Autophagy can degrade aggregate-prone proteins, but excessive autophagy can have adverse effects. It would be beneficial if autophagy could be enhanced in a cell typespecific manner, but this has been difficult because the basic mechanism of autophagy is common. In the present study we found that inhibition of Niemann-Pick-type C1-like 1 (NPC1L1) by ezetimibe activates autophagy only in hepatocytes and small intestinal epithelia, but not in other cells. Ezetimibe induced accumulation of free cholesterol in the late endosome/lysosome and increased partitioning of a Ragulator component, LAMTOR1, in rafts. The latter change led to down-regulation of mammalian target of rapamycin (mTOR)C1 activity by decreasing mTOR recruitment to the late endosome/lysosome and activated autophagy. A primary effect of ezetimibe was found to be a decrease of free cholesterol in the plasma membrane, because all the results caused by ezetimibe were suppressed by supplementation of cholesterol as a methyl-b-cyclodextrin complex. By enhancing autophagy in human primary hepatocytes with ezetimibe, insoluble mutant a1-antitrypsin Z was reduced significantly. Conclusion: Inhibition of NPC1L1 by ezetimibe activates autophagy in human hepatocytes by modulating cholesterol homeostasis. Ezetimibe may be used to ameliorate liver degeneration in a1-antitrypsin deficiency. (HEPATOLOGY 2014;59:1591-1599 M acroautophagy (hereafter referred to as autophagy) is a degradation process that is conserved in all eukaryotes, in which cytoplasmic portions are enclosed within the autophagosome and eventually digested in the lysosome. The primary role of autophagy is to maintain cellular homeostasis against various stresses, but recent studies revealed that autophagy is involved in a wider range of activities. 1 With regard to diseases, activation of autophagy was shown to reduce potentially toxic protein aggregates. 2,3 It is notable that a lack of constitutive autophagy induced the deposition of protein aggregates and caused neurodegeneration. 1 The results suggest that autophagy activators may be used to treat degenerative diseases. 4 To harness the benefit of autophagy and avoid unwanted side effects to the furthest extent possible, it would be useful if autophagy could be activated in restricted cell types. This may not be an easy task, however, because the basic molecular mechanism for autophagic induction and progression appears to be common in all mammalian cells.We supposed that cellular cholesterol may be a possible point of manipulation to induce cell type-specific autophagy. In a previous study, we found that cholesterol depletion activates autophagy, suggesting that the Abbreviations: ATZ, a1-antitrypsin Z; CBZ, carbamazepine; DeLS, delipidated serum; DMEM, Dulbecco's modified Eagle's medium; DRM, detergent-resistant membrane; FC, free cholesterol; FCS, fetal calf serum; LE/Ly, late endosomes/lysosome; LPDS, lipoprotein-deficient serum; MbCD, methyl-b-cyclodextrin; NPC1L1, Niemann-Pick-type C1-like1; S6K, p70 ribosomal S6 kinase.From the