Lysosomal acid lipase is essential in cholesterol-mediated mTORC1 signaling activation by maintaining a balance between cholesterol ester and free cholesterol in zebrafish

Cholesterol, as a signaling molecule, plays a critical role in regulating the mTORC1 signaling pathway within cells. Lysosomal acid lipase (LAL) is responsible for hydrolyzing cholesterol ester and triglyceride in lysosomes. However, the involvement of LAL in the regulation of the mTORC1 signaling pathway in animals remains controversial. Our study found that lal-deficient zebrafish exhibited retarded growth and reduced body protein content compared to wild type zebrafish. Correspondingly, the mTORC1 signaling pathway was significantly inhibited in the liver of lal−/− zebrafish. Additionally, both lal knockdown and lalistat (LAL inhibitor) treatments led to the accumulation of cholesterol ester (CE) and a decrease in free cholesterol (FC) within lysosomes of zebrafish liver (ZFL) cells. This imbalance inhibited the recruitment of mTORC1 to the lysosomal surface and suppressed the mTORC1 signaling pathway. Moreover, FC treatment promoted the recruitment of mTORC1 to the lysosomal surface and activated the mTORC1 signaling pathway regardless of LAL, whereas LDL-dependent mTORC1 activation required LAL. However, knockdown of slc38a9 (a cholesterol sensor) blocked the recruitment of mTORC1 in ZFL cells treated with FC or LDL. Furthermore, the interaction between P14 (a component of Ragulator complex) and RagA/C was weakened in ZFL cells following treatment with FC or LDL but was enhanced upon slc38a9 knockdown. In addition, both lal siRNA and lalistat treatments increased the interaction between P14 and RagA/C. Our findings indicated that LAL dysfunction hindered the recruitment and activation of mTORC1 through the Slc38a9-mediated lysosomal mTORC1-scaffolding complex in ZFL cells. This inhibition was associated with a decrease in lysosomal FC, which is detected by cholesterol sensor SLC38A9. Therefore, interventions targeting the role of LAL role in the hydrolysis of cholesterol esters within lysosomes could offer promising therapeutic strategies for diseases associated with dysregulated mTORC1 signaling in animals.