GS mRNA levels by 2.8-fold but increased GS protein levels by an average of only 40%. MSO diminished muscle GLN levels by 68% and caused GS protein levels to rise in accordance with GS mRNA. Chronic stress was mimicked using 6 days of MSO treatment, which produced anorexia, 23% loss of body weight, and 64% decrease in muscle Gln levels, as well as pronounced increases in both muscle GS mRNA (26-fold) and protein levels (35-fold) without elevation of plasma glucocorticoid levels. Calorie-restricted pair-fed animals exhibited lesser increases in muscle GS mRNA (8-fold) and protein levels (5-fold) without a decline in muscle Gln content. Thus regulation of GS expression in both acute and chronic stress involved both transcriptional and posttranscriptional mechanisms, perhaps affected by muscle Gln content.

Kinetic impairment of nitrogen and muscle glutamine metabolisms in old glucocorticoid-treated rats.

Aged rats are more sensitive to injury, possibly through an impairment of nitrogen and glutamine (Gln) metabolisms mediated by glucocorticoids. We studied the metabolic kinetic response of adult and old rats during glucocorticoid treatment. The male Sprague-Dawley rats were 24 or 3 mo old. Both adult and old rats were divided into 7 groups. Groups labeled G3, G5, and G7 received, by intraperitoneal injection, 1.50 mg/kg of dexamethasone (Dex) for 3, 5, and 7 days, respectively. Groups labeled G3PF, G5PF, and G7PF were pair fed to the G3, G5, or G7 groups and were injected with an isovolumic solution of NaCl. One control group comprised healthy rats fed ad libitum. The response to aggression induced specifically by Dex (i.e., allowing for variations in pair-fed controls) appeared later in the aged rats (decrease in nitrogen balance from day 1 in adults but only from day 4 in old rats). The adult rats rapidly adapted to Dex treatment, whereas the catabolic state worsened until the end of treatment in the old rats. Gln homeostasis was not maintained in the aged rats; despite an early increase in muscular Gln synthetase activity, the Gln pool was depleted. These results suggest a kinetic impairment of both nitrogen and muscle Gln metabolisms in response to Dex with aging.