Abstract

Background: Chronic EtOH insult is known to lead to an accumulation of lipid droplets (LDs) in hepatocytes resulting in hepatic steatosis. Our previous study reported that EtOH resulted in a 10-fold reduction of the LD-associated segregase VCP/p97, a protein implicated in LD protein clearance. Concomitant with this decrease was a 6-fold increase in the association of the hydroxysteroid enzyme HSD17β13 known to play an important role in hepatic steatosis. From these findings we predict that this alteration in the LD-proteome can lead to a disruption of LD catabolism and hepatocellular steatosis in vivo. The GOAL of this current study was to experimentally reduce hepatocellular VCP levels via a targeted knock out in mice to test the effects on LD-associated HSD17β13 levels and hepatic steatosis.

Methods: A hepatocyte specific knockout of VCP was used along with histology, immunofluorescence microscopy, western blot analysis in combination with transient knockdown and manipulation of cultured hepatocytes.

Results: The liver-specific knockout of VCP resulted in a dramatic >10-fold increase in liver triglycerides and hepatic LDs in just 7days on a normal chow diet. Importantly, 13-day KO mice displayed livers with a 5-fold increase in HSD17β13. Parallel studies using primary isolated mouse hepatocytes showed that acute inhibition of VCP activity with either the drug DBeQ, or siRNA mediated knockdown, resulted in an elevation of HSD17β13 levels and a 5-fold increase in LD content. We have found that HSD17β13 is a mono-ubiquitinated protein predicted to be removed from the LD surface to the proteosome by VCP. Surprisingly, treatment of hepatoctyes with the proteasome inhibitor MG132 had no effect on HSD17β13 levels while inhibition of lysosome function with chloroquine resulted in a 6-fold increase of HSD17β13. In support of this finding IF staining of these hepatocytes shows a marked colocalization of lysosomes with HSD17β13-associated LDs.

Conclusion: This study suggests that a reduction in the levels of the VCP segregase by EtOH insult, or targeted KO, results in a substantial increase in the hepatocellular levels, and LD association of HSD17β13. Hepatocytes without VCP are unable to target ubiquitylated HSD17β13 to the lysosome for degradation leading to hepatic steatosis. These findings provide new insights into the cellular mechanisms by which EtOH exposure disrupts normal hepatic lipid catabolism. Supported by R01AA020735 (to M.A.M. and C.A.C.).