Introduction

Endothelial cell dysfunction is a hallmark of cardiovascular disease (CVD), the leading cause of mortality among subjects with diabetes (Kannel 1979; Haffner et al. 1998; Dubsky et al. 2023; Yang et al. 2024a). Endothelial dysfunction is tightly linked to metabolic activity (De Vriese et al., 2000; Marfella et al., 2001; Rohlenova et al., 2018; Citrin et al., 2025). While endothelial cells rely on glycolysis for up to 85% of their ATP production during normal function, excess glucose metabolism can lead to dysfunction by increasing oxidative stress, permeability, and inflammation (Nishikawa et al., 2000; Du et al., 2001; Hoshiyama et al., 2004; Clyne, 2021; Xiao et al., 2021). However, the way in which excess glucose metabolism in conditions such as diabetic hyperglycemia interacts with other metabolic pathways is not fully understood.

Glutamine is the most abundant amino acid in the body, and in healthy endothelial cells, glutamine metabolism is crucial for biomass production during vascular sprouting and for maintaining redox homeostasis (Mirveis et al., 2023). Endothelial cells import glutamine through the Na⁺-dependent antiporter SLC1A5 (ASCT2), which exchanges extracellular glutamine for an intracellular neutral amino acid, as well as the Na⁺-dependent symporters SLC38A1 and SLC38A2 (Liu et al., 2018), which use the inward sodium electrochemical gradient to bring glutamine into the cell (Mann et al., 2003; Bröer & Gauthier-Coles, 2022).

Once glutamine enters the endothelial cell, glutaminase 1 (GLS-1) hydrolyzes glutamine into glutamate and ammonia (Wu et al., 2000; Huang et al., 2017; Zheng et al., 2026). Glutamate is further metabolized into α-ketoglutarate (α-KG), either through glutamate dehydrogenase (GLUD1) or via transamination reactions. α-KG then enters the tricarboxylic acid (TCA) cycle for ATP and macromolecule production (Hinca et al., 2021). Glutamate derived from glutamine contributes to the formation of other amino acids, including asparagine via asparagine synthetase and proline from pyrroline-5-carboxylate (P5C) via P5C reductase (Wu et al., 2000; Li et al., 2022). Glutamine-derived glutamate is also essential to the endothelial cell antioxidant system via glutathione (GSH) synthesis. Glutamate, through the enzyme glutamate–cysteine ligase (GCL), conjugates with cysteine to form the dipeptide γ-glutamylcysteine. Glutathione synthetase (GS) then adds glycine to γ-glutamylcysteine, resulting in the final tripeptide, γ-L-glutamyl-L-cysteinylglycine, known as GSH (Lu, 2013). Thus, glutamine could impact endothelial function via central carbon metabolism and biosynthetic pathway and by boosting antioxidant defense.

Glutamine also interacts with other metabolic pathways such as the hexosamine biosynthetic pathway (HBP). The HBP integrates glucose and glutamine metabolism to generate UDP-N-acetylglucosamine (UDP-GlcNAc), a substrate required for protein O-GlcNAcylation and cell signaling regulation (Wellen et al., 2010; Paneque et al., 2023). Glutamine directly impacts UDP-GlcNAc production by providing the essential amide group required for glutamine: fructose-6-phosphate aminotransferase (GFAT) activity. In addition, glutamine provides carbon substrates necessary for UDP-GlcNAc synthesis, including acetyl-CoA and UTP (Darley-Usmar et al., 2012; Yang et al., 2026). UDP-GlcNAc influences cell signaling through post-translational protein modification via GlcNAcylation, which often occurs at the same serine and threonine residues targeted by phosphorylation (Sessa et al., 1990; Aulak et al., 2020; Basehore et al., 2021).

Finally, glutamine contributes to the ornithine cycle and one carbon (1 C) metabolism. Glutamine‑derived glutamate can be converted to ornithine via pyrroline‑5‑carboxylate synthase. When this ornithine is metabolized, endothelial cells synthesize polyamines, which are essential for cell proliferation, as well as proline, which is essential for collagen synthesis and vascular extracellular matrix integrity (Wu et al., 2000; Sivashanmugam et al. 2017; Li et al., 2026). Glutamine metabolism in the TCA cycle supplies precursors for serine and glycine, the main 1 C donors to 1 C metabolism (Ducker & Rabinowitz, 2017; Hwang et al., 2023). This glutamine‑supported 1 C flux sustains nucleotide synthesis, redox balance, and methylation capacity, thereby promoting endothelial cell proliferation, survival, and genetic regulation (Ducker & Rabinowitz, 2017; Petrova et al., 2023).

Glutamine supplementation may protect against CVD and related complications through several metabolic and anti-inflammatory mechanisms. In pulmonary artery endothelial cells, glutamine supplementation reduced hydrogen peroxide–induced injury, helping maintain cellular ATP and viability during high oxidative stress (Hinshaw & Burger, 1990). In animal models of endotoxin shock and severe injury, glutamine supplementation improved survival, enhanced immune function, and supported gut barrier integrity (Wischmeyer et al., 2001). However, glutamine shows inconsistent effects on endothelial function, with beneficial effects in some studies and detrimental effects in others (Hecker et al.; Meininger & Guoyao, 1990; Ellis et al., 2016; Kheradmand et al., 2026).

Although glutamine supplementation is used clinically to enhance vascular health, the endothelial cell metabolic adaptations in response to altered glutamine are not fully understood. In this study, we therefore examined how physiological and supplemented glutamine affect endothelial metabolism. We hypothesized that increasing glutamine concentration will increase TCA cycle flux in the forward and reverse directions, as well as amino acid synthesis and HBP flux. We supplemented endothelial cell culture media with 0–10 mM glutamine and measured changes in glutamine and glutamate levels. We then used a Seahorse Mito Stress Test to measure oxidative respiration and heavy isotope-labeled glutamine to track glutamine-derived metabolites by liquid chromatography-mass spectrometry. Understanding how glutamine systematically impacts endothelial cell metabolism may help identify novel metabolic targets for cardiovascular disease prevention.