A proteomic analysis of liver after ethanol binge in chronically ethanol treated rats
© Aroor et al.; licensee BioMed Central Ltd. 2012
Received: 2 December 2011
Accepted: 30 April 2012
Published: 30 April 2012
Binge ethanol in rats after chronic ethanol exposure augments necrosis and steatosis in the liver. In this study, two-dimensional gel electrophoresis proteomic profiles of liver of control, chronic ethanol, control-binge, and chronic ethanol- binge were compared.
The proteomic analysis identified changes in protein abundance among the groups. The levels of carbonic anhydrase 3 (CA3) were decreased after chronic ethanol and decreased further after chronic ethanol-binge. Ethanol binge alone in control rats had no effect on this protein suggesting its possible role in increased susceptibility to injury by binge after chonic ethanol treatment. A protein spot, in which both cytosolic isocitrate dehydrogenase (IDH1) and glutamine synthetase (GS) were identified, showed a small decrease after chronic ethanol binge but western blot demonstrated significant decrease only for glutamine synthetase in chronic ethanol treated rats. The level of gluathione S-transferase mu isoform (GSTM1) increased after chronic ethanol but was lower after chronic ethanol-binge compared to chronic ethanol treatment. The protein levels of the basic form of protein disulfide isomerase associated protein 3 (PDIA3) were significantly decreased and the acidic forms were increased after chronic ethanol- binge but not in chronic ethanol treated rats or ethanol binge in control rats. The significant changes in proteome profile in chronic ethanol binge were accompanied by a marked increase in liver injury as evidenced by enhanced steatosis, necrosis, increased 4-hydroxynonenal labeled proteins, CYP2E1 expression, and decreased histone H2AX phosphorylation.
Given the role of CA3, IDH1 and GST in oxidative stress; PDIA3 in protein quality control, apoptosis and DNA repair and decreased glutamine synthetase as a sensitive marker of pericentral liver injury this proteome study of chronic ethanol-binge rat model identifies these proteins for the first time as molecular targets with potential role in progression of liver injury by binge ethanol drinking.
KeywordsBinge ethanol Chronic ethanol Liver proteomics
Alcoholic liver disease is a worldwide health problem [1, 2]. The mechanisms that cause progression of liver injury are complex. Healthy liver is resistant to the action of ethanol and most individuals consuming alcohol have steatosis but not steatohepatitis . The progression of steatosis to steatohepatitis has been shown to be dependent on additional factors such as endotoxin, nutritional factors, and other disease states such as hepatitis C viral infection [4–7]. In this regard, binge drinking habit in chronic alcoholics is one of the most important factors contributing to the progression of alcoholic liver injury [8–11]. We have recently developed a chronic ethanol-binge rat model in which short term chronic ethanol treatment for 4 weeks does not result in significant liver injury. When these rats were subjected to 3 episodes of repeat ethanol binge it dramatically amplified liver injury. This rat model mimics findings similar to humans particularly during early alcoholic liver injury . Therefore, it offers an opportunity to determine the effects of binge after chronic ethanol intake and to explore the underlying mechanisms of enhancement of liver injury by binge ethanol.
Recent developments of systems biology approaches including genomics, metabolomics and proteomics offer a better insight into the mechanisms of cellular injury and identification of protein targets without a prior knowledge of the specific underlying molecular pathway . Although gene expression has provided valuable information on transcriptional regulation, significant discrepancy occurs between changes in RNA and protein expression since not all expressed genes are translated into protein products . Two dimensional electrophoresis in combination with mass spectrometry (2DE-MS), has been widely applied for the analyses of protein expression and their post translational modifications . Proteome studies on liver have been useful in elucidating the biomarkers of liver damage and factors that contribute to susceptibility of liver tissue to drug or disease induced liver injury [14–16]. Although, liver proteomic studies have been reported for chronic ethanol treated rats [17–22], proteomics after ethanol binge drinking has not been examined. We have therefore undertaken a 2-dimensional (2D) gel-based proteomics study of liver subjected to binge ethanol administration in vivo.
Chronic ethanol, control-binge and chronic ethanol-binge display differential expression of proteins
Identification of differentially expressed protein spots by mass spectrometry
Total Ion Scorec
No. peptides matched
Carbonic anhydrase 3
Carbonic anhydrase 3
Cytosolic isocitrate dehydrogenased
Glutathione S transferase M1
Protein disulfide isomerase A3
Protein disulfide isomerase A3
Protein disulfide isomerase A3
Protein disulfide isomerase A3
Relationship of proteome changes to liver injury during chronic ethanol and chronic ethanol-binge
This is the first proteomic study of global alterations of proteins in liver from chronic ethanol-binge treated animals. The chronic ethanol binge model developed in this laboratory displays a number of features that are seen during human alcoholic liver injury (12). Therefore this model is useful for the investigation of the mechanism of binge induced enhancement of alcoholic liver injury. Using a proteomics approach, we have demonstrated changes in the levels of a number of liver proteins affected differentially by chronic ethanol, control-binge and chronic ethanol-binge. A notable aspect of this study is the identification of these protein targets in a single model. Previously these protein targets have been reported individually in separate studies. The fact that normal liver is resistant to acute ethanol binge  as seen in control-binge group in this study, we propose that the increased vulnerability to binge after chronic ethanol treatment and the accompanying alterations in proteins contribute to the amplification of injury.
We find a marked decrease in CA3 in chronic ethanol but not control-binge treated rats. Decrease in CA3 protein level after chronic ethanol administration has been reported , but the effects of acute ethanol binge or chronic–ethanol binge were not examined in that study. In another study, chronic ethanol treatment in rats decreased carbonic anhydrase mRNA levels  suggesting that the regulation of CA3 occurs at the level of transcription. CA3 is an enzyme that has negligible carbonic anhydrase activity compared to other isoforms of carbonic anhydrase . It has been proposed to have a cytoprotective action against oxidative stress. In NIH/3T3 cells over-expressing CA3, steady state levels of reactive oxygen species are reduced and these cells were protected against hydrogen peroxide induced oxidative stress . CA3 has also been shown to undergo reversible S-thiolation reactions with glutathione and reversible glutathiolation has been proposed to provide protection to the reactive cysteine residues in proteins from damaging oxidative reactions . This assumes significance from studies supporting antioxidant role of CA3. A decrease in CA3 in the present study and augmentation of liver injury as demonstrated by increased transaminase favors the view that it has a protective role. Moreover, lipid peroxidation and oxidative stress has been shown to be increased in an experimental model of chronic ethanol followed by intraperitoneal administration of ethanol binge . In the present study, we observed a marked decrease in CA3 levels in chronic ethanol-binge group that was accompanied by an increase in protein oxidation of low and high molecular weight proteins. There was a predominant increase of protein oxidation of low molecular weight in chronic ethanol group. Levels of oxidation of high molecular weight proteins increased in the control-binge group. In this regard, it may be noted that carbonic anhydrase levels are far lower in humans and they are more susceptible to alcoholic liver injury than rats. Interestingly, female rats have far lower CA3 than male rats and female rats are more susceptible to alcoholic liver injury . Increase in lipid peroxidation was accompanied by increased expression of CYP2E1 and ethanol induced CYP2E1 has been implicated in oxidative stress and steatosis [26, 27]. However, the magnitude of increase in the CYP2E1 expression was dramatic in chronic-ethanol binge compared to chronic ethanol or binge alone. These findings suggest that chronic ethanol intake sensitizes liver for ethanol binge induced expression of CYP2E1 to a greater extent than direct effects of ethanol alone.
Isocitrate dehydrogenase 1 (IDH1) is a NADP-dependent enzyme that catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) in the cytoplasm/peroxisomes with the concomitant production of NADPH. It is proposed to play an important role in cellular defense against oxidative stress . Treatment of hepatocytes with ethanol or acetaldehyde decreased IDH1 activity, and knock down of IDH1resulted in potentiation of alcohol induced oxidative stress . Decreased activity of IDH1 by oxidative stress has also been reported in liver . In the present study, IDH1 was not significantly decreased after chronic ethanol intake or chronic ethanol-binge.
Glutamine synthetase catalyzes ATP-dependent amidation of glutamate to glutamine. It is responsible for the intrahepatic and inter tissue ammonia elimination/detoxification occurring in the liver . Glutamine synthetase has also been implicated in nucleotide synthesis and amino acid turnover . Glutamine synthetase in normal liver is expressed by a small population of perivenous hepatocytes [42, 43]. Decreased expression and activity of glutamine synthetase has been considered to be the sensitive indicator of liver injury and chronic ethanol intake has been shown to result in decreased glutamine synthetase activity [43, 44]. Although, glutamine synthetase expression is modestly decreased after chronic ethanol, further decrease occurs after chronic-ethanol binge but not in control-binge. Decreased protein levels of glutamine synthetase in liver may contribute to increased plasma ammonia levels and exaggeration of central nervous system toxicity.
Glutathione S transferase (GST) is a superfamily of enzymes comprising several isoenzymes. The major GST subunits expressed in the adult liver are alpha (A1, A2 and A3) and Mu (M1 and M2). The Pi subunit of GST is only expressed in fetal hepatocytes, and during early stages of hepatocarcinogenesis . GST Pi and M forms are drug metabolizing enzymes and play role in suppressing oxidative stress and reducing the levels of 4-hydroxynonenal . Chronic ethanol exposure has been shown to cause both up- and down- regulation of GST M in vivo and in hepatocytes [46–49]. Accumulation of 4–hydroxynonenal after chronic ethanol treatment is considered to mediate liver injury via lipid peroxidation. Therefore, increased expression of GST that conjugates glutathione to a host of electrophiles  may represent an adaptive response induced by chronic ethanol. In this regard, GST M null phenotype has been shown to be associated with increased susceptibility to alcoholic liver injury . In the present study, the expression of GST M1 was marginally increased after chronic ethanol or control ethanol binge, but the levels did not increase after chronic ethanol- binge suggesting compromised GST M1 after chronic ethanol-binge.
An intriguing finding in the present study is the change in the levels of different isoforms of PDIA3 in the chronic ethanol-binge group compared to either chronic ethanol treated or control ethanol binge groups. PDIA3 belongs to the endoplasmic reticular chaperone protein family of protein disulfide isomerases . These more negatively charged isoforms arise by either acetylation or phosphorylation of PDIA3. Recently, increased accumulation of phosphorylated forms of PDIA3 was shown after angiotensin II stimulation of vascular cells  or by prolonged fasting in rat liver . Increased carbonylation of liver PDIA3 has been reported after chronic ethanol treatment in rats and this was accompanied by decreased function of the isomerase . PDIA3 is emerging as an important regulator of cell function at multiple levels. It has been implicated in sorting of glycoproteins, quality control of endoplasmic reticular proteins, antigen processing by MHC proteins, control of STAT3 function, induction of apoptosis, and suppression of steatosis [23, 31, 54, 55]. We have observed increased steatosis and suppressed apoptosis after chronic ethanol binge (Figure 7) with concomitant increases in the levels of PDIA3 acidic isoforms (Figure 5). Therefore, these results favor the role of PDIA3 in augmentation of liver injury by ethanol binge. Although suppression of apoptosis is considered as a survival mechanism, such phenomenon under the conditions of toxic insult such as ethanol may be accompanied by disruption of cell repair mechanisms such as repair of damaged DNA. DNA damage and impairment of DNA repair have been reported in animal and human alcoholic liver damage. Relevantly, knockdown of PDIA3 expression impaired phosphorylation of H2AX and DNA repair . We have observed both decreased phosphorylation of H2AX and increase in the levels of acidic isoform of PDIA3 in chronic ethanol-binge group. It is also known that increase in acidic (phosphorylated) isoform of PDIA3 is related to decreased protein folding function of PDIA3 . It is therefore plausible that in chronic ethanol-binge condition changes in PDIA3 disrupt protein folding, decrease H2AX phosphorylation resulting in a compromised DNA repair.
Male Sprague–Dawley rats (250–300 g) were purchased from Harlan Laboratories (Indianapolis, IN). ). The antibodies for carbonic anhydrase 3, protein disulfide isomerase associated protein 3, cytosolic isocitrate dehydrogenase, and glutamine synthetase were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies for histone H2AX and cleaved caspase 3 were purchased form Cell Signaling (Beverly, MA). CYP2E1 antibody was from AbCam Cambridge, MA. Other reagents including protease inhibitor cocktail (Sigma p8340) and β-actin antibody were obtained from the Sigma-Aldrich (St. Louis, MO).
Animal feeding for chronic ethanol-binge model of alcoholic liver injury
Rats were housed under a 12-h/12-h light/dark cycle and were permitted ad libitum consumption of standard laboratory rat chow. After a one-week adaptation period, the animals were fed Lieber–DeCarli liquid diet (Dyets, Inc., Bethlehem, PA) . Ethanol was progressively introduced into the liquid diet starting at 1.25% (wt./vol.) for day 1, increased to 1.67% (wt./vol.) for day 2, to 2.5% (wt./vol.) for days 3 and 4 and, finally, maintained at 5% (wt./vol.) for 4 weeks. Weight-matched littermates were pair-fed on the same liquid diet, except that the ethanol was replaced by dextrin/maltose (control) to maintain the isocaloric intake in the two groups. Each day, the previous day's ethanol diet intake was measured, and the pair-fed rats were fed same calorie of diet containing dextrin/maltose. After 4 weeks, rats were divided into four groups: control, chronic ethanol, control-binge , chronic-ethanol binge. Chronic ethanol-binge and control-binge groups had three doses (each 5 g/kg body wt) of binge ethanol administered intragastrically every 12 hr. In the control group for chronic ethanol-binge, ethanol was replaced by water. Liver samples were collected 4 hr after the last binge administration (Aroor et al, 2011), and immediately frozen in liquid nitrogen until further use. The animal care and protocol for their use were approved by the University of Missouri Animal Care & Use Committee.
Two dimensional electrophoresis
Frozen liver samples were pulverized in liquid nitrogen using mortar and pestle. Tris-buffered phenol extraction solution was added to the powdered samples and proteins were extracted using phenol extraction protocol . Protein pellets were resuspended in IEF buffer (8 M Urea, 2 M thiourea, 4% CHAPS, 2% C7BzO, 100 mM DTT, 2.2% 2-HED). Solubilized samples were quantified using the EZQ protocol from Life Technologies (Grand Island, NY). One mg of protein sample was then applied to 24 cm pH 3–10 IPG strips for passive rehydration overnight. The IPG strips were isoelectrically focused on the Protean IEF from Bio-Rad using the following parameters: 250 V for 250Vhr (rapid ramp), 1000 V for 500Vhr (rapid ramp), 8000 V for 2 hours (gradient), 8000 V for 80,000Vhr (rapid ramp), and then hold at 500 V. The IPG strips were then placed on 12% SDS-PAGE gels for the second dimension and ran using the Ettan Dalt 12 at 1 W/gel overnight. After the run, gels were stained with Pro-Q diamond stain and imaged using the Ettan DIGE imager. Gels were then post stained with Coomassie Brilliant Blue (G250) for the total protein and scanned using the UMAX powerlook flatbed scanner. Data were analyzed using Delta 2D system from Decodon after normalization. The identification of spots that showed distinct differences was based on aligning and matching of spots in gels and quantification of matched spots. The spots were also manually inspected to verify the accuracy of matching. We selected a protein spot that showed the least variation between the samples and this spot was used for normalization. All the protein spots chosen were present in all the gels. The results were also analyzed based on normalization to total spot volume and the pattern was similar to data analysis based on normalization to the protein spot with the least variation among the gels. For phosphoprotein analysis, the values were normalized for total protein.
Analysis of proteins by mass spectrometry
Spots were excised from the gel and subjected to trypsin digest using a standard in-gel digestion protocol . The extracted peptides were lyophilized and resuspended in 3 μl of 1% formic acid. A 0.5 μl aliquot of the resulting peptides was mixed 1:1 with CHCA matrix (5 mg/mL in 60% ACN, 0.3% TFA, 10 mM ammonium phosphate) and spotted onto the MALDI target. The instrument (Applied Biosystems 470 Proteomics Analyzer) was operated in positive ion mode and spectra were acquired over a mass range of 700 to 4000 Da. Peptide calibration standards (4700 calibration mix, Applied Biosystems) were used to calibrate the instrument in MS mode using six peptides of known mass. Calibration was achieved by the “plate model and default” mode for MS of six external calibrant spots. Additionally, internal (i.e. within the sample spot) calibration was achieved using trypsin autolysis peptides (where present). Note: although trypsin autolysis ions are useful for obtaining the best mass accuracy, these ions are automatically excluded from the database search conducted using the GPS Explorer.
Following an MS scan of each sample the 8 most abundant peptides were picked automatically for tandem MS (MSMS – peptide fragmentation) acquisition. Following this a database search was performed using V3.6 of GPS Explorer. Ions with s/n ratios >20 (excluding trypsin autolysis) up to a max of 125 per spectrum were submitted for searches against mammal entries in the NCBInr database (last updated September 9th 2010) using the “combined MS + MSMS” function of the GPS Explorer software. The GPS Explorer software is integrated with the MASCOT V2.2 (http://www.matrixscience.com). Search parameters allowed: 1 trypsin miss-cleavage and the following modifications to peptides: carbamidomethyl cysteines, and methionine oxidation. MS mass tolerance was 100 ppm, MSMS mass tolerance was 0.2 Da.
Liquid Chromatography Tandem Mass Spectrometry analysis: Digests were diluted to 5 μl with 1% formic acid and transferred to an autosampler vial and placed in the autosampler which was maintained at 4°C. A portion of the sample (3 μL) was loaded onto a 150 mm C18 CHIP (Agilent Technologies cat# G4240-62002). Peptides were separated and eluted from the analytical column with a continuous gradient of acetonitrile from 3 to 45% (in 0.1% formic acid) over 8 minutes. Following an MS scan of the eluting peptides, each cycle, the five most abundant peptides were subjected to peptide fragmentation (MSMS). Data across 17 minutes were collected. MSMS data were extracted and a peak list file (mgf – mascot generic format file) was generated using the qualitative analysis software. A search of NCBInr-limited to mammals was conducted using a local copy of MASCOT server V2.2. We selected only eight proteins by proteome analysis. The use of Coomassie blue staining and use of broader pH range might have resulted in the detection of lower number of spots. Our objective was to cover wide range of proteins and obtain adequate amount of protein for mass spectrometry analysis. Therefore, we have adopted this protocol. The limited number spot difference has also been reported for other proteome studies in chronic ethanol administration and endotoxin induced liver injury [19, 58].
Data are expressed as means ± S.E.M. Statistical significance of the differences between means was assessed by Student's t test. A probability of less than 5% was considered significant (P < 0.05).
Carbonic anhydrase 3
Coomassie brilliant blue
Cytochrome P 450 2E1
Histone H2A X
Glutathione S-transferase, mu isoform
Cytosolic isocitarate dehydrogenase
Protein disulfide isomerase associated protein 3.
This work was supported in part by NIH grant AA11962 and AA16347.
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