Alcohol Disrupts Endoplasmic Reticulum Function and Protein Secretion in Hepatocytes

• Published in: Alcoholism, clinical and experimental research Vol. 36; no. 1; pp. 14 - 23
• Main Authors: Howarth, Deanna L., Vacaru, Ana M., Tsedensodnom, Orkhontuya, Mormone, Elisabetta, Nieto, Natalia, Costantini, Lindsey M., Snapp, Erik L., Sadler, Kirsten C.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-01-2012; Wiley; Wiley Subscription Services, Inc
• Subjects: Alcohol; Alcoholic Liver Disease; Alcoholism and acute alcohol poisoning; Animals; Biological and medical sciences; Endoplasmic reticulum; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - ultrastructure; Ethanol; Ethanol - toxicity; Fluorescence Recovery After Photobleaching; Gastroenterology. Liver. Pancreas. Abdomen; Hep G2 Cells; Hepatocytes - drug effects; Hepatocytes - secretion; Humans; Liver; Liver cirrhosis; Liver. Biliary tract. Portal circulation. Exocrine pancreas; Medical sciences; Mice; Other diseases. Semiology; Protein folding; Toxicology; Unfolded Protein Response; Unfolded Protein Response - drug effects; Zebrafish; Ethanol; Fluorescence Recovery After Photobleaching; Digestive system; Alcoholic hepatitis; Secretion; Liver; Fluorescence; Hepatic disease; Zebrafish; Unfolded Protein Response; Recovery; Protein; Alcoholic beverage; Vertebrata; Brachydanio rerio; Hepatocyte; Pisces; Alcoholic Liver Disease; Digestive diseases; Endoplasmic reticulum
• ISSN: 0145-6008; 1530-0277
• DOI: 10.1111/j.1530-0277.2011.01602.x

Background:  Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo.
Methods:  HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL‐17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP).
Results:  VL‐17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced.
Conclusions:  Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.