Updated project metadata. Nitric oxide (NO) is a crucial gaseous signaling molecule engaged in a variety of physiological and pathological processes. It is produced by three isoenzymes called nitric oxide synthases (NOS): neuronal, inducible and endothelial NOS (eNOS). eNOS-derived NO plays an important role in endothelium-dependent vasodilation as well as in lipid and glucose homeostasis in the liver. In addition, it has been shown that NO exert a beneficial effect on mitochondrial biogenesis and respiration. Thus, the aim of our study was to used iTRAQ-based quantitative proteomics to investigate the changes in protein expression in the mitochondrial and cytosolic fractions isolated from the liver of the double (apolipoprotein E (apoE) and eNOS) knockout (apoE/eNOS-DKO) mice as compared to apoE-/- mice – an animal model of atherosclerosis and hepatic steatosis. Collectively, our proteomic approach revealed the increased expression of proteins related to gluconeogenesis, fatty acids and cholesterol biosynthesis as well as the decreased expression of proteins participated in triglyceride breakdown, cholesterol transport, protein transcription & translation and processing in endoplasmic reticulum (ER). Moreover, one of the most down-regulated proteins were major urinary proteins (MUPs), which are abundantly expressed in the liver and are involved in the regulation of lipid and glucose metabolism as well as mitochondrial function. However, the exact functional consequences of the revealed alterations require further investigation.