Hepatic NPC1L1 overexpression attenuates alcoholic autophagy in mice

Wang,Youlin; Yang,Pan; Zhang,Bo; Ding,Yao; Lei,Shun; Hou,Yuning; Guan,Xiaoqing; Li,Qingwang

doi:10.3892/mmr.2019.10549

Hepatic NPC1L1 overexpression attenuates alcoholic autophagy in mice

Authors:
- Youlin Wang
- Pan Yang
- Bo Zhang
- Yao Ding
- Shun Lei
- Yuning Hou
- Xiaoqing Guan
- Qingwang Li
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Affiliations: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China, Center for Molecular and Translational Medicine, Georgia State University, Atlanta, GA 30303, USA, Department of Oncology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, P.R. China, Department of Oncology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, P.R. China
Published online on: August 1, 2019 https://doi.org/10.3892/mmr.2019.10549
Pages: 3224-3232
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Alcohol consumption causes liver steatosis in humans. Metabolic disorders of lipids are one of the factors that cause liver steatosis in hepatocytes. Hepatic Niemann‑Pick C1‑like 1 (NPC1L1) regulates lipid homeostasis in mammals. The relationship between NPC1L1 and autophagy in those with a history of alcohol abuse is unclear. The present study aimed to investigate the function of NPC1L1 in the activation of hepatic autophagy in a mouse model with a human (h)NPC1L1 transgene under alcohol feeding conditions. The mice expressing hNPC1L1 (Ad‑L1) or controls (Ad‑null) were created by retro‑orbital adenovirus injection. The Ad‑L1 and Ad‑null mice were fed with alcohol or a non‑alcoholic diet to mimic chronic alcohol consumption in humans. Hepatic autophagy was demonstrated in isolated primary hepatocytes by monitoring autophagic vacuoles under fluorescence microscopy, and by western blotting for autophagic makers. Isolated hepatocytes from the livers of Ad‑L1 mice were treated with different doses of ezetimibe to study the restoration of autophagy. Chronic alcohol feeding caused liver injury and steatosis, shown by significantly higher levels of plasma alanine transaminase and aspartate transaminase activity, and by hematoxylin and eosin staining in Ad‑L1 and Ad‑null mice. Compared to Ad‑null control mice, the microtubule‑associated proteins 1A/1B light chain 3 (LC3) particles in the isolated hepatocytes of Ad‑L1 mice were decreased, both under alcohol and non‑alcoholic feeding. The ratio of LC3II/LC3I was significantly decreased, and the level of p62/sequestosome‑1 protein was significantly increased in Ad‑L1 mice compared with Ad‑null mice after alcohol feeding. Levels of LC3II protein were statistically increased in hepatocytes isolated from Ad‑L1 mice with ezetimibe treatment. The increase in LC3II expression was dose dependent. Within the tested range, it reached its highest level at 40 µM. The livers of Ad‑L1 mice represent a more human‑like state for the study of hepatic autophagy. Hepatic expression of human NPC1L1 resulted in an inhibition of autophagy; it may contribute to alcoholic fatty liver disease in humans.

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1	Ishak KG, Zimmerman HJ and Ray MB: Alcoholic liver disease: Pathologic, pathogenetic and clinical aspects. Alcohol Clin Exp Res. 15:45–66. 1991. View Article : Google Scholar : PubMed/NCBI
2	Ramaiah S, Rivera C and Arteel G: Early-phase alcoholic liver disease: An update on animal models, pathology, and pathogenesis. Int J Toxicol. 23:217–231. 2004. View Article : Google Scholar : PubMed/NCBI
3	Walther TC and Farese RV Jr: Lipid droplets and cellular lipid metabolism. Ann Rev Biochem. 81:687–714. 2012. View Article : Google Scholar : PubMed/NCBI
4	Lass A, Zimmermann R, Haemmerle G, Riederer M, Schoiswohl G, Schweiger M, Kienesberger P, Strauss JG, Gorkiewicz G and Zechner R: Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in chanarin-dorfman syndrome. Cell Metab. 3:309–319. 2006. View Article : Google Scholar : PubMed/NCBI
5	Ding WX, Li M, Chen X, Ni HM, Lin CW, Gao W, Lu B, Stolz DB, Clemens DL and Yin XM: Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice. Gastroenterology. 139:1740–1752. 2010. View Article : Google Scholar : PubMed/NCBI
6	Noh BK, Lee JK, Jun HJ, Lee JH, Jia Y, Hoang MH, Kim JW, Park KH and Lee SJ: Restoration of autophagy by puerarin in ethanol-treated hepatocytes via the activation of AMP-activated protein kinase. Biochem Biophys Res Commun. 414:361–366. 2011. View Article : Google Scholar : PubMed/NCBI
7	Thomes PG, Ehlers RA, Trambly CS, Clemens DL, Fox HS, Tuma DJ and Donohue TM: Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells. Autophagy. 9:63–73. 2013. View Article : Google Scholar : PubMed/NCBI
8	Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo AM and Czaja MJ: Autophagy regulates lipid metabolism. Nature. 458:1131–1135. 2009. View Article : Google Scholar : PubMed/NCBI
9	Li Y, Wang S, Ni HM, Huang H and Ding WX: Autophagy in alcohol-induced multiorgan injury: Mechanisms and potential therapeutic targets. Biomed Res Int. 2014:4984912014. View Article : Google Scholar : PubMed/NCBI
10	Rasineni K, Donohue TM Jr, Thomes PG, Yang L, Tuma DJ, McNiven MA and Casey CA: Ethanol-induced steatosis involves impairment of lipophagy, associated with reduced Dynamin2 activity. Hepatol Commun. 1:501–512. 2017. View Article : Google Scholar : PubMed/NCBI
11	Ni HM, Du K, You M and Ding WX: Critical role of FoxO3a in alcohol-induced autophagy and hepatotoxicity. Am J Pathol. 183:1815–1825. 2013. View Article : Google Scholar : PubMed/NCBI
12	Cheng J, Ohsaki Y, Tauchi-Sato K, Fujita A and Fujimoto T: Cholesterol depletion induces autophagy. Biochem Biophys Res Commun. 351:246–252. 2006. View Article : Google Scholar : PubMed/NCBI
13	Altmann SW, Davis HR, Zhu LJ, Yao X, Hoos LM, Tetzloff G, Iyer SP, Maguire M, Golovko A, Zeng M, et al: Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science. 303:1201–1204. 2004. View Article : Google Scholar : PubMed/NCBI
14	Davis HR Jr, Zhu LJ, Hoos LM, Tetzloff G, Maguire M, Liu J, Yao X, Iyer SP, Lam MH, Lund EG, et al: Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J Biol Chem. 279:33586–33592. 2004. View Article : Google Scholar : PubMed/NCBI
15	Temel RE, Tang W, Ma Y, Rudel LL, Willingham MC, Ioannou YA, Davies JP, Nilsson LM and Yu L: Hepatic Niemann-Pick C1-like 1 regulates biliary cholesterol concentration and is a target of ezetimibe. J Clin Invest. 117:1968–1978. 2007. View Article : Google Scholar : PubMed/NCBI
16	Davis HR Jr and Altmann SW: Niemann-Pick C1 like 1 (NPC1L1) an intestinal sterol transporter. Biochim Biophys Acta. 1791:679–683. 2009. View Article : Google Scholar : PubMed/NCBI
17	Wang Y, Tang W, Yang P, Shin H and Li Q: Hepatic NPC1L1 promotes hyperlipidemia in LDL receptor deficient mice. Biochem Biophys Res Commun. 499:626–633. 2018. View Article : Google Scholar : PubMed/NCBI
18	Tang W, Jia L, Ma Y, Xie P, Haywood J, Dawson PA, Li J and Yu L: Ezetimibe restores biliary cholesterol excretion in mice expressing Niemann-Pick C1-like 1 only in liver. Biochim Biophys Acta. 1811:549–555. 2011. View Article : Google Scholar : PubMed/NCBI
19	Kurano M, Hara M, Tsuneyama K, Okamoto K, Iso-O N, Matsushima T, Koike K and Tsukamoto K: Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver. J Lipid Res. 53:2275–2285. 2012. View Article : Google Scholar : PubMed/NCBI
20	Yamamura T, Ohsaki Y, Suzuki M, Shinohara Y, Tatematsu T, Cheng J, Okada M, Ohmiya N, Hirooka Y, Goto H and Fujimoto T: Inhibition of niemann-pick-type C1-like1 by ezetimibe activates autophagy in human hepatocytes and reduces mutant α1-antitrypsin Z deposition. Hepatology. 59:1591–1599. 2014. View Article : Google Scholar : PubMed/NCBI
21	Simpson IA and Sonne O: A simple, rapid, and sensitive method for measuring protein concentration in subcellular membrane fractions prepared by sucrose density ultracentrifugation. Anal Biochem. 119:424–427. 1982. View Article : Google Scholar : PubMed/NCBI
22	Fischer AH, Jacobson KA, Rose J and Zeller R: Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc 2008: pdb.prot4986. 2008.
23	Li WC, Ralphs KL and Tosh D: Isolation and culture of adult mouse hepatocytes. Methods Mol Biol. 633:185–196. 2010. View Article : Google Scholar : PubMed/NCBI
24	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
25	Ding W X, Manley S and Ni HM: The emerging role of autophagy in alcoholic liver disease. Exp Biol Med (Maywood). 236:546–556. 2011. View Article : Google Scholar : PubMed/NCBI
26	Dolganiuc A, Thomes PG, Ding WX, Lemasters JJ and Donohue TM Jr: Autophagy in alcohol-induced liver diseases. Alcohol Clin Exp Res. 36:1301–1308. 2012. View Article : Google Scholar : PubMed/NCBI
27	Tang W, Ma Y and Yu L: Plasma cholesterol is hyperresponsive to statin in ABCG5/ABCG8 transgenic mice. Hepatology. 44:1259–1266. 2006. View Article : Google Scholar : PubMed/NCBI
28	Ki SH, Park O, Zheng M, Morales-Ibanez O, Kolls JK, Bataller R and Gao B: Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: Role of signal transducer and activator of transcription 3. Hepatology. 52:1291–1300. 2010. View Article : Google Scholar : PubMed/NCBI
29	Bertola A, Mathews S, Ki SH, Wang H and Gao B: Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc. 8:627–637. 2013. View Article : Google Scholar : PubMed/NCBI
30	Betters JL and Yu L: NPC1L1 and cholesterol transport. FEBS Lett. 584:2740–2747. 2010. View Article : Google Scholar : PubMed/NCBI