ABSTRACT Liver is the major hub that is responsible for regulating both plasma cholesterol and triglyceride levels as it is involved in their synthesis, secretion, and clearance. Several studies have reported that any errors in these processes will lead to metabolic diseases such as non-alcoholic fatty liver disease and hypercholesterolemia. Hypercholesterolemia will eventually result in atherosclerosis that is the leading cause of death in US. Also, plasma cholesterol and triglyceride levels are highly heritable and several human genome-wide association studies have identified hundreds of genetic loci that could control the levels of these plasma lipids. Unfortunately, only a small number of these loci have been translated into a gene target and validated through experimental studies. Therefore, there is a critical need for identifying and validating novel biologically causal candidate genes that could serve as a therapeutic target. To this end, we used an integrative genomics approach in a panel of diverse inbred strains of mice to integrate information on natural genetic variations (genomics) with molecular phenotypes (transcriptomics) and clinical phenotypes (phenomics) and identified a liver gene, liver pyruvate kinase (L-PK), in regulating hepatic steatosis via mitochondrial involvement. When L-PK was downregulated, both insulin sensitivity and liver triglyceride levels improved, while L-PK overexpression overloaded the mitochondrial function, augmenting the disease condition. More importantly, all these observations were made in male but not female mice. Serendipitously, we also reported that L-PK was associated with regulating plasma cholesterol levels. Over the next five years, we aim to mechanistically dissect this relationship and their underlying pathways in a sex-specific manner. The studies proposed in this application aim to address this by using a combination of an array of experimental approaches including gonadectomy, lipidomic, metabolomic, transcriptomic and bioenergetic studies, and a novel humanized hyperlipidemia-induced atherosclerosis animal model. We propose to determine which of the cellular mechanisms (cholesterol synthesis, secretion, and clearance) are altered by L-PK in altering plasma cholesterol levels and finally, test the hypothesis that L-PK regulates hyperlipidemia-induced atherosclerosis in a sex-specific manner (Aim 1), and determine the sex- specific signaling pathways (AR and PTEN-PI3K-AKT signaling) altered by L-PK in mediating this phenomenon (Aim 2). Upon completion of this grant, we will be able to define how L-PK reprograms cholesterol metabolism, thereby influencing atherosclerosis progression in a sex-specific manner. Ultimately, this information will aid in identifying personalized therapeutic implications of L-PK in hypercholesterolemia.