Lopresti, Michael2022-02-152022-02-152021-12https://hdl.handle.net/11299/226390University of Minnesota Ph.D. dissertation. December 2021. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Douglas Mashek. 1 computer file (PDF); vi, 104 pages.Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid droplets (LD) in hepatocytes. NAFLD development and progression is associated with an increase in hepatic cholesterol levels and decreased autophagy and lipophagy flux. Previous studies have shown that the expression of lysosomal acid lipase (LAL, encoded by the gene LIPA), which can hydrolyze both triglyceride and cholesteryl esters, is inversely correlated with the severity of NAFLD. Here, we examine how LAL dysfunction promotes the unregulated synthesis of cholesterol and whether LAL overexpression protects against the development of NAFLD. We used a combination of LAL inhibition and knockdown to evaluate how cholesterol liberated from LAL regulates sterol regulatory element binding protein 2 (SREBP2). Our data indicates that the lysosomal cholesterol pool that regulates SREBP2 is heavily reliant on exogenous lipid sources and is converted into oxysterols to provide negative feedback. We also predicted that overexpressing LIPA in the livers of mice fed a Western diet would prevent the development of NAFLD. As expected, mice fed the Western diet exhibited numerous markers of NAFLD, including hepatomegaly, lipid accumulation, and inflammation. Unexpectedly, LAL overexpression did not attenuate steatosis and had only minor effects on neutral lipid composition. However, LAL overexpression exacerbated inflammatory gene expression and infiltration of immune cells in mice fed the Western diet. LAL overexpression also resulted in abnormal phagosome accumulation and lysosomal lipid accumulation depending upon the dietary treatment. Overall, we show a disconnect in the regulation of cholesterol resulting from LAL knockdown versus overexpression. While knockdown results in a broken feedback loop, the overexpression of LAL does not significantly alter cholesterol pools. Ultimately, hepatic overexpression of LAL drives immune cell infiltration and inflammation and does not attenuate the development of NAFLD, suggesting that targeting LAL expression may not be a viable route to treat NAFLD in humans.enThesis or Dissertation