SUN-042 Loss of the 18-Kda Translocator Protein Tspo Destabilizes Cholesterol Homeostasis in a Hepatic Steatosis Cell Model

The 18-kDa translocator protein TSPO is a conserved ubiquitous high affinity cholesterol binding protein localized in the outer mitochondrial membrane. It plays a critical role in the segregation and transport of free cholesterol (FC) from the outer to the inner mitochondrial membrane in steroidogenic cells. Numerous studies have shown the presence of TSPO in liver. Imaging studies in humans, using specific TSPO ligands, showed that TSPO is highly expressed and accurately mirrors the histological picture of non-alcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH). The function of TSPO in simple steatosis and NASH is unknown. Wild type Huh7 (WT) is a well-differentiated human hepatocyte-derived carcinoma cell line, which contains TSPO in mitochondria. In the present study, we generated a stable TSPO knockdown (TSPO KD) in the Huh7 cell line. To mimic hepatic steatosis in vitro, we treated WT and TSPO KD cells with either acetylated LDL (AcLDL), to deliver cholesterol into the cells, or AcLDL plus 58035, an Acetyl-CoA Acetyltransferase (ACAT) and thus cholesterol esterification inhibitor, to induce the accumulation of FC. AcLDL treatment significantly increased FC levels in WT cells; this effect was more pronounced in TSPO KD cells. In contrast, treatment of the cells with AcLDL and 58035 resulted in reduced FC levels likely due to the damage induced by FC accumulation that led to reduced cell proliferation. Considering that TSPO binds FC with high affinity, we investigated the underlying mechanism/link between TSPO KD and FC accumulation by examining the conversion between FC and cholesterol ester (CE) upon AcLDL treatment. The results obtained showed that knocking down TSPO expression reduced ACAT2 (transforming FC to CE) levels, but not cholesterol ester hydrolase (CEH) converting CE to FC, expression. TSPO was found to directly interact with ACAT2. Under these conditions, the accumulation of FC led to the appearance of lipid droplets (LD) of increased size in TSPO KD compared to WT cells, suggesting that TSPO plays a role in LD formation and/or dynamics in liver cells. GRP78, IRE1A, and PERK endoplasmic reticulum stress markers were also elevated in TSPO KD cells following AcLDL treatment while the mitochondrial membrane potential decreased compared to WT cells. Moreover, increased numbers of lysosomes and autophagosomes were observed in TSPO KD cells, an effect further potentiated by the treatment with AcLDL plus 58035. This finding was validated by the up regulation of the LAMP1 and LC3B, lysosome and autophagic structure markers, respectively. Taken together, these data suggest that under cholesterol treatment, TSPO loss would trigger ER stress, mitochondrial dysfunction, and lipid droplet accumulation via FC accumulation, and ultimately cause intracellular protein and organelle degradation, hallmarks of hepatic steatosis.