Abstract
Autophagy is a conserved mechanism that plays a housekeeping role by eliminating protein aggregates and damaged organelles. Recent studies have demonstrated that acute ethanol intoxication induces hepatic autophagy in mice. The effect of dietary zinc intake on hepatic autophagic flux during ethanol intoxication has not been evaluated using animal models. Herein, we investigated whether zinc deficiency and excess can affect autophagic flux in the liver in mice and in human hepatoma cells acutely exposed to ethanol. A mouse model of binge ethanol feeding was utilized to analyze the effect of low, adequate, and high zinc intake on hepatic autophagic flux during ethanol intoxication. Autophagic flux was inferred by analyzing LC3II/LC3I ratio, protein levels of p62/SQSTM1, Beclin1 and Atg7, and phosphorylation of 4EBP1. In addition, the degradation of the fusion protein LC3-GFP and the formation of autophagosomes and autolysosomes were evaluated in cells. Ethanol treatment stimulated autophagy in mice and cells. High zinc intake resulted in enhanced autophagy in mice exposed to ethanol. Conversely, zinc deficiency was consistently associated with impaired ethanol-induced autophagy in mice and cells. Zinc-deficient mice exhibited a high degree of ethanol-driven steatosis. Furthermore, zinc depletion increased apoptosis in cells exposed to ethanol. The results of this study suggest that adequate zinc intake is necessary for proper stimulation of autophagy by ethanol. Poor zinc status is commonly found among alcoholics and could likely contribute to faulty autophagy.
Similar content being viewed by others
References
Arigony ALV, de Oliveira IM, Machado M et al (2013) The influence of micronutrients in cell culture: a reflection on viability and genomic stability. Biomed Res Int. https://doi.org/10.1155/2013/597282
Blankenship JR, Heitman J (2005) Calcineurin is required for Candida albicans to survive calcium stress in serum. Infect Immun 73:5767–5774
Bode JC, Hanisch P, Henning H, Koenig W, Richter FW, Bode C (1988) Hepatic zinc content in patients with various stages of alcoholic liver disease and in patients with chronic active and chronic persistent hepatitis. Hepatology 8:1605–1609
Chan LL-Y, Shen D, Wilkinson AR et al (2012) A novel image-based cytometry method for autophagy detection in living cells. Autophagy 8:1371–1382. https://doi.org/10.4161/auto.21028
Chen Y, Klionsky DJ (2011) The regulation of autophagy-unanswered questions. J Cell Sci 124(Pt 2):161–170
Cho KS, Yoon YH, Choi JA, Lee SJ, Koh JY (2012) Induction of autophagy and cell death by tamoxifen in cultured retinal pigment epithelial and photoreceptor cells. Investig Ophthalmol Vis Sci 53:5344–5353. https://doi.org/10.1167/iovs.12-9827
Cho HI, Choi JW, Lee SM (2014) Impairment of autophagosome-lysosome fusion contributes to chronic ethanol-induced liver injury. Alcohol 48:717–725. https://doi.org/10.1016/j.alcohol.2014.08.006
Davis SR, Cousins RJ (2000) Metallothionein expression in animals: a physiological perspective on function. J Nutr 130:1085–1088
Ding WX, Li M, Chen X, Ni HM, Lin CW, Gao W, Lu B, Stolz DB, Clemens DL, Yin XM (2010) Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice. Gastroenterology 139:1740–1752
Dinsmore W, Callender ME, McMaster D, Todd SJ, Love AH (1985) Zinc absorption in alcoholics using zinc-65. Digestion 32:238–242
Donohue TM, Osna NA, Clemens DL (2006) Recombinant HepG2 cells that express alcohol dehydrogenase and cytochrome P450 2E1 as a model of ethanol-elicited cytotoxicity. Int J Biochem Cell Biol 38:92–101
Guo S, Liang Y, Murphy SF et al (2015) A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications. Autophagy 11:560–572. https://doi.org/10.1080/15548627.2015.1017181
Ho E, Ames BN (2002) Low intracellular zinc induces oxidative DNA damage, disrupts p53, NFκB, and AP1 DNA binding, and affects DNA repair in a rat glioma cell line. Proc Natl Acad Sci USA 99:16770–16775. https://doi.org/10.1073/pnas.222679399
Hung HH, Huang WP, Pan CY (2013) Dopamine- and zinc-induced autophagosome formation facilitates PC12 cell survival. Cell Biol Toxicol 29:415–429
Hwang JJ, Kim HN, Kim J, Cho DH, Kim MJ, Kim YS, Kim Y, Park SJ, Koh JY (2010) Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line. Biometals 23:997–1013
Joachimiak R, Kaźnica A, Drewa T (2007) Influence of sodium butyrate on hepatocellular carcinoma (hepG2) and glioblastoma (C6) cell lines in vitro. Acta Pol Pharm 64:561–563
Jung CH, Ro S-H, Cao J, Otto NM, Kim D-H (2010) mTOR regulation of autophagy. FEBS Lett 584:1287–1295. https://doi.org/10.1016/j.febslet.2010.01.017
Kang X, Zhong W, Liu J, Song Z, McClain CJ, Kang YJ, Zhou Z (2009) Zinc supplementation reverses alcohol-induced steatosis in mice through reactivating hepatocyte nuclear factor-4alpha and peroxisome proliferator-activated receptor-alpha. Hepatology 50:1241–1250
Kawamata T et al (2017) Zinc starvation induces autophagy in yeast. J Biol Chem 292:8520–8530. https://doi.org/10.1074/jbc.M116.762948
Klionsky DJ et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1–222. https://doi.org/10.1080/15548627.2015.1100356
Lee SJ, Cho KS, Koh JY (2009) Oxidative injury triggers autophagy in astrocytes: the role of endogenous zinc. Glia 57:1351–1361. https://doi.org/10.1002/glia.20854
Lin CW, Zhang H, Li M, Xiong X, Chen X, Chen X, Dong XC, Yin XM (2013) Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice. J Hepatol 58:993–999
Liuzzi JP, Yoo C (2013) Role of zinc in the regulation of autophagy during ethanol exposure in human hepatoma cells. Biol Trace Elem Res 156:350–356. https://doi.org/10.1007/s12011-013-9816-3
Liuzzi JP, Bobo JA, Lichten LA, Samuelson DA, Cousins RJ (2004) Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis. Proc Natl Acad Sci USA 101:14355–14360
Massey VL, Arteel GE (2012) Acute alcohol-induced liver injury. Front Physiol 3:193
McClain CJ, Su LC (1983) Zinc deficiency in the alcoholic: a review. Alcohol Clin Exp Res 7:5–10
McClain CJ, Antonow DR, Cohen DA, Shedlofsky SI (1986) Zinc metabolism in alcoholic liver disease. Alcohol Clin Exp Res 10:582–589
Ni H-M, Du K, You M, Ding W-X (2013) Critical role of FoxO3a in alcohol-induced autophagy and hepatotoxicity. Am J Pathol 183:1815–1825
Pan R, Timmins GS, Liu W, Liu KJ (2015) Autophagy mediates astrocyte death during zinc-potentiated ischemia–reperfusion injury. Biol Trace Elem Res 166:89–95. https://doi.org/10.1007/s12011-015-0287-6
Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H et al (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282:24121–24145
Sakiyama T, Musch MW, Ropeleski MJ, Tsubouchi H, Chang EB (2009) Glutamine increases autophagy under basal and stressed conditions in intestinal epithelial cells. Gastroenterology 136:924–932. https://doi.org/10.1053/j.gastro.2008.12.002
Saliani N, Darabi M, Yousefi B et al (2013) PPARγ agonist-induced alterations in Δ6-desaturase and stearoyl-CoA desaturase 1: role of MEK/ERK1/2 pathway. World J Hepatol 5:220–225. https://doi.org/10.4254/wjh.v5.i4.220
Shay NF, Mangian HF (2000) Neurobiology of zinc-influenced eating behavior. J Nutr 130:1493S–1499S
Stirn FE, Elvehjem CA, Hart EB (1935) The indispensability of zinc in the nutrition of the rat. J Biol Chem 109:347–359
Substance Abuse and Mental Health Services Administration (2011) Results from the 2010 national survey on drug use and health: summary of national findings. Substance Abuse and Mental Health Services Administration, Rockville, MD. https://www.samhsa.gov/data/sites/default/files/NSDUHNationalFindingsResults2010-web/2k10ResultsRev/NSDUHresultsRev2010.pdf. Accessed 22 Dec 2017
Tanida I, Yamaji T, Ueno T, Ishiura S, Kominami E, Hanada K (2008) Consideration about negative controls for LC3 and expression vectors for four colored fluorescent protein-LC3 negative controls. Autophagy 4:131–134
Thomes PG, Trambly CS, Fox HS, Tuma DJ, Donohue TM Jr (2015) Acute and chronic ethanol administration differentially modulate hepatic autophagy and transcription factor EB. Alcohol Clin Exp Res 39:2354–2363. https://doi.org/10.1111/acer.12904
Wei CC, Luo Z, Song YF, Pan YX, Wu K, You WJ (2017) Identification of autophagy related genes LC3 and ATG4 from yellow catfish Pelteobagrus fulvidraco and their transcriptional responses to waterborne and diet borne zinc exposure. Chemosphere 175:228–238. https://doi.org/10.1016/j.chemosphere.2017.02.042
Wong E, Cuervo AM (2010) Integration of clearance mechanisms: the proteasome and autophagy. Cold Spring Harb Perspect Biol 2:a006734. https://doi.org/10.1101/cshperspect.a006734
Xue J, Moyer A, Peng B, Wu J, Hannafon BN, Ding W-Q (2014) Chloroquine is a zinc ionophore. PLoS ONE 9:e109180. https://doi.org/10.1371/journal.pone.0109180
Zhou Z, Wang L, Song Z, Saari JT, McClain CJ, Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress. Am J Pathol 166:1681–1690
Zoico E, Franceschetti G, Chirumbolo S, Rossi AP, Mazzali G, Rizzatti V, Budui S, Zamboni M (2014) Phenotypic shift of adipocytes by cholecalciferol and 1α,25 dihydroxycholecalciferol in relation to inflammatory status and calcium content. Endocrinology 155:4178–4188. https://doi.org/10.1210/en.2013-1969
Acknowledgements
This study was supported by NIH 1R03AA022451-01.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Liuzzi, J.P., Narayanan, V., Doan, H. et al. Effect of zinc intake on hepatic autophagy during acute alcohol intoxication. Biometals 31, 217–232 (2018). https://doi.org/10.1007/s10534-018-0077-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10534-018-0077-7