AbstractTriglycerides (TG) are the most important energy source in mammals. Adipose Triglyceride Lipase (ATGL) is the rate limiting enzyme in the catabolism of TG to generate diglycerides (DG) and free fatty acids (FFA). ATGL-mediated TG hydrolysis is activated by Comparative Gene Identification 58 (CGI-58). However, humans and mice lacking either CGI-58 or ATGL show phenotypic differences in organ lipid homeostasis including the liver and skin, suggesting an ATGL independent function of CGI-58 at least in these organs.To investigate the potential ATGL-independent function of CGI-58 in the liver, we examined the impact of liver-specific CGI-58 or ATGL-deficiency on hepatic lipid homeostasis and metabolism in mice. Hepatic TG accumulation was more pronounced in mice lacking CGI-58 in the liver (livCGI-58ko) compared to mice with hepatic ATGL-deficiency (livATGLko). Notably, cholesterol ester (CE) levels were exclusively increased in the liver of livCGI-58ko mice suggesting an ATGL-independent role of CGI-58 in liver lipid catabolism. Cytosolic lipid hydrolytic activities, however, were comparable in livCGI-58ko and livATGLko mice. Hepatic CGI-58-deficiency interferes with the peroxisomal pathway, as mRNA levels of peroxisomal genes were significantly decreased. Impaired expression or absence of lysosomal acid lipase (LAL) provokes marked TG and CE accumulation in the liver, which prompted us to measure LAL-activities in livCGI-58ko mice. Liver-specific CGI-58-deficiency decreases acid TGH and CEH activities in liver preparations, whereas no changes were observed in preparations of livATGLko mice. Furthermore, analysis of mRNA levels showed a decrease in investigated lysosomal genes in livCGI-58ko compared to livATGLko mice. These findings suggest that hepatic CGI-58 deficiency impacts lysosomal lipid catabolism and function.