(LB02-06-25) Betaine Improves Endothelial Cell Function by Increasing Nitric Oxide Levels Through Activation of eNOS and Inhibition of ET-1 Expression in an In Vitro System

Objectives: Betaine, or trimethylglycine, is a methyl donor with a crucial role in one-carbon metabolism and is known for lowering homocysteine levels, which may help cardiovascular health by preventing atherosclerosis. This study aimed to investigate if betaine supplementation can directly affect endothelial function using an in vitro model.

Methods: This study utilized a Human Pulmonary Microvascular Endothelial (HPMEC) cell line as an in vitro model. HPMEC cells were treated with betaine, alongside controls including VEGF (Vascular endothelial growth factor as a positive control), and L-NAME (define L-NAME as a negative control) for a specific duration. Supernatants were used to measure NO levels using Nitrate/Nitrite colorimetric assay, and endothelin-1 levels using ELISA. The cells were harvested for western blot analysis to assess eNOS and phosphorylated eNOS protein expression.

Results: Betaine treatment led to an increase in NO production (16.2% increase, p >0.05) in HPMEC cells as early as 10 minutes post-treatment, with a significant increase (172% increase, p< 0.01) observed at 1 hour. The cotreatment with L-NAME, a known inhibitor of NO production, significantly attenuated (40% decrease, p< 0.05) but did not fully inhibit the increase in NO production induced by betaine. These findings were corroborated by western blot analysis, which showed that betaine treatment increased the activating phosphorylation levels of eNOS, with phosphorylation increasing over time. The activation effect of eNOS by betaine was diminished when cotreated with L-NAME. In addition, betaine treatment reduced the expression of ET-1, a well-known potent vasoconstrictor, which is elevated by L-NAME treatment.

Conclusions: Betaine effectively enhances NO production and eNOS activation in HPMEC cells, demonstrating its potential role in modulating endothelial function. L-NAME partially inhibits this effect, indicating that NO production and eNOS activation are critical pathways influenced by betaine. Furthermore, betaine's ability to reduce ET-1 expression suggests its potential to counteract vasoconstrictive forces. These findings highlight betaine's promising therapeutic implications in vascular health management.

Funding Sources: This study is funded by Abbott Nutrition.