Project Summary/Abstract Cardiovascular disease (CVD) arising from atherosclerosis remains a leading cause of death. Current lipid- lowering therapies cannot completely eliminate CVD risk, likely due to lack of influence on other major risk factors beyond dyslipidemia. Dysregulated metabolism of amino acids (AA) was reported in cardiometabolic diseases with lower circulating glycine as a common denominator in acute myocardial infarction, type 2 diabetes (T2D), obesity and nonalcoholic fatty liver disease (NAFLD). While potential mechanisms by which glycine protects from T2D and NAFLD have been identified, the role of glycine in cholesterol metabolism and atherosclerosis is unknown. We recently reported glycine as the most potent AA in lowering lipid accumulation in macrophages. In our preliminary studies here, suppression of key pathways driving glycine biosynthesis was evident in atherogenic conditions in humans and mice. Glycine deprivation using our newly developed AA- modified Western diets (WD) enhanced hypercholesterolemia and atherosclerosis in apoE-/- mice. In contrast, glycine treatment was protective of those phenotypes, while lowering hyperglycemia and hepatic steatosis, which are other major CVD risk factors. This was associated with lower lipid peroxidation and induction of pathways driving glutathione biosynthesis and transport of cholesterol into bile. Furthermore, we identified a glycine-based compound (DT-109) with dual cholesterol- and glucose-lowering properties in mice. Our findings led us to hypothesize that glycine-based treatments are atheroprotective by inducing glutathione-mediated antioxidant defense and hepatic-intestinal cholesterol excretion. The long-term objectives of this study are to ascertain impaired glycine metabolism in atherogenesis, establish antiatherogenic glycine-based therapy able to reduce other CVD risk factors and uncover the underlying mechanisms. Aim 1 will assess impaired synthesis and increased utilization of glycine in atherogenesis. We will use transcriptomics and metabolomics in apoE-/-, our new AGXT1-/- mice and human fatty liver samples. The link between variants in genes driving glycine metabolism and atherosclerotic disease will be determined by human GWAS. Aim 2 will determine the effects of lower and higher glycine availability on atherosclerosis development. We will use apoE-/- mice fed WD with or without glycine or treated with DT-109. AGXT1-/- mice crossbred with apoE-/- mice will be used to determine the role endogenous glycine. Aim 3 will establish DT-109 as a glycine-based therapy for atherosclerosis, its pharmacokinetics and the underlying mechanisms. Atherosclerotic apoE-/- mice will be used to assess glycine incorporation to glutathione by metabolomics and fluxomics and hepatic-intestinal cholesterol excretion. We will study the ability of DT-109, compared to glycine, to regress established atherosclerosis in apoE-/- mice. Completing these studies will set the basis for novel glycine-based treatments for atherosclerosis, while the outlined career development plan, including hands-on training, coursework and seminars, will allow me to develop my long-term goal of becoming an independent investigator in cardiometabolic research.