Ubiquitously expressed lipid droplet protein PLIN2 has been used as a protein marker for intracellular lipid droplets. The expression of Plin2 correlates positively with the amount of lipids in the cells. Plin2 mice have an ~60% reduction in hepatic triglyceride (TG) content, and are protected against fatty liver disease. Our recent study indicates that Plin2 deficiency elevates the autophagy flux, establishing a connection between lipid catabolism and autophagy in which PLIN2 plays a negative regulatory role. In the absence of PLIN2, many other proteins are found enriched on the lipid droplet, and among them, ABHD5 is known to involve in lipolysis elicited by neutral lipase as well as autophagy. Using the CRISPR technology we established Abhd5-null, Plin2-null and Abhd5/Plin2-null murine AML12 hepatocyte models to study whether and how ABHD5 mediates the TG reduction in Plin2-deficiency. Plin2-null cells have reduced (~50%) intracellular TG content compared to the WT while Abhd5-null cells have three times as much. Abhd5/Plin2-null cells have similar level of intracellular TG as the Abhd5-null cells. Western blot analyses reveal a blockage of autophagy flux in the Abhd5-null cells. By reintroducing, through lentiviral expression vectors, the WT as well as several mutant Abhd5 proteins back to these hepatocyte models we discovered that some Abhd5 mutants that cause human Chanarin-Dorfman Syndrome results in the translocation of the ABHD5 protein from cytosol to nucleus. Using these models we examine the dynamics of cellular TG contents, neutral lipase activities, and autophagy flux in order to delineate the role of ABHD5 in Plin2-deficiency mediated autophagy augmentation.1