l glutamine  (Millipore)

Journal: Journal of Clinical Medicine

Article Title: Targeting Endothelial Cell Metabolism by Inhibition of Pyruvate Dehydrogenase Kinase and Glutaminase-1

doi: 10.3390/jcm9103308

Figure Lengend Snippet: Inhibition of glutamine conversion to glutamate reduced HUVECs proliferation, migration, and respiration without change in apoptosis induction and tube formation. ( A – D ), HUVECs were exposed during 24 h to 10 mM of l -glutamine-5- 13 C and co-incubated with (or without) BPTES. Intra- and extracellular metabolites were quantified by 13 C NMR spectroscopy ( n = 5). ( A ), glutamate production from glutamine (intracellular concentration of glutamate) ( B ), glutamine uptake (extracellular concentration of glutamine at T0 minus extracellular concentration of glutamine after 24 h). ( C ), intracellular glutamine concentration. ( D ), glutamine index, (ratio of glutamate production on glutamine uptake). ( E ), proliferation of HUVECs (% of control) treated with increasing doses of BPTES (0–10 µM) ( n = 4). ( F ), oxygen consumption rate (OCR), measured in HUVECs treated with or without 1 µM of BPTES ( n = 3). ( G ) percentage of apoptotic and dead HUVECs treated with or without 1 µM of BPTES ( n = 4). ( H ) percentage of wound closure after 14 h calculated from each T0 counterparts. ( I ) quantitative analysis of specific parameters (total tube length, total number of tubes, total branching point, and total loops) of HUVECs tube formation after 4 h incubation on Matrigel in the presence (or not) of 1 µM of BPTES. Statistical analysis: Paired t -test ( A – D , F , H , I ), One-way ( E ), or Two-way ANOVA ( G ) (Sidak’s multiple comparison test), comparison from control, * p

Article Snippet: Instead of the glutamine component, 10 mM of l -glutamine-5-13 C (Sigma-Aldrich) was supplemented in the isotope-labeled glutamine medium.

Techniques: Inhibition, Migration, Incubation, Nuclear Magnetic Resonance, Spectroscopy, Concentration Assay

Journal: Metabolites

Article Title: Staphylococcus aureus Infection Reduces Nutrition Uptake and Nucleotide Biosynthesis in a Human Airway Epithelial Cell Line

doi: 10.3390/metabo6040041

Figure Lengend Snippet: Incorporation of labelled precursor metabolites into purine and pyrimidine synthesis is strongly reduced during infection. Control cells and infected cells were cultured with either ( A ) l -glutamine-amide- 15 N or ( B ) d -glucose- 13 C 6 for 12 h. Mean percentages of incorporation levels of labelled precursors are presented ( n = 3). Proportions of complete labelled nucleotides and nucleotide-sugars are black, labelled sugar moieties are presented as grey pattern and unlabelled metabolite amounts are displayed as light grey.

Article Snippet: Metabolic Inhibitors and Labelled Precursors To inhibit glutamine dependent metabolic reactions, we applied two glutamine analogues namely 6-diazo-5-oxo-l -norleucine (DON) with a final concentration of 0.5 mmol/L and azaserine (both obtained from Sigma Aldrich) with a final concentration of 0.028 mmol/L to the incubation medium for 12 h. In our 15 N labelling approach we used RPMI 1640 R7509 medium as incubation medium as described above but replaced unlabelled glutamine with 2 mmol/L of l -glutamine-(amide-15 N) (obtained from Sigma Aldrich).

Techniques: Infection, Cell Culture

Journal: PLoS ONE

Article Title: T Helper Cell Activation and Expansion Is Sensitive to Glutaminase Inhibition under Both Hypoxic and Normoxic Conditions

doi: 10.1371/journal.pone.0160291

Figure Lengend Snippet: BPTES and 968 attenuated CD4 + T cell proliferation and activation under normoxia and hypoxia. (A) The proliferation index (PI) or average number of cell divisions, was calculated from CFSE dilutions of α-CD3/CD28 stimulated CD4 + T cells by flow cytometry at days 0, 3 and 5 in the absence (0 μM) and presence of (25 μM) BPTES and 968. Cell proliferation in 0.33% DMSO-containing media was used as control under normoxia and hypoxia. BPTES and 968 both reduced PI compared to control under normoxia. Under hypoxia, only 968 decreased the PI significantly (Mean ± SD, n = 4). (B) Expression of the cell surface activation markers CD226 and CD25 was lowered on day 3 by 968 and BPTES under both normoxic and hypoxic conditions. (C) On day 5, CD25 and CD226 expression was reduced by 968 but not significantly by BPTES both under normoxia and hypoxia. (D and E) Endogenous levels of [13C5-15N2]- glutamine was measured by ESI-LC MS after 3 (D) and 5 days of stimulation (E). [13C5-15N2] -glutamine was significantly increased in the presence of BPTES and 968 under normoxia both on day 3 and day 5. Under hypoxic conditions, only BPTES showed significant increase of intracellular glutamine on day 5. Data were normalized according to internal control (see material and methods ). Data presented results of three separate experiments (n = 3) with 5 parallels in each experiments. (****p˂0.001, ***p˂0.005, **p ˂0.01, *p˂0.05)

Article Snippet: Metabolic Tracing One million cells were incubated in the presence of 0.5 mM 15N2-13C5- Glutamine (Sigma Aldrich) and 5mM D-glucose (Sigma Aldrich) in DMEM no glucose,no glutamine medium and collected at 2 h by centrifugation (15000 rpm, 4°C).

Techniques: Activation Assay, Flow Cytometry, Cytometry, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Brain interstitial fluid glutamine homeostasis is controlled by blood–brain barrier SLC7A5/LAT1 amino acid transporter

doi: 10.1177/0271678X15609331

Figure Lengend Snippet: Impact of brain perfusion with system A and L competitive inhibitors on amino acids in brain microdialysates. Competitive inhibitors (20 mM) were introduced in the brain by perfusion via the microdialysis probe (as indicated by the grey shaded area) for 3 h. For each panel the indicated amino acid responses in ISF are plotted normalized to baseline values vs. time in brain (adjusted for the time samples take to reach fraction collector). In panel a, Glycine (Gly), L-valine (Val), and in panel b, L-glutamine (Gln), and taurine (Tau) responses following 20 mM methylaminoisobutyric acid (MeAIB) brain perfusion are shown. Data are means ± SD from three animals. (c) Val, Gln, L-glutamate (Glu) and Tau concentrations as a result of brain perfusion of 20 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). Data are means ± SD from four animals. (d) Gln levels in ISF normalized to the mean baseline values prior to MeAIB or BCH perfusion, respectively, and compared to Gln levels at 50 and 80 minutes of perfusion with the indicated inhibitor. For all panels, baseline samples are indicated by the solid line. Perfusate equilibration without sample collection is indicated by the break in the X axis (time in brain). Inhibitor presence in perfusate is indicated by the shaded area of the graph and underlined X axis region. Statistical comparisons were performed by two-way Anova with Dunnett’s post-test for the comparison of values obtained with inhibitor versus baseline (a,b,c) and with Bonferroni post-test for the comparison of values obtained with different inhibitors and with baseline (d). Tracked amino acids are indicated in the individual panel keys. Statistical significance for all panels are: **** = p

Article Snippet: Norleucine (NLeu), α-(methylamino)-isobutyric acid (MeAIB), 2-amino-2-norbornanecarboxylic acid (BCH), L-glutamic acid-γ-monohydroxamate (GAH), L-glutamine (Gln), L-valine (Val), sulphosalicylic acid (SSA), and cresyl violet acetate were purchased from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland).

Techniques: