Inhibition of Niemann-Pick-type C1-like1 by ezetimibe activates autophagy in human hepatocytes and reduces mutant α1-antitrypsin Z deposition

Abstract

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

Capitalizing on the Autophagic Response for Treatment of Liver Disease Caused by Alpha-1-Antitrypsin Deficiency and Other Genetic Diseases

“…Although the mechanism of this effect was not elucidated, it was independent of mTOR pathway. Another drug that has recently been shown to activate autophagy specifically is the cholesterol-lowering agent ezetimibe [28]. This drug was investigated by Yamamura et al because cholesterol depletion had an effect on autophagy.…”

“…The studies suggest that, by inhibiting NPC1L1, ezetimibe reduces the recruitment of mTOR to the lysosome and therein inhibits mTORC1 activity to activate autophagy. Most exciting, the authors found that ezetimibe reduces cellular accumulation of ATZ in primary cultures of human hepatocytes engineered for expression of ATZ [28]. …”

“…This complex is suppressed by specific inhibitors such as rapamycin and its analogs, which therefore enhance autophagy [43]. A recent study showed that ezetimibe perturbed the cholesterol homeostasis and decreased mTOR recruitment to late endosome/lysosome; thus ezetimibe is thought to induce autophagy by suppressing mTORC1 [28]. The mTOR signaling pathway can also be regulated by specific inhibitors target to the upstream of mTOR, for example, PI3K inhibitor [44].…”

The Interpretation of Cholesterol Balance Derived Synthesis Data and Surrogate Noncholesterol Plasma Markers for Cholesterol Synthesis under Lipid Lowering Therapies

“…Therefore, it must be assumed that EZE may exert a so far unknown hepatic interaction leading to increased DNCS and reduced biliary chol secretion. This may or may not be related to the observation of Yamamura et al [51] that EZE treatment is associated with increased autophagy in human hepatocytes.…”

Relevance of autophagy to fatty liver diseases and potential therapeutic applications

“…During starvation, about 30% of hepatic proteins are decreased in wild-type mice in 24h of fasting, which becomes insignificant in conditional knockout mice of Atg7 (Mizushima and Klionsky 2007; Komatsu et al 2005). In fact, protein turnover via autophagy is related to multiple hepatic pathophysiological conditions, including the clearance of misfolded mutant proteins in alpha-1-antitrypsin deficiency (Puri and Chandra 2014; Hidvegi et al 2010; Yamamura et al 2014), and in the clearance of the inclusion bodies (Strnad et al 2008; Zatloukal et al 2007; Harada et al 2008). …”