Poster T12 - Glutamine as a potential therapeutic target for inflammatory diabetic cardiovascular complications.

Introduction: Diabetes presents a significant global health challenge, with approximately 537 million affected adults today and an anticipated rise to 643 million by 2030. Individuals with diabetes are at higher risk of micro- and macrovascular complications, emphasizing the urgent need to develop effective therapeutic strategies to prevent diabetic cardiovascular disease (CVD). CVD initiation involves dysfunction of endothelial cells (EC), which line the vascular wall and regulate vascular homeostasis. Hyperglycemia induces EC dysfunction through increased oxidative stress and inflammation, along with reduced availability of the vasodilator nitric oxide. Furthermore, diabetic patients exhibit decreased serum glutamine, which could also affect immune function and oxidative balance, to induce vascular complications. Clinical trials have shown that oral glutamine supplementation enhances vascular health by reducing CVD risk factors, including inflammatory modulators like interleukin 6 (IL-6), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1). Despite these findings, the specific impact of glutamine on cardiovascular health in diabetes remains largely unexplored. I hypothesize that glutamine reduces endothelial cells’ oxidative stress in hyperglycemia, improves inflammatory response, and increases nitric oxide bioavailability, positioning glutamine as a potential therapeutic agent for prevention of diabetic cardiovascular complications.

Materials and

Methods: Male human coronary artery endothelial cells (HCAEC; passages 5-9) were cultured in Endothelial Growth Medium-2 (EGM-2) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine. Once confluent, HCAECs were treated with Dulbecco's Modified Eagle Medium (DMEM) supplemented with the EGM-2 additives, 10% FBS, 1% penicillin-streptomycin, either 5.5 mM or 15 mM glucose, and varied glutamine concentrations (0, 0.5 mM, or 2 mM). We measured glutamine consumption and glutamate production after 48 hours using a YSI Bioanalyzer. The Seahorse XF Mito stress test was used to determine the oxygen consumption rate (OCR) in real time as a measure of mitochondrial respiration. Cells were incubated with Seahorse DMEM media with the appropriate glutamine concentration for an hour before the assay, and the media was refreshed right before starting the assay. Finally, an inflammatory endothelial phenotype was induced by adding 10 ng/mL of the inflammatory cytokine TNF-α to confluent HCAEC for 4 hours. Leukocytes were isolated from freshly drawn blood using an Acrodisc syringe and stained with 1 μM of Calcein for 30 minutes. Then, ECs were incubated with 2 million leukocytes for 10 minutes and rinsed to remove unadhered leukocytes. Samples were imaged using the FITC channel in a fluorescent microscope, and the number of green fluorescent particles was counted using ImageJ software. Nuclei were stained with Hoechst for 10 minutes before incubation with leukocytes for cell count. Data were plotted using GraphPad Prism, and statistical analysis was performed using one-way ANOVA. p-values less than 0.05 were considered statistically significant.

Results, Conclusions, and Discussions: Results and discussion: HCAECs took up more glutamine (up to 5 fold higher) and secreted more glutamate (up to 2 fold higher) as glutamine concentration increased from 0 to 2 mM in both normal and high glucose media (Figs 1A-D). In normal but not high glucose media, glutamine also increased the oxygen consumption rate (OCR) (Fig 1E, F), which correlates with basal respiration, by 125% at 2 mM glutamine (p < 0.0001). The number of leukocytes adhered to the ECs increased by 2-fold (Fig 1F) in both normal (5 mM) and high (15 mM) glucose conditions as extracellular glutamine concentration increased from 0 to 2 mM. Additionally, HCAECs treated with 0.5 or 2 mM glutamine had higher cell counts compared to HCAEC without glutamine (Fig 1-G), emphasizing that glutamine promotes EC survival when stressed by the pro-inflammatory cytokine TNF-α.

Conclusion: Glutamine plays a critical role in endothelial cell (EC) metabolism and survival. Our research indicates that glutamine may be required for EC survival and inflammatory signaling when EC are stressed with pro-inflammatory cytokines like TNF-α. Thus, glutamine may modulate inflammatory signaling pathways in the context of diseases like diabetes. This research enhances our understanding of how glutamine impacts ECs under hyperglycemic conditions and highlights the promise of glutamine as a novel therapeutic strategy for preventing diabetic CVD.

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