Targeting Glutamine Synthesis Inhibits Stem Cell Adipogenesis in Vitro
Ksenija Velickovica Hilda Anaid Lugo Leijaa Amal Surratia Dong-Hyun Kimb
Harold Sacksc Michael E. Symondsd,e Virginie Sottilea,f
aSchool of
Medicine, The University of Nottingham, Nottingham, UK, bCentre for Analytical
Bioscience, Advanced Materials and Healthcare Technologies Division, School of
Pharmacy, The University of Nottingham, Nottingham, UK, cVA Endocrinology and
Diabetes Division, Department of Medicine, University of California, Los
Angeles, CA, USA, dThe Early Life Research Unit, Division of Child
Health, Obstetrics and Gynaecology, The University of Nottingham, Nottingham, UK, eNottingham
Digestive Disease Centre and Biomedical Research Centre, School of Medicine,
The University of Nottingham, Nottingham, UK, fDepartment of Molecular Medicine,
The University of Pavia, Pavia, Italy
Key Words
Glutamine • Adipogenesis • Lipogenesis • Methionine sulfoximine
Abstract
Background/Aims: Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on the differentiation of adipocytes and lipid droplet accumulation. Methods: Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine – MSO) during differentiation and their response was compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). Results: Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration,and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. Conclusion: These results suggest that the glutamine pathway may have a previously over-looked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity.
Introduction
Glutamine is the most abundant extracellular amino acid in the body, that is primarily derived from skeletal muscle, from where it is transported to target tissues [1, 2]. The importance of glutamine for cell proliferation, survival and immune response is well recognized, especially in rapidly dividing cells [3-6]. Once inside the cell, glutamine can be converted into glutamate by the glutaminase enzyme and enter the tricarboxylic acid (TCA) cycle to maintain oxidative phosphorylation, where in addition to glucose, it is an important carbon source for de novo lipogenesis [7-9]. This process involves reverse transfer through the TCA cycle and reductive carboxylation by isocitrate dehydrogenases (IDHs) to generate acetyl-CoA [10]. In addition to being an important precursor for proteins, amino sugars, purines and pyrimidines, glutamine can also regulate gene expression for some transcriptional factors, enzymes, channels and receptors [11-13]. Furthermore, low glutamine availability has been reported to increase the expression of pro-inflammatory genes in white adipose tissue [14], while a number of benefits have been reported in human clinical studies with supplemental glutamine [2], particularly on weight loss [15] and insulin plasma concentration [16] in obese people and for the maintenance for optimal health [2].
A primary function of adipose tissue is to store excess energy in the form of lipids [17]. There are three main types of adipocytes, white, brown and beige [18], which despite their different morphological and functional characteristics, are all mesenchymal in origin with lipid droplet(s) formation, a hallmark of adipogenesis [19, 20]. Unlike white fat, brown and beige adipocytes can dissipate energy in the form of heat through a unique property of the mitochondrial uncoupling protein 1 (UCP1) which uncouples respiration from ATP synthesis [21]. The metabolic requirements of adipocytes for glutamine are mainly dependent on glutamine synthetase (GS), a mitochondrial enzyme that catalyses the reaction between ammonia and glutamate [22, 23]. GS activity increases during adipocyte differentiation [24, 25], pointing its possible role in adipocyte maturation, although effects of manipulating glutamine availability during adipogenesis are not known. The present study therefore aimed to examine whether glutamine has a regulatory role in adipose tissue development by examining the impact of glutamine deprivation or supplementation in the adipogenic differentiation of mesenchymal stem cells (MSCs), an established model of adipogenesis [26, 27].
Materials and Methods
Mouse MSCs characterised as beige cells [26, 27] were grown and maintained routinely in a standard (ST) medium containing low glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 1% non-essential amino acids and 5 mM glutamine. When cells reached confluency, adipogenic differentiation was started using adipogenic (AD) medium. For this purpose, glutamine-free standard medium was used, adding 1 μM dexamethasone (Cayman Chemicals, USA), 100 µM isobutylmethylxanthine (IBMX) (Sigma-Aldrich, UK), 1 μM rosiglitazone (Cayman Chemicals, USA), 10 μg/ml insulin (Sigma-Aldrich, UK), and 1 nM triiodothyronine (T3) (Sigma-Aldrich, UK). All supplements were prepared according to the manufacturer’s guidelines. Adipogenic cultures were divided in two groups (Supplementary Fig. 1 – for all supplementary material see www.cellphysiolbiochem.com): either treated with L-methionine sulfoximine (MSO, 3 mM) (Acros Organics, UK), an irreversible glutamine synthetase inhibitor [28-31], or with low (5 mM) medium (10 mM) and high (20 mM) glutamine supplementation. Parallel MSCs kept in ST medium were used as undifferentiated controls (Ctrl). The analyses performed in this study were performed on 3 batches of cells (each one done with three technical replicates), cells were treated for 7 days prior analysis.
Cell Viability Assay and Oil Red O staining
Both cell viability and the presence of lipid droplets were determined by Presto Blue Viability assay and Oil Red O staining (ORO), respectively, as previously described [26, 27].
Intracellular glutamine concentration
Gene expression analysis
Extraction of total RNA and quantitative PCR were performed as previously described [26]. Gene expression was determined using the GeNorm normalization algorithm against two selected reference genes (stability value M = 0.46), TATA sequence binding protein (TBP) and acidic ribosomal protein subunit P0 (RPLP0) using GeNorm software (version 3.5; Primer Design Ltd). The following murine-specific oligonucleotide primers (Eurofins) were used. For Adiponectin: forward 5’-AACTTGTGCAGGTTGGATGGC-3’ and reverse 5’-TTCTCTCCCTTCTCTCCAGGA-3’. For FABP4: forward 5’-TGGAAGCTTGTCTCCAGTGA-3’ and reverse 5’-AATCCCCATTTACGCTGATG-3’. For PPARγ: forward 5’-GCCGAGTCTGTGGGGATAAA-3’ and reverse 5’-GTGAGACATCCCCACAGCAA-3’. For TBP: forward 5’-CCTTGTACCCTTCACCAATGAC-3’ and reverse 5’-ACAGCCAAGATTCACGGTAGA-3’. For RPLP0: forward 5’-CTGGAGAAACTGCTGCCTCA-3’ and reverse 5’-AGTGTTCTGAGCTGGCACA-3’. For IDH1: forward 5’-ATGCAAGGAGATGAAATGACACG-3’ and reverse 5’-GCATCACGATTCTCTATGCCTAA-3’. For IDH2: forward 5’-ATCAAGGAGAAGCTCATCCTGC-3’ and reverse 5’-TCTGTGGCCTTGTACTGGTCG-3’. For IDH3: forward 5’-CCTCCTGCTTAGTGCTGTGA-3’ and reverse 5’-CGTTGCCTCCCAGATCTTT-3’.
Extracellular glutamine extraction and LC-MS analysis
Extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). For this purpose, MSCs were cultured in ST medium, while adipogenic cells were differentiated in AD medium supplemented with 5 mM glutamine over 10 days. Medium samples from untreated, MSCs control (ST medium), AD-treated cultures, and AD blank media (no cells) were collected at day 0, 5 and 10 of treatment. Glutamine was extracted from 250 mL of each culture medium sample by adding 750 mL of cold methanol in a ratio of 1 : 4 for protein precipitation. Samples were then mixed vigorously and incubated at -20°C for 20 min. After the incubation period, samples were centrifuged at 13,200 g at 4°C for 10 min and transferred to pre-cooled tubes at 4°C and then stored at -80°C prior to LC-MS analysis. The relative quantification of glutamine was performed on LC-MS as previously described [35]. Briefly, extracts of spent culture media were analysed on an Accela system coupled to an Exactive MS (Thermo Fisher Scientific, Hemel Hempstead, UK). Spectral data was acquired in full scan ion mode (m/z 70-1400, resolution 50 000) and chromatographic separation was achieved using a ZIC-pHILIC (150 mm x 4.6 mm, 5 mm column, Merck Sequant) maintained at 45°C with a flow rate of 0.3 ml/min. Datasets were then processed with XCMS for untargeted peaks and analysed using mzMatch [36, 37]. Identification of glutamine was based on matching the accurate mass and retention time of the detected glutamine peak with those of the authentic standard of glutamine which was co-analysed with the samples under identical experimental conditions. Univariate one-way ANOVA testing was performed to determine significant compounds changes in the level of glutamine consumed and excreted by MSCs (both in untreated and AD treatment) compared to their medium-only blanks, over time.
All datasets were analysed by one-way analysis of variance (ANOVA) followed by Tukey post hoc test, unless otherwise stated. Statistical significance was accepted at p < 0.05, with *p < 0.05; **p < 0.01; ***p < 0.001. Error bars plotted on graphs represented the mean ± SEM. Data were analysed using Graph Pad Prism Software (https://www.graphpad.com).
Results
The effect of glucose and/or glutamine restriction on adipocyte differentiation
Before analysing the effect of glutamine supplementation, it was necessary to determine whether glucose, as a source of carbon atoms, might compensate for glutamine during adipogenesis [38] and to examine possible effects of different glucose concentrations on differentiation. To this end, the lipid content was measured in cultures differentiated under 3 medium conditions: (i) glucose-free medium, (ii) low glucose (5.5 mM) medium, and (iii) high glucose (25 mM) medium (Fig. 1). None of the conditions triggered lipid accumulation in the absence of adipogenic supplements on the control medium. In glucose-free AD medium, MSCs were unable to differentiate into adipocytes and accumulate lipids, indicating glucose was required for adipogenic differentiation, even when glutamine was added. In the presence of glucose, at both low and high concentrations, AD medium containing glutamine did support differentiation as seen by the significant increase in lipid content compared to the undifferentiated group, confirming adipocyte differentiation. However, when glutamine synthesis was blocked using MSO treatment, cells were not able to differentiate in response to AD medium even in the presence of glucose supplementation at low or high concentration, showing that adipogenic differentiation is glutamine-dependent.
Effects of glutamine availability on cell viability and adipogenic differentiation
Measurement of the intracellular glutamine concentration (Fig. 2) showed a significant increase in conditions where cells were exposed to medium with glutamine supplementation, while it remained close to undetectable with MSO exposure. Cell viability was not significantly altered in any of the treatment groups (Supplementary Fig. 2), however ORO staining of accumulated lipids confirmed the presence of numerous differentiated cells with lipid droplets in glutamine-treated groups, in a dose dependent manner, which was blocked with MSO treatment (Fig. 3).
Glutamine concentration affects gene expression of common adipogenic markers and isocitrate dehydrogenase enzymes
Gene expression of the adipogenic markers PPARγ, adiponectin and FABP4 was decreased with exposure to MSO, whereas glutamine supplementation increased gene expression of PPARγ and adiponectin, while FABP4 was unchanged (Fig. 4A). Exposure to MSO decreased gene expression of IDH1 and IDH3, but not IDH2 (Fig. 4B). Glutamine supplementation to 20 mM (high) had no significant effect on IDH1-3 expression levels.
Changes in glutamine concentration in differentiating culture
The level of extracellular glutamine concentration was measured over 10 days culture in order to evaluate glutamine uptake from the medium containing 5 mM glutamine (Fig. 5). The level of glutamine detected in medium for control cultures (MSCs) was observed to decline over time in undifferentiated cultures, however this decrease was significantly attenuated when cells were treated in AD conditions.
Discussion
The differentiation of MSCs into mature adipocytes is a complex process accompanied by diverse morphological changes, with lipogenesis providing the primary structural lipid component [20, 39]. The recognition that glutamine contributes carbon to lipogenic acetyl-CoA [8, 40-42] raises the question of its importance in adipogenic development.
The present study provides new insights on the role of glutamine in adipogenic differentiation of a mesenchymal progenitor cell line. Glutamine deprivation mediated by the irreversible GS inhibitor MSO [30, 31] was observed to prevent adipogenic differentiation, whereas the presence of exogenous glutamine had the opposite effect and increased lipid content. Glucose restriction abolished adipogenesis in a process that could not be compensated by glutamine supplementation to provide acetyl-CoA for lipogenesis. Furthermore, glucose supplementation could not compensate for the glutamine deprivation by MSO treatment, as differentiation was also suppressed, confirming the necessity for both glucose and glutamine in lipogenesis. These findings are thus in accord with Yoo et al, who highlighted distinct roles for glucose and glutamine in lipid synthesis during differentiation in brown adipocyte cultures [38]. They reported that although glucose, rather than glutamine, was the sole carbon source of the glycerol backbone used for the de novo lipogenesis, the contribution of glutamine to lipogenic acetyl-CoA was unexpectedly greater than that of glucose [38]. As shown here, glutamine is necessary for AD differentiation and lipogenesis, with lipid accumulation showing a dose-dependent increase. This was accompanied by increased gene expression of common adipogenic markers PPARγ and AdipoQ, suggesting a regulatory role of glutamine in beige adipocyte differentiation. Further experiments looking at different adipogenic subtypes could determine whether the role of glutamine differs in brown and white lineages. Glutamine could therefore promote PPARγ transcription in adipocytes as shown in intestinal epithelial cells [11, 43]. Since PPARγ and AdipoQ are not only involved in differentiation [20] but also in glucose and lipid metabolism [44, 45], mitochondriogenesis [46] and thermogenic function [47], glutamine may also affect other aspects of adipocyte function. The study of glutaminase knockout/inhibitor models could provide additional information on the contribution of glutamine to adipogenesis, in light of recent studies showing a role for glutaminase in skeletal stem cell differentiation to the adipogenic lineage [48].
The importance of glutamine in AD differentiation was further supported by measurements of both intracellular and extracellular glutamine concentrations. A decrease in extracellular levels as differentiation progresses suggests adipogenic cells could take up extracellular glutamine during adipogenesis via glutamine transporters as identified in rodent and human adipocytes [49, 50]. Furthermore, based on current literature, it seems that the effects of MSO on intra- and extracellular amino acids/metabolites might differ depending on the cell and/or organ type [31, 51-54], which should be considered in future studies.
Although all three IDH isoforms catalyse the same essential conversion of isocitrate to α-ketoglutarate [55], each has a unique role in maintaining cellular redox and energetic status [56]. Our results indicate that IDH1 may play a central role in lipid metabolism, extending earlier studies showing both IDH1 and IDH2 contribute to lipid synthesis [7, 57]. IDH1 was found to be the dominant regulator in lipid synthesis in both non-malignant and malignant cells [58-60]. In contrast, IDH3 is a mitochondrial enzyme that plays a central role in the TCA cycle, promoting oxidative phosphorylation to generate ATP [55]. Cells with IDH3 mutations exhibit reduced maximal and reserve capacity [61], which are direct measures of mitochondrial function and ability to adapt to respiratory stress [61, 62]. Based on the present IDH3 gene expression results, we hypothesise that glutamine deprivation has adverse effects on mitochondrial function, while the upregulation of both IDH1 and IDH3 gene expression may be indicative of glutamine supporting both lipogenesis and ATP production during AD differentiation. Further experiments are now required to determine the contribution of glutamine to both pathways and other aspects of adipogenesis. Analysis of intra- and extracellular metabolites, in particular glutamate, α-ketoglutarate and isocitrate could provide useful information on the link between glutamine and the processes of energy production and lipid synthesis during adipogenesis.
Conclusion
In summary, these latest results provide first evidence that glutamine restriction abolishes adipocyte differentiation in vitro and is thus essential for this process. Future studies will examine whether glutamine can contribute to uncoupled respiration and could evaluate whether manipulating glutamine metabolism may represent a possible therapeutic strategy to reduce obesity.
Acknowledgements
VS thanks E. Gherardi and members of the DMM (University of Pavia) for their support and useful discussions.
Author contributions
KV, HALL, MS and VS designed the experiments. KV and HALL carried out cellular and molecular analyses. AS and DHK performed the experimental study on extracellular metabolite detection. KV, HALL, MS and VS finalized the manuscript with input from HS, AS and DHK. All authors contributed to the scientific review and discussion of the study.
Funding
This work was funded by the EU-CASCADE fellowship scheme funded by the EU’s 7th FP PCOFUND-GA-2012-600181 (KV), by CONACyT Mexico (HALL), and received support from The Cardiometabolic Disease Research Foundation (Los Angeles, USA). Part of the methods utilised were developed through work that was supported by the BBSRC Doctoral Training Partnership [grant number BB/F017014/1]. VS is supported by a grant from the Italian Ministry of Education, University and Research (MIUR) to the Department of Molecular Medicine of the University of Pavia under the initiative “Dipartimenti di Eccellenza (2018–2022)”.
Disclosure Statement
The authors have no conflicts of interest to declare.
References
|
1
Newsholme P, Procopio J, Lima MM, Pithon-Curi TC, Curi R: Glutamine and
glutamate--their central role in cell metabolism and function. Cell Biochem
Funct 2003;21:1-9. |
|
|
|
|
|
2 Watford M: Glutamine metabolism and function
in relation to proline synthesis and the safety of glutamine and proline
supplementation. J Nutr 2008;138:2003S-2007S. |
|
|
|
|
|
3 Curi R, Lagranha CJ, Doi SQ, Sellitti DF,
Procopio J, Pithon-Curi TC, et al.: Molecular mechanisms of glutamine action.
J Cell Physiol 2005;204:392-401. |
|
|
|
|
|
4 Ardawi MS, Newsholme EA: Maximum activities of
some enzymes of glycolysis, the tricarboxylic acid cycle and ketone-body and
glutamine utilization pathways in lymphocytes of the rat. Biochem J
1982;208:743-748. |
|
|
|
|
|
5 Newsholme P, Curi R, Gordon S, Newsholme EA:
Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by
murine macrophages. Biochem J 1986;239:121-125. |
|
|
|
|
|
6 Vander Heiden MG, Cantley LC, Thompson CB:
Understanding the Warburg effect: the metabolic requirements of cell
proliferation. Science 2009;324:1029-1033. |
|
|
|
|
|
7 Wise DR, Thompson CB: Glutamine addiction: a
new therapeutic target in cancer. Trends Biochem Sci 2010;35:427-433. |
|
|
|
|
|
8 Lopez-Soriano FJ, Alemany M: Effect of
cold-temperature exposure and acclimation on amino acid pool changes and enzyme
activities of rat brown adipose tissue. Biochim Biophys Acta
1987;925:265-271. |
|
|
|
|
|
9 Green CR, Wallace M, Divakaruni AS, Phillips SA,
Murphy AN, Ciaraldi TP, et al.: Branched-chain amino acid catabolism fuels
adipocyte differentiation and lipogenesis. Nat Chem Biol 2016;12:15-21. |
|
|
|
|
|
10 Dalziel K, Londesborough JC: The mechanisms
of reductive carboxylation reactions. Carbon dioxide or bicarbonate as
substrate of nicotinamide-adenine dinucleotide phosphate-linked isocitrate
dehydrogenase and malic enzyme. Biochem J 1968;110:223-230. |
|
|
|
|
|
11 Ban K, Sprunt JM, Martin S, Yang P, Kozar RA:
Glutamine activates peroxisome proliferator-activated receptor-gamma in
intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism.
Am J Physiol Gastrointest Liver Physiol 2011;301:G547-554. |
|
|
|
|
|
12 Brasse-Lagnel C, Fairand A, Lavoinne A,
Husson A: Glutamine stimulates argininosuccinate synthetase gene expression
through cytosolic O-glycosylation of Sp1 in Caco-2 cells. J Biol Chem
2003;278:52504-52510. |
|
|
|
|
|
13 Corless M, Kiely A, McClenaghan NH, Flatt PR,
Newsholme P: Glutamine regulates expression of key transcription factor,
signal transduction, metabolic gene, and protein expression in a clonal
pancreatic beta-cell line. J Endocrinol 2006;190:719-727. |
|
|
|
|
|
14 Petrus P, Lecoutre S, Dollet L, Wiel C, Sulen
A, Gao H, et al.: Glutamine Links Obesity to Inflammation in Human White
Adipose Tissue. Cell Metab 2020;31:375-390 e311. |
|
|
|
|
|
15 Laviano A, Molfino A, Lacaria MT, Canelli A,
De Leo S, Preziosa I, et al.: Glutamine supplementation favors weight loss in
nondieting obese female patients. A pilot study. Eur J Clin Nutr
2014;68:1264-1266. |
|
|
|
|
|
16 Greenfield JR, Farooqi IS, Keogh JM, Henning
E, Habib AM, Blackwood A, et al.: Oral glutamine increases circulating
glucagon-like peptide 1, glucagon, and insulin concentrations in lean, obese,
and type 2 diabetic subjects. Am J Clin Nutr 2009;89:106-113. |
|
|
|
|
|
17 Cannon B, Nedergaard J: Brown adipose tissue:
function and physiological significance. Physiol Rev 2004;84:277-359. |
|
|
|
|
|
18 Wu J, Cohen P, Spiegelman BM: Adaptive
thermogenesis in adipocytes: is beige the new brown? Genes Dev
2013;27:234-250. |
|
|
|
|
|
19 Chen Q, Shou P, Zheng C, Jiang M, Cao G, Yang
Q, et al.: Fate decision of mesenchymal stem cells: adipocytes or
osteoblasts? Cell Death Differ 2016;23:1128-1139. |
|
|
|
|
|
20 Gregoire FM, Smas CM, Sul HS: Understanding
adipocyte differentiation. Physiol Rev 1998;78:783-809. |
|
|
|
|
|
21 Giralt M, Villarroya F: White, brown,
beige/brite: different adipose cells for different functions? Endocrinology
2013;154:2992-3000. |
|
|
|
|
|
22 Labow BI, Souba WW, Abcouwer SF: Mechanisms
governing the expression of the enzymes of glutamine metabolism--glutaminase
and glutamine synthetase. J Nutr 2001;131:2467S-2474S; discussion
2486S-2487S. |
|
|
|
|
|
23 Bolzoni M, Chiu M, Accardi F, Vescovini R,
Airoldi I, Storti P, et al.: Dependence on glutamine uptake and glutamine
addiction characterize myeloma cells: a new attractive target. Blood
2016;128:667-679. |
|
|
|
|
|
24 Bhandari B, Burns DM, Hoffman RC, Miller RE:
Glutamine synthetase mRNA in cultured 3T3-L1 adipocytes. Complexity, content
and hormonal regulation. Mol Cell Endocrinol 1986;47:49-57. |
|
|
|
|
|
25 Roberts LD, Virtue S, Vidal-Puig A, Nicholls
AW, Griffin JL: Metabolic phenotyping of a model of adipocyte
differentiation. Physiol Genomics 2009;39:109-119. |
|
|
|
|
|
26 Velickovic K, Lugo Leija HA, Bloor I, Law J,
Sacks H, Symonds M, et al.: Low temperature exposure induces browning of bone
marrow stem cell derived adipocytes in vitro. Sci Rep 2018;8:4974. |
|
|
|
|
|
27 Velickovic K, Wayne D, Leija HAL, Bloor I,
Morris DE, Law J, et al.: Caffeine exposure induces browning features in
adipose tissue in vitro and in vivo. Sci Rep 2019;9:9104. |
|
|
|
|
|
28 Pace J, Mc DE: Methionine sulphoximine and
some enzyme systems in volving glutamine. Nature 1952;169:415-416. |
|
|
|
|
|
29 Ronzio RA, Rowe WB, Meister A: Studies on the
mechanism of inhibition of glutamine synthetase by methionine sulfoximine.
Biochemistry 1969;8:1066-1075. |
|
|
|
|
|
30 Berlicki L: Inhibitors of glutamine
synthetase and their potential application in medicine. Mini Rev Med Chem
2008;8:869-878. |
|
|
|
|
|
31 Issaq SH, Mendoza A, Fox SD, Helman LJ:
Glutamine synthetase is necessary for sarcoma adaptation to glutamine
deprivation and tumor growth. Oncogenesis 2019;8:20. |
|
|
|
|
|
32 Yang Y, Ishak Gabra MB, Hanse EA, Lowman XH,
Tran TQ, Li H, et al.: MiR-135 suppresses glycolysis and promotes pancreatic
cancer cell adaptation to metabolic stress by targeting phosphofructokinase-1.
Nat Commun 2019;10:809. |
|
|
|
|
|
33 Shen G, Zhang H, Jia P, Li G, Wang X, Zhou X,
et al.: GOLM1 Stimulation of Glutamine Metabolism Promotes Osteoporosis via
Inhibiting Osteogenic Differentiation of BMSCs. Cell Physiol Biochem
2018;50:1916-1928. |
|
|
|
|
|
34 Dill V, Zimmer A, Beer M, Eschbaumer M: Investigation
of cell culture conditions for optimal foot-and-mouth disease virus
production. BMC Biotechnol 2019;19:33. |
|
|
|
|
|
35 Surrati A, Linforth R, Fisk ID, Sottile V,
Kim DH: Non-destructive characterisation of mesenchymal stem cell
differentiation using LC-MS-based metabolite footprinting. The Analyst
2016;141:3776-3787. |
|
|
|
|
|
36 Scheltema RA, Jankevics A, Jansen RC, Swertz
MA, Breitling R: PeakML/mzMatch: a file format, Java library, R library, and
tool-chain for mass spectrometry data analysis. Anal Chem 2011;83:2786-2793. |
|
|
|
|
|
37 Tautenhahn R, Bottcher C, Neumann S: Highly
sensitive feature detection for high resolution LC/MS. BMC Bioinformatics
2008;9:504. |
|
|
|
|
|
38 Yoo H, Stephanopoulos G, Kelleher JK:
Quantifying carbon sources for de novo lipogenesis in wild-type and IRS-1
knockout brown adipocytes. J Lipid Res 2004;45:1324-1332. |
|
|
|
|
|
39 Bourin P, Bunnell BA, Casteilla L, Dominici
M, Katz AJ, March KL, et al.: Stromal cells from the adipose tissue-derived
stromal vascular fraction and culture expanded adipose tissue-derived
stromal/stem cells: a joint statement of the International Federation for
Adipose Therapeutics and Science (IFATS) and the International Society for
Cellular Therapy (ISCT). Cytotherapy 2013;15:641-648. |
|
|
|
|
|
40 Kowalchuk JM, Curi R, Newsholme EA: Glutamine
metabolism in isolated incubated adipocytes of the rat. Biochem J
1988;249:705-708. |
|
|
|
|
|
41 Cooney G, Curi R, Mitchelson A, Newsholme P,
Simpson M, Newsholme EA: Activities of some key enzymes of carbohydrate,
ketone body, adenosine and glutamine metabolism in liver, and brown and white
adipose tissues of the rat. Biochem Biophys Res Commun 1986;138:687-692. |
|
|
|
|
|
42 Yoo H, Antoniewicz MR, Stephanopoulos G,
Kelleher JK: Quantifying reductive carboxylation flux of glutamine to lipid
in a brown adipocyte cell line. J Biol Chem 2008;283:20621-20627. |
|
|
|
|
|
43 Sato N, Moore FA, Kone BC, Zou L, Smith MA,
Childs MA, et al.: Differential induction of PPAR-gamma by luminal glutamine
and iNOS by luminal arginine in the rodent postischemic small bowel. Am J
Physiol Gastrointest Liver Physiol 2006;290:G616-623. |
|
|
|
|
|
44 Berthiaume M, Sell H, Lalonde J, Gelinas Y,
Tchernof A, Richard D, et al.: Actions of PPARgamma agonism on adipose tissue
remodeling, insulin sensitivity, and lipemia in absence of glucocorticoids.
Am J Physiol Regul Integr Comp Physiol 2004;287:R1116-1123. |
|
|
|
|
|
45 He W, Barak Y, Hevener A, Olson P, Liao D, Le
J, et al.: Adipose-specific peroxisome proliferator-activated receptor gamma
knockout causes insulin resistance in fat and liver but not in muscle. Proc
Natl Acad Sci U S A 2003;100:15712-15717. |
|
|
|
|
|
46 Klingenspor M: Cold-induced recruitment of
brown adipose tissue thermogenesis. Exp Physiol 2003;88:141-148. |
|
|
|
|
|
47 Rosen ED, MacDougald OA: Adipocyte
differentiation from the inside out. Nat Rev Mol Cell Biol 2006;7:885-896. |
|
|
|
|
|
48 Yu Y, Newman H, Shen L, Sharma D, Hu G,
Mirando AJ, et al.: Glutamine Metabolism Regulates Proliferation and Lineage
Allocation in Skeletal Stem Cells. Cell Metab 2019;29:966-978 e964. |
|
|
|
|
|
49 Ritchie JW, Baird FE, Christie GR, Stewart A,
Low SY, Hundal HS, et al.: Mechanisms of glutamine transport in rat
adipocytes and acute regulation by cell swelling. Cell Physiol Biochem
2001;11:259-270. |
|
|
|
|
|
50 Rubio-Aliaga I, Wagner CA: Regulation and
function of the SLC38A3/SNAT3 glutamine transporter. Channels (Austin)
2016;10:440-452. |
|
|
|
|
|
51 Ghoddoussi F, Galloway MP, Jambekar A, Bame
M, Needleman R, Brusilow WS: Methionine sulfoximine, an inhibitor of
glutamine synthetase, lowers brain glutamine and glutamate in a mouse model
of ALS. J Neurol Sci 2010;290:41-47. |
|
|
|
|
|
52 Dadsetan S, Bak LK, Sorensen M, Keiding S,
Vilstrup H, Ott P, et al.: Inhibition of glutamine synthesis induces
glutamate dehydrogenase-dependent ammonia fixation into alanine in
co-cultures of astrocytes and neurons. Neurochem Int 2011;59:482-488. |
|
|
|
|
|
53 Fries AW, Dadsetan S, Keiding S, Bak LK,
Schousboe A, Waagepetersen HS, et al.: Effect of glutamine synthetase
inhibition on brain and interorgan ammonia metabolism in bile duct ligated
rats. J Cereb Blood Flow Metab 2014;34:460-466. |
|
|
|
|
|
54 Palmieri EM, Spera I, Menga A, Infantino V,
Iacobazzi V, Castegna A: Glutamine synthetase desensitizes differentiated
adipocytes to proinflammatory stimuli by raising intracellular glutamine
levels. FEBS Lett 2014;588:4807-4814. |
|
|
|
|
|
55 Al-Khallaf H: Isocitrate dehydrogenases in
physiology and cancer: biochemical and molecular insight. Cell Biosci
2017;7:37. |
|
|
|
|
|
56 Grelli KN, Palubinsky AM, Kale AC,
Lizama-Manibusan BN, Stankowski JN, Milne GL, et al.: Alteration of
isocitrate dehydrogenase following acute ischemic injury as a means to
improve cellular energetic status in neuroadaptation. CNS Neurol Disord Drug
Targets 2013;12:849-860. |
|
|
|
|
|
57 Filipp FV, Scott DA, Ronai ZA, Osterman AL,
Smith JW: Reverse TCA cycle flux through isocitrate dehydrogenases 1 and 2 is
required for lipogenesis in hypoxic melanoma cells. Pigment Cell Melanoma Res
2012;25:375-383. |
|
|
|
|
|
58 Jo SH, Son MK, Koh HJ, Lee SM, Song IH, Kim
YO, et al.: Control of mitochondrial redox balance and cellular defense
against oxidative damage by mitochondrial NADP+-dependent isocitrate
dehydrogenase. J Biol Chem 2001;276:16168-16176. |
|
|
|
|
|
59 Metallo CM, Gameiro PA, Bell EL, Mattaini KR,
Yang J, Hiller K, et al.: Reductive glutamine metabolism by IDH1 mediates
lipogenesis under hypoxia. Nature 2011;481:380-384. |
|
|
|
|
|
60 Smolkova K, Jezek P: The Role of
Mitochondrial NADPH-Dependent Isocitrate Dehydrogenase in Cancer Cells. Int J
Cell Biol 2012;2012:273947. |
|
|
|
|
|
61 Findlay AS, Carter RN, Starbuck B, McKie L,
Novakova K, Budd PS, et al.: Mouse Idh3a mutations cause retinal degeneration
and reduced mitochondrial function. Dis Model Mech 2018;11:dmm036426. |
|
|
|
|
|
62 Brand MD, Nicholls DG: Assessing
mitochondrial dysfunction in cells. Biochem J 2011;435:297-312. |
|