Materials and methods

Endothelial cell culture

Primary human coronary artery endothelial cells (HCAEC, passage 5–9) were purchased from Lifeline Cell Technology. HCAECs were cultured in Endothelial Growth Medium-2 (EGM-2; Lonza) supplemented with 1% penicillin-streptomycin (PS; Thermo Fisher Scientific, 15140163), 10% fetal bovine serum (FBS; Cytiva, SH30088) and 1% glutamine (Fisher Scientific, 25–030-081). Cells were passaged into appropriate well plates and incubated in EGM-2 media until confluent. Cells were then switched to Dulbecco’s Modified Eagle Medium (DMEM) without phenol red, glucose, or glutamine (Gibco, A1443001) supplemented with Endothelial SingleQuots (Lonza, CC-4176), 5.5 or 15 mM D-glucose (Sigma-Aldrich, G8270), and 0–10 mM glutamine for 24 h. Low glutamine concentrations (0.5–2 mM) were used to simulate in vivo circulating glutamine, while higher glutamine concentrations were used to replicate the elevated glutamine in some in vitro cell culture media. L-glucose or mannitol were used as osmotic controls to match the osmolarity of the corresponding glutamine and glucose additions.

Glutamine uptake and glutamate secretion

A YSI 2950 Biochemistry Analyzer (Yellow Springs Instruments, 527690) was used to measure media glucose, lactate, glutamine, and glutamate concentration. At 0 and 24 h of glutamine treatment, 150 µL media was collected and placed in a 96 well-plate (CELLTREAT; 229196) for analysis by YSI. Glucose and glutamine uptake and lactate and glutamate secretion were calculated as the difference between the 0 and 24 h concentrations.

Glutaminase analysis

A Western blot was used to detect changes in HCAEC glutaminase. After glutamine treatment, cells were washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific; 70011069) and lysed with RIPA buffer (Thermo Fisher Scientific; 89901) containing Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific; 78440) and ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich; E9884). The cell lysate was centrifuged for 15 min at 17,000 × g at 4 °C to remove cellular debris. The supernatant was collected, and the protein concentration was quantified using a BCA assay (Thermo Fisher Scientific; 23225). 3.5 µg protein was loaded into each well of a NuPAGE 4%−12% Bis-Tris gel (Thermo Fisher Scientific; NP0323BOX) for separation by SDS-PAGE. Proteins were then transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific; IB34001 × 3) using an iBlot 3 (Thermo Fisher Scientific). Membranes were blocked in 5% bovine serum albumin (BSA; Sigma-Aldrich; A7906) in tris buffered saline (TBS; Fisher Scientific; BP24711) with 0.5% Tween (TBS-T; Thermo Fisher Scientific; 85115) for 1 h at room temperature. Blots were then incubated with primary antibodies in 1% BSA in TBS-T overnight at 4 °C. Primary antibodies included glutaminase-1 (1:1000; Abcam; EP7212) and β-actin (1:1000; Santa Cruz Biotechnology; sc-47778). The next day, membranes were washed with TBS-T and incubated with the appropriate anti-rabbit (Promega; W4011) or anti-mouse (Promega; W4021) secondary antibodies (1:2000) for 2 h. Protein bands were visualized by SuperSignal West Pico PLUS Chemiluminescent Substrate kit (Thermo Fisher Scientific; 34578) and imaged using an Alpha Innotech Fluorchem Imager. AlphaView SA 3.4.0 was used to quantify band intensity.

Oxidative respiration

HCAEC oxidative respiration was measured using a Seahorse Mito Stress Test (Agilent; 103015). HCAEC were seeded at 50,000 cells/well in Seahorse XF96 cell culture plates and incubated overnight to allow cells to adhere to the plate. The next day, DMEM with 0, 0.5, or 2 mM glutamine and 5.5 or 15 mM D-glucose or the appropriate osmotic controls was added to the wells. After 24 h, the assay was performed on a Seahorse XFe96 (Agilent) according to the manufacturer’s protocols. Wave software (Agilent) was used to calculate basal respiration, maximal respiration, spare capacity, and non-mitochondrial oxygen consumption from the measured oxygen consumption rate (OCR). Following the assay, cells were stained with DAPI for 10 min, and cell nuclei were counted. Seahorse values were normalized to cell count for each well. Experiments performed on different days were normalized to the average OCR of the 0 mM glutamine condition.

Isotope tracing via liquid chromatography-tandem mass spectrometry (LC-MS/MS)

To gain detailed insights into intracellular glutamine metabolism, we designed a parallel isotope-labeling experiment with 1-13 C and 5-13 C glutamine. 1-13C-glutamine was used to trace glutamine entry into the reductive (reverse) carboxylation pathway, as the C1 label is released as CO₂ during forward TCA cycle flux. 5-13C-glutamine was used to identify forward TCA–derived metabolites. 0, 1, 2, or 5 mM 1-13C-glutamine (CLM-3612, Cambridge Isotope Laboratory) or 5-13 C-glutamine (CLM-1166, Cambridge Isotope Laboratory) were added to confluent HCAECs in 6 well-plates in supplemented DMEM (5.5 mM glucose) for 24 h. Endothelial cells were previously determined to reach isotopic steady state by this time(Moiz et al., 2021). Metabolites were then extracted in 500 µL ice-cold 80:20 methanol: water at −80 °C for 15 min. Cell lysates were scraped in the extraction solvent, transferred to 1.7 mL micro centrifuge tubes, and centrifuged at 17,000 × g for 15 min at 4 °C to pellet cell proteins. Finally, the pellet was lysed in 50 µL RIPA buffer, and protein concentration was measured by BCA for normalization. The supernatant was collected, stored at −80 °C, and analyzed by LC-MS/MS in the University of Colorado School of Medicine Metabolomics Core. Metabolomics analysis was performed using a Vanquish UHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific). Samples (10 µL) were injected onto a 2.1 × 100 mm, 1.7 μm particle size Waters Acquity BEH C18 column at 45 °C and separated using a 5-minute reversed-phase gradient, based on a previously described method (Nemkov et al., 2019). For negative ion mode, mobile phase A consisted of water with 10 mM ammonium acetate (NH4OAc), and mobile phase B was a 1:1 mixture of methanol and acetonitrile with 10 mM NH4OAc. For positive ion mode, mobile phase A was water with 0.1% formic acid, while mobile phase B was acetonitrile with 0.1% formic acid.

In both ionization modes, the UHPLC separation followed the same gradient: 100% A (0.00 to 0.50 min); increased to 100% B (0.50 to 1.10 min); held at 100% B (1.10 to 2.75 min); returned to 100% A (2.75 to 3.00 min); and held at 100% A (3.00 to 5.00 min) for re-equilibration. The flow rate was maintained at 0.45 mL/min throughout the analysis. The mass spectrometer was run independently in both negative and positive ion modes, acquiring full MS scans over an m/z range of 65–975 at a resolution of 120,000. Source conditions included a sheath gas flow of 50 arbitrary units (Arb), auxiliary gas flow of 10 Arb, and spray voltages of 3 kV (negative mode) and 3.4 kV (positive mode). The instrument was calibrated prior to analysis using the Easy-IC internal standard (Thermo Fisher Scientific).

Sample run order was randomized. Technical replicates were injected throughout each sequence to assess analytical reproducibility and quality control. Raw files were converted to.mzXML format using RawConverter and analyzed with El-Maven (Elucidata) using the KEGG database for metabolite identification and peak integration, as previously described (Nemkov et al., 2017).

Mass spectrometry data analysis

Natural abundance correction, multivariate analysis, and univariate analysis were performed in RStudio. The IsoCorrector 1.22.0 package was first used to correct the isotopologue natural abundance for each metabolite from the LC–MS/MS raw data. As we used one carbon labeled glutamine, labeling was expected to result primarily in M + 1 isotopologues. Consistent with this, higher order isotopologues (M + 2 and above) were not detected for most metabolites. Under these conditions, natural abundance correction using IsoCorrectoR may overcorrect the data. Therefore, both raw and natural abundance–corrected mass isotopologue distributions were analyzed. As both approaches showed consistent trends, raw mass isotopologue distributions are included in the supplementary data (Online resources 8 and 9), and the corrected mass isotopologue distributions are presented in the main figures. After correction, the labeled fraction was calculated by dividing the signal from labeled metabolites by the total signal (labeled + unlabeled). The mixOmics 6.28.0 package was then used for unsupervised multivariate analysis principal component analysis (PCA) as an exploratory tool. PCA score and loading plots were generated using the ggplot2 package in RStudio, and stackplots and barplots were created using GraphPad Prism 10.

Media succinate

Media was collected from HCAEC cultured in 5.5 mM D-glucose DMEM with 0 or 5 mM glutamine for 24 h. Media was centrifuged for 10 min at 17,000 × g at 4 °C to remove cell debris. 200 µL supernatant was added to 800 µL ice cold 100% HPLC grade methanol, vortexed, and centrifuged at 17,000 × g at 4 °C for 15 min. The supernatant was transferred to a new tube and dried overnight using a CentriVap Vacuum Concentrator (Labconco). Samples were resuspended in 100 µL 80% methanol (Sigma-Aldrich, G6025-11VL) and transferred to mass spectrometry vials (Waters, 600000670). Extracellular succinate was analyzed using a Bruker maXis-II QTOF mass spectrometer (Bruker Daltonics) coupled to a Waters ACQUITY UPLC system. Chromatographic separation was performed on an Atlantis BEH C18 AX column (2.1 × 100 mm, 1.7 μm; Waters) maintained at 30 °C, with the sample manager held at 6 °C. A 5 µL injection volume was used. The mobile phases consisted of solvent A: water containing 0.1% formic acid and solvent B: acetonitrile containing 0.1% formic acid. The flow rate was 0.25 mL/min. The gradient program was: 0–2 min, 2% A; 2–11 min, linear increase to 90% A; 11–16 min, hold at 90% A; 16–17 min, return to 2% A; 17–20 min, re-equilibration at 2% A. Mass spectrometric detection was performed in negative electrospray ionization (ESI−) mode with a capillary voltage of 4,500 V. Data were acquired in full scan mode over an m/z range of 80–400 without MS/MS fragmentation. A 10 µM sodium succinate dibasic hexahydrate standard (Sigma-Aldrich; S2378) was injected at the beginning of each sequence to confirm both mass and retention time prior to sample analysis. Succinate was identified based on accurate mass and retention time relative to the standard. Extracted ion chromatograms were generated at m/z 116.9967 ± 0.02 Da, and the succinate peak eluted at 1.4 min.

Succinate dehydrogenase activity assay

Succinate dehydrogenase (SDH) activity was measured using a commercially available SDH Activity Assay Kit (Abcam; ab228560) according to the manufacturer’s instructions. HCAECs were seeded at equal density in 100 mm culture dishes and grown to confluency in EGM-2 prior to each experiment. Cells were rinsed with PBS and then switched to DMEM supplemented with 1% PS, 10% FBS, 5.5 mM glucose, and either 0 or 5 mM glutamine for 24 h. Following treatment, cells were detached using trypsin, washed with cold PBS, and resuspended in 100 µL SDH assay buffer supplemented with 1% protease inhibitor cocktail and EDTA. Cells were lysed using a D1000 homogenizer (Benchmark Scientific), incubated on ice for 10 min, and then centrifuged at 10,000 × g for 5 min at 4 °C. The supernatant was collected for analysis. Protein concentration was determined using a BCA assay. 75 µg protein for each sample was loaded into a 96-well plate and adjusted to a final volume of 50 µL. The standard curve and reaction mixture were prepared according to the manufacturer’s protocol and added to the designated wells. Absorbance was measured at 600 nm in kinetic mode for 60 min at 2 min intervals using a Spark multimode plate reader (TECAN). SDH activity was calculated according to the manufacturer’s instructions using the linear portion of the kinetic curve.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 10. Data are presented as mean ± standard deviation (SD) unless otherwise indicated. For experiments involving multiple glutamine concentrations, statistical significance was determined using an ordinary one-way ANOVA. When all samples were compared to each other, a Tukey’s multiple comparisons test was used. When comparisons were made to a single control group (e.g., 0 mM glutamine), a Dunnett’s multiple comparisons test was used. For comparisons between two groups, a non-parametric Mann Whitney test was used. Statistical significance was defined as p < 0.05. The number of biological replicates (n) and the specific statistical test for each dataset are indicated in the corresponding figure legend.