Discussion

Glutamine supplementation shows potential both in vitro and in vivo to preserve endothelial function under diverse stress conditions. However, how extracellular glutamine reprograms the endothelial metabolic network remains poorly defined. In this study, we systematically examined the metabolic fate of glutamine in endothelial cells over increasing glutamine concentrations (0 to 10 mM). We showed that endothelial cells increase glutamine uptake as extracellular glutamine concentration increases, but glutamate secretion and oxidative respiration saturate above 2 mM glutamine, suggesting a limited capacity for glutaminolysis. Glutamine’s carbons primarily enter the forward TCA cycle, with a small contribution to reverse carboxylation. Glutamine carbons then support glutathione and non-essential amino acid synthesis. Although most TCA metabolite abundance increased with extracellular glutamine, succinate, unsaturated fatty acid, and 1 C metabolite total abundance declined. Together, these findings indicate that while endothelial cells take up more glutamine when it is available, excess glutamine is stored rather than metabolized. The presence of glutamine, but not its excess, sustains the TCA cycle, amino acid synthesis, and redox balance.

In both normal and high glucose, glutamine uptake and intracellular glutamine increased with extracellular glutamine concentration. While others have shown that increasing extracellular glutamine from 0 to 2 mM increased intracellular glutamine, for example in bovine venular endothelial cells (Meininger & Guoyao, 1997) and in human diploid fibroblasts (Bannai & Ishii, 1988), our study is the first to our knowledge to examine the extremely high glutamine concentrations often used in in vitro cell culture. Excess glutamine uptake would come at a cost to the endothelial cell, given that the primary glutamine importer ASCT2 (SLC1A5) imports sodium and exports a neutral amino acid (e.g., serine, threonine). Glutamine uptake would increase energy use, since the cell must use the Na⁺/K⁺ ATPase to maintain the sodium gradient. This excess energy likely comes from oxidative respiration, since we did not observe an increase in glycolysis. The net export of amino acids by ASCT2 would also need to be countered by amino acid import from system A and N transporters (SNAT1-5).

In contrast, intracellular glutamate and glutamate secretion plateaued above 2 mM glutamine, suggesting that excess glutamine is stored rather than metabolized. Intracellular glutamate saturation may occur due to limitations in its production, while extracellular glutamate saturation may occur due to limitations in its export. Glutamine is metabolized to glutamate by GLS-1. We measured an increase in GLS-1 as glutamine increased from 0 to 0.5 mM but no further increase up to 2 mM glutamine. Furthermore, intracellular glutamate binds to and reduces GLS-1 activity (Shapiro et al., 1982; Cyriac & Lee, 2024). Thus, GLS-1 availability and activity may limit glutamine conversion to glutamate despite continued glutamine uptake. The observed extracellular glutamate plateau may reflect limits in amino acid exchange. Glutamate export is functionally coupled to cystine uptake through the cystine–glutamate antiporter xCT (Jyotsana et al., 2022). In human diploid fibroblasts, extracellular glutamate increased with extracellular cystine, indicating that extracellular cystine is essential for glutamate efflux (Bannai & Ishii, 1988). Therefore, glutamine-derived intracellular glutamate could be exported in exchange for extracellular cystine up to the point at which cystine availability becomes limiting. These data suggest that glutamine supplementation alone may not be sufficient to fully exploit the protective effects of glutamine, including enhanced antioxidant capacity and inflammation reduction (Jiang et al., 2017; Peyton et al., 2018; Kheradmand et al., 2026). Increasing GLS-1 expression or activity or promoting glutamate efflux through increased extracellular cystine may be required.

Glutamine availability may support elevated endothelial metabolic activity, since we observed increased oxidative respiration without a decrease in glycolysis. Indeed, glutamine supplementation actually increased lactate secretion. This may occur because glutamine provides carbons to the TCA cycle to support oxidative respiration. Glucose-derived pyruvate can then be reduced to lactate and secreted, rather than oxidized in the TCA cycle. Alternatively, glutamine could act as a signaling metabolite that activates mechanistic target of rapamycin (mTOR) and thereby promotes glycolytic metabolism (Szwed et al., 2021). Further experiments are needed to confirm this observation and determine the impact of glutamine-induced elevated metabolism on endothelial function.

Our isotope enrichment data confirmed work by others showing that glutamine is a major mitochondrial substrate for endothelial cells (Huang et al., 2017; Kim et al., 2017; Kaczara et al., 2024). Parallel labeling with 1-13C- and 5-13C-glutamine showed that endothelial cells metabolize glutamine primarily in the forward oxidative TCA cycle but also in reductive carboxylation. Oxidative glutamine metabolism supports the production of ATP and TCA intermediates for energy production, while reductive glutamine metabolism supports the production of citrate and acetyl-CoA for lipid synthesis (Metallo et al., 2012). Interestingly, excess glutamine did not change the percentage of glutamine that underwent reductive carboxylation, suggesting that glutamine does not shift endothelial cell energy vs. biomass requirements or affect mitochondrial activity. Instead, reductive carboxylation seems to be driven by hypoxia. In the A549 cell line, reductive carboxylation increased from ~ 10% to 80% in hypoxia (Metallo et al., 2012). We measured lower glutamine reductive carboxylation than in other studies (5% vs. 13%) (Kim et al., 2017), which could relate to variations in cell type (HUVEC vs. HCAEC) or nutrient and oxygen availability.

In contrast to other TCA metabolites, which showed increased total abundance as extracellular glutamine increased, succinate total abundance decreased as glutamine concentration rose. We measured a large unlabeled intracellular succinate pool in our HCAEC, which is consistent with studies in HUVEC and iPSC-derived brain microvascular endothelial cells (Moiz et al., 2021; Weber et al., 2025). In HepG2 cells, glutamine deprivation promoted deSUMOylation of succinate dehydrogenase substrate A (SDHA), impairing SDH assembly and activity (Liu et al., 2023). Consistent with this, our data showed that glutamine enhanced SDH activity, which likely increased succinate consumption and contributed to the reduced intracellular succinate pool. However, the change in succinate could also be caused by reduced succinate production, altered compartmentalization, changes in succinate turnover, or dilution (Abdullah et al., 2023). These alternative mechanisms would also need to be tested via targeted or dynamic isotope tracing.

We and others demonstrated that glutamine reduces intracellular oxidative stress through GSH synthesis (Peyton et al., 2018; Kheradmand et al., 2026). However, increasing glutamine above 1 mM did not further increase GSH total abundance. The first step of GSH synthesis, in which glutamate and cysteine are combined in a reaction catalyzed by glutamate cysteine ligase (GCL), is rate-limiting and may explain the plateau in GSH production (Zhang & Forman, 2012; Li et al., 2022). The glutamate concentration at which this reaction rate is half of its maximum velocity (Km) is 1.8 mM, which is much lower than the estimated intracellular glutamate concentration (~ 21 mM in fibroblasts). Thus, GCL is likely already saturated with glutamate, so additional glutamine-derived glutamate would not increase GSH production (Lu, 2013). Furthermore, GSH itself binds to the glutamate site on GCL to inhibit its activity (Ki=2.3 mM), meaning that high GSH can also reduce GSH production (Richman & Meister, 1975; Franklin et al., 2009). Thus, high extracellular glutamine alone is not sufficient to increase endothelial GSH.

Ornithine cycle metabolites were not labeled with glutamine-derived carbons, but both citrulline and arginine decreased in abundance with extracellular glutamine. Ornithine aminotransferase (OAT) catalyzes the reversible reaction between P5C and ornithine. Given the lack of 13C labelled ornithine, this reaction appears to be primarily in the direction of P5C production from ornithine in our endothelial cells. Reduction of citrulline and arginine abundance may be explained by inhibited citrulline transport and reduced citrulline to arginine conversion. In bovine endothelial cells, extracellular glutamine reduced intracellular citrulline by inhibiting citrulline transport, likely because they share some of the same neutral amino acid transporters (Wu & Meininger, 1993). Furthermore, 2 mM glutamine inhibited arginine production from citrulline via argininosuccinate in bovine aortic endothelial cells (Sessa et al., 1990). Some studies also suggest that cultured endothelial cells do not have detectable carbamoyl-phosphate synthase I (CPS-I) activity, which is essential to transform ornithine into arginine (Wu et al., 2000).

The observed increase in UDP-GlcNAc abundance and isotopic enrichment from 5-13C-glutamine shows that glutamine and its carbons contribute to the endothelial cell HBP. Glutamine provides a nitrogen to convert fructose-6-phosphate into glucosamine-6-phosphate early in the HBP (Kornfeld, 1967; Wellen et al., 2010; Yang et al., 2026), and glutamine abundance correlates with UDP-GlcNAc synthesis (Paneque et al., 2023). However, UDP-GlcNAc synthesis integrates many metabolic inputs, one of which is uridine supplied by pyrimidine nucleotide metabolism (Bond and Hanover 2015; Paneque et al. 2023; Yang et al. 2024b). We observed increased aspartate total abundance and isotopic enrichment, suggesting that glutamine-derived aspartate contributes to UDP-GlcNAc formation (Oberkersch et al., 2022).

Finally, increasing glutamine decreased folate and histidine abundance, suggesting altered 1 C metabolism flux. 1 C metabolism is essential for nucleotide and amino acid biosynthesis, methylation reactions, and redox balance (Ducker & Rabinowitz, 2017; Petrova et al., 2023). In the 1 C pathway, folate serves as the central carrier by accepting and transferring single-carbon units. Histidine and serine contribute 1 C units to folate in the cytosol, while serine and glycine contribute 1 C units to folate in mitochondria (Ducker & Rabinowitz, 2017; Lin et al., 2022). The altered 1 C metabolism flux could relate to glutamine-induced enhanced cell proliferation via nucleotide synthesis (Peyton et al., 2018) or to enhanced antioxidants (Kheradmand et al., 2026). Additional experiments would be required to investigate this mechanism.

While our study shows the impact of glutamine on endothelial cells in culture, it is not without limitations. We treated endothelial cells at confluence, when endothelial cells are predominantly quiescent; however, we did not directly control and assess cell cycle phase. Because metabolic fluxes and isotopic labeling patterns differ between proliferating and quiescent endothelial cells (Kalucka et al., 2018), it would be interesting to compare glutamine metabolism across defined cell cycle states. In addition, while stable isotope tracing provided insight into glutamine carbon use in the forward and reductive carboxylation, the use of single-position ¹³C tracers limited our ability to resolve all possible carbon and nitrogen entry routes into downstream pathways. Finally, our analyses were performed at isotopic steady state following 24 h incubations and therefore reflect net metabolic outcomes rather than dynamic flux changes. Dynamic tracing experiments would be useful to capture transient metabolic reprogramming with glutamine availability.