Abstract 1092 Hepatic nonvesicular cholesterol transport is critical for systemic lipid homeostasis

Objectives: In cell models, nonvesicular cholesterol transport by the Aster protein family has been linked to changes in the 'accessible' pool of plasma membrane cholesterol and the regulation of endoplasmic reticulum sterol metabolism. However, the relevance of these mechanisms in vivo is unknown. Methods: To study Aster function in the liver, we generated mice with hepatocyte-specific deletion of either Aster-A (L-A knockout (KO)), Aster-C (L-C KO) or both (L-A/C KO) and conducted lipidomic analysis, accessible cholesterol staining with in vivo tracer studies with [14C] cholesterol. Results: We show that Aster-dependent nonvesicular transport mediates the missing terminal step in HDL reverse cholesterol transport (RCT): the flux of cholesterol through the liver into the gut for excretion. Loss of Aster expression blocks this movement and consequently raises plasma and tissue cholesterol levels. Furthermore, our results provide a molecular explanation for the longstanding mystery of how HDL cholesterol is transported in the liver. Mechanistically, we reveal that sphingomyelin-sequestered cholesterol and accessible cholesterol interchange under physiological conditions through a novel fatty acid-sphingomyelinase-Aster axis. Conclusions: The importance of vesicular cholesterol transport (e.g. the LDL receptor endocytosis pathway) in physiology is well established. By contrast, the importance of nonvesicular pathways (direct protein-mediated transfer of lipids between membranes) in systemic metabolism is completely unknown. Our paper implicates the Aster family of nonvesicular transporters in fundamental mechanisms critical for whole body cholesterol homeostasis. Abstract Body: In cell models, changes in the 'accessible' pool of plasma membrane (PM) cholesterol are linked with the regulation of endoplasmic reticulum sterol synthesis and metabolism by the Aster family of nonvesicular transporters; however, the relevance of such nonvesicular transport mechanisms for lipid homeostasis in vivo has not been defined. Here we reveal two physiological contexts that generate accessible PM cholesterol and engage the Aster pathway in the liver: fasting and reverse cholesterol transport. During fasting, adipose-tissue-derived fatty acids activate hepatocyte sphingomyelinase to liberate sequestered PM cholesterol. Aster-dependent cholesterol transport during fasting facilitates cholesteryl ester formation, cholesterol movement into bile and very low-density lipoprotein production. During reverse cholesterol transport, high-density lipoprotein delivers excess cholesterol to the hepatocyte PM through scavenger receptor class B member 1. Loss of hepatic Asters impairs cholesterol movement into feces, raises plasma cholesterol levels and causes cholesterol accumulation in peripheral tissues. These results reveal fundamental mechanisms by which Aster cholesterol flux contributes to hepatic and systemic lipid homeostasis. This work was supported by NIH grant R01 DK126779 and Fondation Leducq Transatlantic Network of Excellence (19CVD04). Xu Xiao was supported by AHA Postdoctoral Fellowship (18POST34030388). John Paul Kennelly is supported by AHA Postdoctoral Fellowship (903306).