(E-187) Exogenous glutamine supplementation increases glycolytic and mitochondrial activity in endothelial cells.

Introduction:: Endothelial cells (EC), which line the inner blood vessels, become dysfunctional in metabolic disorders such as diabetes. EC dysfunction then contributes to accelerated cardiovascular disease. Both human and EC metabolism are complex networks of interconnected pathways, and changes in one metabolite can have cascading effects on others. For instance, in diabetes, blood glucose concentrations increase while glutamine concentrations decrease. Glutamine is a nonessential amino acid that is present in high quantities in the human body and, in some studies, has been shown to enhance vascular health. ECs use glutamine for energy and biomass (protein) production, as well as to produce antioxidants that mitigate oxidative stress. We therefore hypothesize that increasing exogenous glutamine will enhance EC function by increasing glycolysis and oxidative phosphorylation.


Materials and Methods:: Male human coronary artery endothelial cells (HCAEC; passage 5-9) were cultured in endothelial growth media-2 (EGM-2) media supplemented with 10% fetal bovine serum (FBS), 1% penicillin streptomycin, and 1% L-glutamine. Once confluent, HCAECs were treated with Dulbecco's Modified Eagle Medium (DMEM) supplemented with EGM-2 kit, 10% FBS, 1% penicillin streptomycin, 5.5 mM glucose, and varied glutamine concentrations (0, 0.2, 0.5, 1, and 2 mM) for 24-48 hours. We then measured glutamine, glutamate, glucose and lactate consumption and production after 24 and 48 hours using a YSI Bioanalyzer. Finally, the Seahorse XF Glycolytic Rate Assay was used to determine the extracellular acidification and oxygen consumption rate in real time as a measure of glycolytic activity and mitochondrial respiration, respectively. Cells were incubated with DMEM media with the appropriate glutamine concentration for an hour before the assay in a non-CO₂ 37 ^oC incubator, and the media was refreshed right before starting the assay. Statistic analysis: Baseline OCR and glycoPER were analyzed with a one-way ANOVA only comparing each data point with the 0 mM glutamine condition. P-values smaller than 0.05 are considered significant and the P-values not shown are not statistically significant.


Results, Conclusions, and Discussions:: Results and discussions: YSI analysis showed that HCAECs take up more glutamine and glucose as glutamine concentration increased from 0 mM to 2 mM (Fig 1-A). We also found that the glutamate and lactate concentration in the media increased with increasing glutamine concentration (Fig 1-B), which indicates an increase in EC oxidative respiration and glycolysis. Seahorse data further confirmed increased metabolism by showing that increasing glutamine concentration increased the oxygen consumption rate (OCR) and glycolytic proton efflux rate (GlycoPER), indicating that both mitochondrial respiration and glycolytic activity increase (Fig 1-C & D).


Conclusions: Glutamine plays a significant role in EC metabolic activity. Specifically, glycolysis and mitochondrial oxidative respiration increase as exogenous glutamine concentration increases. In some metabolic disorders, glutamine concentration in the serum is reduced, which may contribute to EC dysfunction. However, our research suggests that glutamine supplementation can increase the metabolic activity of endothelial cells and potentially restore their function.




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