Am J Physiol Gastrointest Liver Physiol 306: G37–G47, 2014. First published October 31, 2013; doi:10.1152/ajpgi.00085.2013.
Acute ethanol preexposure promotes liver regeneration after partial hepatectomy in mice by activating ALDH2 Xiang Ding,1,2 Juliane I. Beier,1,2 Keegan J. Baldauf,1,2 Jenny D. Jokinen,1,2 Hai Zhong,1,2 and Gavin E. Arteel1,2 1
Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Louisville; 2University of Louisville Alcohol Research Center, Louisville, Kentucky Submitted 18 March 2013; accepted in final form 23 October 2013
Ding X, Beier JI, Baldauf KJ, Jokinen JD, Zhong H, Arteel GE. Acute ethanol preexposure promotes liver regeneration after partial hepatectomy in mice by activating ALDH2. Am J Physiol Gastrointest Liver Physiol 306: G37–G47, 2014. First published October 31, 2013; doi:10.1152/ajpgi.00085.2013.—It is known that chronic ethanol significantly impairs liver regeneration. However, the effect of acute ethanol exposure on liver regeneration remains largely unknown. To address this question, C57Bl6/J mice were exposed to acute ethanol (6 g/kg intragastrically) for 3 days, and partial hepatectomy (PHx) was performed 24 h after the last dose. Surprisingly, acute ethanol preexposure promoted liver regeneration. This effect of ethanol did not correlate with changes in expression of cell cycle regulatory genes (e.g., cyclin D1, p21, and p27) but did correlate with protection against the effect of PHx on indices of impaired lipid and carbohydrate metabolism. Ethanol preexposure protected against inhibition of the oxidant-sensitive mitochondrial enzyme, aconitase. The activity of aldehyde dehydrogenase 2 (ALDH2) was significantly increased by ethanol preexposure. The effect of ethanol was blocked by inhibiting (Daidzin) and was mimicked by activating (Alda-1) ALDH2. Lipid peroxides are also substrates for ALDH2; indeed, alcohol preexposure blunted the increase in lipid peroxidation (4OH-nonenal adducts) caused by PHx. Taken together, these data suggest that acute preoperative ethanol exposure “preconditions” the liver to respond more rapidly to regenerate after PHx by activating mitochondrial ALDH2, which prevents oxidative stress in this compartment. aldehyde dehydrogenase 2; alcohol; liver regeneration; oxidative stress THE LIVER HAS TREMENDOUS REGENERATIVE capacity that distinguishes it from other vital organs (e.g., the brain, heart, and lungs) that are far less able to replace functional tissue. As the main detoxifying organ in the body, the liver is prone to toxic injury. Due to its regenerative properties, however, the liver is able to restore to full size and ensure survival. In experimental models (e.g., mice), full regeneration occurs within 7–10 days (36). Although hepatocytes rarely proliferate in the healthy adult liver, virtually all surviving hepatocytes replicate at least once after 70% partial hepatectomy (PHx). In addition to hepatocyte proliferation, there is a tightly coordinated response to complement the regenerative process (34). Perturbations of this complex and synchronized response can impact normal tissue recovery from injury or damage. Indeed, it is now clear that impaired regeneration and/or restitution is critical to the chronicity of numerous hepatic diseases. Studies have shown that chronic ethanol (EtOH)
Address for reprint requests and other correspondence: G. E. Arteel, Dept. of Pharmacology and Toxicology, 505 S Hancock St., CTRB, Rm 506, Univ. of Louisville Health Sciences Center, Louisville, KY, 40292 (e-mail: [email protected]). http://www.ajpgi.org
exposure delays the induction of hepatocyte DNA synthesis in response to PHx (20). This effect of chronic ethanol may contribute to impaired regeneration and restitution from damage associated with alcoholic liver disease. In contrast to chronic exposure, little is known about the effect of acute ethanol administration on liver regeneration. Under some conditions, acute and chronic ethanol exposure mediate similar responses in the organ. For example, both acute and chronic ethanol exposure enhance lipopolysaccharide-induced liver damage (1, 6). Should acute ethanol exposure also impair the hepatic regenerative response, this model could be employed as a screening tool for subsequent chronic studies. The purpose of the present study was a priori to test the hypothesis that acute alcohol exposure will impair hepatic regeneration after PHx, analogous to findings with chronic ethanol exposure (20). Surprisingly, it was found that acute ethanol exposure enhanced hepatic regeneration after PHx. The purpose of this study was then to determine the mechanisms behind this effect. MATERIALS AND METHODS
Animals and treatments. Male C57BL/6J mice, 6 – 8 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a pathogen-free barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and procedures were approved by the University of Louisville Institutional Animal Care and Use Committee. Food and tap water were allowed ad libitum. Animals received ethanol (6 g/kg intragastrically) or isocaloric/isovolumetric maltose-dextrin solution for 3 days (Fig. 1A) (1). With this dose of alcohol, peak blood levels are ⬃300 mg/dl, and mice are sluggish but conscious; the animals regain normal behavior after ⬃6 h of alcohol dosing (2). Some maltosedextrin-exposed mice were injected with a specific aldehyde dehydrogenase 2 (ALDH2) agonist: Alda-1 (20 mg/kg ip; EMD Chemicals, Gibbstown, NJ), or vehicle (50% DMSO and 50% PEG) for 3 days before PHx, and the last dose of Alda-1 was given during surgery. Some ethanol-exposed mice were given 7-(-D-Glucopyranosyloxy)-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (75 mg/kg ip, Daidzin; Sigma, St. Louis, MO) or vehicle (50% DMSO and 50% PEG) during PHx surgery to inhibit ALDH2 activity. Seventy percent PHxs were performed 24 h after the last ethanol dose under isofluorane anesthesia (8) with minor modifications. Mice were anesthetized with ketamine/xylazine (100/15 mg/kg intramuscularly) at select time points up to 96 h after PHx (see timeline in Fig. 1A) and samples collected (1). Hepatocytes were isolated from some ethanol- or maltose-dextran-exposed mice 24 h after the last administration (see below). Biochemical analyses and histology. The capacity of liver regeneration was assessed by liver weight/body weight ratio; cell cycle progression (per 1,000 hepatocytes) was estimated using specific proliferating chain nuclear antigen (PCNA) staining patterns and cell morphology as described previously (41, 42). Briefly, G0 cells do not
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Fig. 1. Effect of acute ethanol (EtOH) preexposure on liver regeneration after partial hepatectomy (PHx). A: experimental timeline. B: plasma transaminases (left) and liver weight/body weight ratios (right) at the time of death. ALT, alanine aminotransferase; AST, aspartate aminotransferase. C: representative photomicrographs (⫻200) depicting proliferating chain nuclear antigen (PCNA) immunostaining 12 h and 24 h after PHx in maltose-dextrin (MD)-exposed (left) and ethanolexposed (right) groups. D: cell cycle progression (per 1,000 hepatocytes) at different time points. aP ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
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stain for PCNA; G1 cells are lightly stained in the nucleus, whereas S-phase cell nuclei are darkly stained; G2 cells have a cytoplasmic staining pattern with or without nuclear staining; M cells have diffuse cytoplasmic and deep blue chromosomal staining and a clear presence of mitotic bodies. Frozen sections of liver (10 m) were stained with oil red O (2). Hepatic lipids were determined in extracted liver samples (2). Hepatic glycogen reserves were determined by periodic acid (0.5%) Schiff (PAS) staining (12) and by anthrone reagent (32). Mitochondrial protein for the ALDH2 and aconitase activity assays were extracted from liver, as described by Wiekowski et al. (43). Enzymatic activity of ALDHs and ALDH2 was determined spectrophotometrically by monitoring the reduction of NAD⫹ to NADH at 340 nm (15). To determine the role of PKC-ε on activating ALDH2, the effect of knocking down this enzyme with antisense oligonucleotides (ASO) against PKC-ε was determined, as described previously by Kaiser et al. (17). Briefly, mice received the PKC-ε ASO or vehicle saline at a dose of 25 mg/kg ip twice a week before ethanol or maltose dextrin administration (see above). Mice were killed 24 h after the last
ethanol gavage. Mitochondrial aconitase activity was determined by monitoring the reduction of NAD⫹ to NADH at 340 nm (26). Plasma levels of aminotransferases (alanine aminotransferase and aspartate aminotransferase, ALT and AST, respectively) were determined using standard kits (Thermotrace, Melbourne, Australia). Adducts of 4-hydroxynonenal (4-HNE) were detected by immunohistochemistry using a rabbit polyclonal 4-HNE antibody (Alpha Diagnostic, San Antonio, TX) (10). RNA isolation and real-time RT-PCR. RNA extraction and realtime RT-PCR was performed as described previously (2). The PCR primers and probes PCK1, Fas, and G6P were designed using Primer 3 (Whitehead Institute for Biomedical Research, Cambridge, MA). Primers were designed to cross exons to ensure that only cDNA and not genomic DNA was amplified. Other PCR primers and probes (e.g., cyclin D1, p21, and p27) were purchased premade (Applied Biosystems, Carlsbad, CA). The comparative CT method was used to determine fold differences between samples and the calibrator gene (-actin), and purity of PCR products was verified by gel electropho-
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resis. The comparative CT method determines the amount of target, normalized to an endogenous reference (-actin) and relative to a calibrator (2⫺⌬⌬Ct). Immunoblots. Liver samples were homogenized in RIPA buffer [20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (wt/vol) Triton X-100], containing protease and phosphatase inhibitor cocktails (Sigma). Samples were loaded onto SDS-polyacrylamide gels of 12% (wt/vol) acrylamide followed by electrophoresis and Western blotting onto nitrocellulose membranes (Hybond P; GE Healthcare, Piscataway, NJ). Primary antibodies against phosphorylated and total extracellular-signal-regulated kinases1/2 (ERK1/ 2), p38, and c-Jun NH2-terminal kinases (JNK1/2) were from Cell Signaling (Billerica, MA). Primary antibodies against PKC-ε and ALDH2 (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Bands were visualized using an ECL kit (Pierce, Rockford, IL) and Hyperfilm (GE Healthcare). Densitometric analysis was performed using UN-SCAN-IT Gel (Silk Scientific, Orem, UT) software. Hepatocyte isolation and culture. Hepatocytes were isolated and separated from liver by collagenase perfusion (3, 33), with minor modifications. Isolated hepatocytes were cultured (10,000 cells/well) overnight on collagen-coated 96-well plates optimized for fluorescent microscopy (NUNC, Rochester, NY). Cells were then exposed to 4-HNE (0 –1 mM) for up to 24 h. Some cells were exposed to Alda-1 (1.5 M) and/or Daidzin (60 M) 30 min before 4-HNE addition. After the predetermined incubation period with 4-HNE, the media was removed, and media containing Hoechst 33342 (1.5 M; nuclear fluorescence), TMRM (20 nM; mitochondrial membrane potential), and TOTO-3 (1 M; cell membrane permeability) dyes was added to the wells. Following a 1-h incubation with the dyes, the plate was placed into the Cellomics Array Scan VTI HCS reader and analyzed, analogous to what has been previously described by O’Brien et al. (22). The first of either 800 valid objects (defined by Hoechst 33342 staining) or five microscope fields were analyzed. The average of each 96-well plate was determined, as well as the individual values for all three fluorophores for each valid object. Statistical analyses. ANOVA with Bonferroni’s post hoc test or the Mann-Whitney Rank Sum test was used for the determination of statistical significance among treatment groups, as appropriate. Results are reported as means ⫾ SE (n ⫽ 4 – 6). RESULTS
Effect of acute ethanol preexposure on indices of liver damage after PHx. As observed previously (1), plasma levels of AST and ALT were within normal ranges in mice exposed to maltose-dextrin in the absence of surgery (Fig. 1B, left); acute ethanol exposure in the absence of PHx did not significantly alter AST and ALT levels compared with maltose-dextrinexposed mice. Preoperative acute ethanol exposure did not significantly alter the increase in plasma AST and ALT caused by PHx. Acute ethanol preexposure promotes liver regeneration after PHx. As is well known for this paradigm, PHx rapidly induces a regenerative response in the remnant liver. A crude index of this response is the increase in liver weight/body weight ratio (Fig. 1B, right), which increased rapidly in maltose-dextrinexposed mice after surgery. Acute ethanol preexposure did not significantly affect the liver/body weight ratio in sham-operated animals or in animals killed immediately after PHx (Fig. 1B). However, acute ethanol administration significantly increased liver mass gain compared with maltose-dextrin controls at later time points (⬎48 h). The effect of ethanol on hepatocyte proliferation after PHx was further analyzed by PCNA immunostaining (Fig. 1C). In maltose-dextrin controls, the amount of PCNA-positive cells
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increased dramatically 24 h after PHx and returned to G0 by the 96-h time point. Acute ethanol preexposure dramatically enhanced the rate of entry into the cell cycle (Fig. 1D). Indeed, ethanol preexposure increased the number of hepatocytes in G1 phase (88 ⫾ 24 per 1,000 hepatocytes) 12 h after PHx, a time point at which few hepatocytes in the control PHx group had entered G1 (3 ⫾ 1 per 1,000 hepatocytes). The rate of progression through the cell cycle was also apparently enhanced by acute ethanol preexposure. For example, although the number of cells in G1 was ⬃18% lower in ethanol-exposed mice compared with control mice (688 ⫾ 63 vs. 843 ⫾ 26, respectively) 24 h after PHx, the number of cells in S phase was ⬃2.6-fold higher (233 ⫾ 60 vs. 87 ⫾ 27, respectively) in the ethanol group. This effect greatly decreased the G1/S ratio in the ethanol group (3 vs. 10), which is indicative of an accelerated G1/S transition. The G1/S ratio was similarly affected by ethanol preexposure 48 h after PHx. Acute ethanol preexposure did not alter the expression of key cell cycle-regulating genes. The rate of hepatocyte division is regulated, not only at the level of entry into the cell cycle, but also at key cell cycle checkpoints (e.g., G1/S transition). As the PCNA results (see Fig. 1D) suggested, an accelerated progression through the G1/S transition in ethanol-exposed mice and the expression of key regulators (cyclin D1, p21 and p27) of the G1/S transition were determined (Fig. 2). PHx surgery increased the expression of all three genes, but with different temporal patterns. Acute ethanol exposure did not significantly change hepatic expression of any of these genes after PHx. Effect of acute ethanol preexposure on the activation of MAPKs caused by PHx. The mitogenic effects of growth factors during hepatic regeneration are mediated, at least in part, by SAPK (e.g., p38, JNK1/2, and ERK1/2). The phosphorylation status of these proteins was therefore assessed by immunoblot (Fig. 3). The phosphorylation of p38, JNK1/2, and ERK1/2 was significantly increased by PHx and peaked 24 h after surgery in the maltose-dextrin group. Although the increase of ERK1/2 and JNK1 phosphorylation caused by PHx was not significantly different between the maltose-dextrin- and ethanol-exposed mice, the increase of p38 and JNK2 phosphorylation was significantly attenuated in ethanol-exposed mice at the 24-h time point after PHx by ⬃10% and 30%, respectively (Fig. 3). Acute ethanol preexposure alters hepatic lipid and glycogen metabolism after PHx. Ethanol is well known to alter hepatic lipid and carbohydrate metabolism. As changes in these pools can potentially impact hepatic regeneration (13), the effect of acute ethanol and PHx on indices of these pools was determined. Figure 4 shows representative photomicrographs depicting lipid and glycogen accumulation in the liver (Oil red O stain and PAS stain, respectively) and quantitative analysis of the pools of hepatic lipids. As observed previously (1), lipid and glycogen levels were within normal ranges in mice exposed to maltose-dextrin. PHx robustly and transiently increased Oil red O staining (Fig. 4A) and hepatic lipid pools in maltose-dextrin-treated animals (Fig. 4B). Acute ethanol exposure did not significantly alter most lipid pools before PHx, except cholesterol, which was approximately twofold higher than maltose-dextrin-treated controls (Fig. 4B). However, the increase in free fatty acids, phospholipids, and triglycerides caused by PHx was almost completely attenuated by acute alcohol preexposure (Fig. 4B).
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00085.2013 • www.ajpgi.org
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alone decreased PCK1 expression by ⬃50% before PHx (P ⬍ 0.05) but did not significantly affect the expression of this gene at other time points. PHx significantly decreased Fas gene expression in the maltose-dextrin group at the 12-h time point after PHx; this effect was significantly attenuated in acute ethanol-exposed mice. GLUT4 expression was also suppressed in the maltose-dextrin group 12–24 h after PHx; in contrast, acute ethanol exposure significantly increased the expression of this gene at these time points after PHx. Acute ethanol preexposure attenuated oxidative stress caused by PHx. PHx is known to cause oxidative stress, which delays liver regeneration (9). As a general index of oxidative stress, the effect of PHx and ethanol preexposure on the accumulation of 4-HNE protein adducts was determined immunohistochemically (Fig. 6). Ethanol preexposure did not affect the amount of adducts in livers before PHx (0 h). PHx dramatically increased the number of 4-HNE adducts in the liver lobule, an effect that peaked 24 h after surgery. Ethanol preexposure decreased the amount of 4-HNE adducts as early as 6 h after PHx (not shown), but the effect was most dramatic at the 24-h time point (Fig. 6).
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Fig. 2. Effect of ethanol preexposure on the expression of key cell cycle control genes after PHx. Animal treatments and methods are as described in MATERIALS AND METHODS. Syclin D1, p21, and p27 gene expression at different time points after PHx, as determined by real-time RT-PCR are shown. Data are means ⫾ SE (n ⫽ 4 – 6); aP ⬍ 0.05 compared with t ⫽ 0 h.
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Hepatic PAS staining (Fig. 4A) and glycogen content decreased with time after PHx. Acute ethanol exposure alone did not significantly alter hepatic glycogen levels before PHx although relatively diminished PAS staining could be seen at some hepatic perivascular areas of ethanol-exposed mice (Fig. 4A). Acute ethanol exposure increased the rate of glycogen depletion after PHx. For example, 12 h after PHx, hepatic glycogen values in the maltose-dextrin group were approximately twofold higher compared with ethanol-treated group (13 ⫾ 2 vs. 6. ⫾ 1 g/mg tissue weight, respectively; P ⬍ 0.05). Effect of acute ethanol preexposure on the expression of genes associated with hepatic lipid and carbohydrate metabolism after PHx. One level of regulation of carbohydrate and lipid metabolism is via expression of rate-limiting enzymes in the processes. Therefore, the mRNA expression of genes critical for lipid synthesis (Fas) and carbohydrate metabolism [phosphoenolpyruvate carboxykinase-1 (PCK1), glucose-6 phosphatase (G6P), and insulin-responsive glucose transporter 4 (GLUT4)] was quantified (Fig. 5). G6P expression was not significantly different between ethanol- and maltose-dextrinexposed groups at any time point after PHx. Ethanol exposure
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Fig. 3. Effect of ethanol preexposure on MAPK activation after PHx. Animal treatments and methods are as described in MATERIALS AND METHODS. A: representative immunoblots 24 h after PHx. B: densitometric analysis of immunoblotting at all time points. Quantitative data are means ⫾ SE (n ⫽ 4 – 6); a P ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
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Fig. 4. Effect of preoperative ethanol exposure and PHx on lipid and glycogen content in liver. A: representative photomicrographs (⫻200) depicting Oil Red O (lipids; columns 1 and 3) and periodic acid Schiff (PAS) (glycogen; columns 2 and 4) staining from mouse liver after PHx. B: biochemical determination of lipid pools in liver at various times after PHx. aP ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
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Preexposure to acute ethanol robustly prevented lipid accumulation in the liver after PHx (Fig. 4) but had little effect on the expression of key synthesis genes (Fig. 5). Another mechanism by which lipid metabolism can be impaired is via oxidative stress damage to the mitochondria (28). Aconitase is
an mitochondrial enzyme known to be sensitive to oxidative stress (38). The effect of PHx and acute ethanol preexposure on aconitase activity was therefore determined (Fig. 6, bottom, right). There was no significant difference in mitochondrial aconitase activity in livers from ethanol-exposed mice and
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Fig. 5. Effect of acute ethanol preexposure on expression of key lipid and glucose metabolism genes. Real-time RT-PCR was performed as described in MATERIALS AND METHODS. Data are means ⫾ SE (n ⫽ 4 – 6) and are expressed as fold of control. aP ⬍ 0.05 compared with maltose-dextrin control of the same time-point after PHx; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
maltose-dextrin controls before PHx. In maltose-dextrin controls, aconitase activity was dramatically inhibited by PHx; indeed, as early as 6 h after PHx, aconitase activity was ⬃20% of baseline in this group. Although PHx also inhibited aconitase activity in livers from ethanol-preexposed mice, this inhibition was blunted by ethanol preexposure at early time points. Indeed, 6 h after PHx, aconitase activity was approximately threefold higher in the ethanol group compared with the maltose-dextrin controls. However, 24 h after PHx, the effect of ethanol on this variable was absent. Effect of acute ethanol preexposure on hepatic PKC- expression and mitochondrial ALDH2 activity and total ALDHs activity. In addition to acetaldehyde, lipid aldehydes (e.g., 4-HNE) are also substrates for some ALDHs (e.g., ALDH2; Fig. 6, bottom, left), which may protect the mitochondria against oxidative damage caused by these compounds (23, 39). The effect of ethanol preexposure and PHx on ALDH levels and activity was therefore determined (Fig. 7A). Ethanol exposure did not significantly alter protein levels of ALDH2, as determined by Western blot. However, the activity of ALDH2 was approximately twofold higher in mitochondria from alcohol-exposed mice before PHx (Fig. 7A). As other ALDH isozymes may also play an antioxidant role in the mitochondrion, the total enzyme activity of ALDHs in regenerating liver was also measured (Fig. 7A). Total activity of all ALDHs was significantly suppressed in the livers of maltose-dextrin-exposed mice 12 h and 24 h after PHx. Acute ethanol preexposure significantly blunted this decrease in total ALDH activity caused by PHx. Protein levels of PKC-ε, an upstream activator of ALDH2, was also elevated by ethanol preexposure under these conditions (Fig. 7A). Because previous studies have shown that PKC-ε is activated by acute alcohol exposure (e.g.,
Ref. 17), and that PKC-ε is a known activator of ALDH2 (5), the effect of knocking down PKC-ε with antisense ASO on ALDH2 activity under these conditions was determined. As was observed previously (17), PKC-ε mRNA levels were approximately fourfold lower in knockdown than in control or ethanol-exposure mice given scrambled control ASO. Knockdown of PKC-ε almost completely prevented the increase in ALDH2 activity caused by ethanol (Fig. 7A, bottom, right). ALDH2 mediates the proliferative effect of acute ethanol preexposure on liver regeneration. Increased hepatic regeneration caused by alcohol preexposure correlated with an increase in ALDH2 activity (Fig. 7A) and protection against oxidative stress (Fig. 6). Therefore, the specific role of ALDH2 on hepatic regeneration under these conditions was determined (Fig. 7B) with Alda-1, a specific activator of ALDH2 (5), and Daidzin, an ALDH2 inhibitor (21). Alda-1 administration to maltose-dextrin-treated mice (Fig. 7B, bottom, left) increased hepatic proliferation to an extent similar to ethanol preexposure 24 h after PHx (Fig. 7B, top, right; see also Fig. 1C). Indeed, analogous to ethanol pretreatment, the number of cells in S-phase was ⬃2.5-fold higher in Alda-1-treated mice compared with controls, and the G1/S ratio was also decreased approximately threefold. In contrast, inhibiting ALDH2 with Daidzin (Fig. 7B, bottom, right) completely blocked the increase in PCNA incorporation caused by ethanol preexposure; the distribution pattern of cell cycle phases was similar to maltose-dextrin-treated mice under these conditions. ALDH2 activation protects hepatocytes from 4-HNE cytotoxicity. The in vivo work summarized supported the hypothesis that ALDH2 activation by acute ethanol preexposure enhanced liver regeneration after PHx via protecting against mitochondrial oxidative stress, thereby metabolically allowing the cells to enter the cell cycle more rapidly (see Fig. 6, bottom, left). To further test this hypothesis, the effect of ALDH2 activation on cytotoxicity caused by 4-HNE exposure in primary hepatocytes was determined (Fig. 8). In naïve cells, exposure of cells to 4-HNE for 24 h dose dependently decreased polarized mitochondria (TMRM; Fig. 8A) and concomitantly increased membrane permeability (TOTO-3). The decrease in the latter value at higher 4-HNE concentrations (⬎100 M) is likely due to cell losses before high-content screening analysis (data not shown). These effects of 4-HNE were not coupled with a decrease in mitochondrial size, as would be expected by apoptotic killing. Activation of ALDH2 by Alda-1 significantly blunted the toxicity of 4-HNE (Fig. 8A) and this protective effect of Alda-1 was significantly attenuated by Daidzin coadministration. Ethanol preexposure in vivo conferred similar protection against 4-HNE toxicity in isolated hepatocytes. As summarized in Fig. 8C, ethanol preexposure prevented the loss of TMRM fluorescence and cytotoxicity (TOTO-3 fluorescence) caused by 125 and 250 M 4-HNE. DISCUSSION
Acute ethanol preexposure enhances liver regeneration after PHx. Chronic ethanol exposure induces multiple effects on liver cell proliferation. For example, chronic ethanol exposure increases the basal number of proliferating cells in the liver (16), which may contribute to hepatomegaly caused by ethanol. However, this increase in basal proliferation may be insuffi-
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Fig. 6. Effect of acute ethanol preexposure on 4-hydroxynonenal (4-HNE) accumulation after PHx. Animal treatments and methods are as described in MATERIALS AND METHODS. Top: representative photomicrographs (⫻200) depicting immunohistochemical detection of 4-HNE protein adducts (brown) against a hematoxylin counterstain are shown. Bottom, left: working hypothesis. Bottom, right: mitochondrial aconitase activity at select times after PHx. aP ⬍ 0.05 compared with maltosedextrin control of the same time-point after PHx; bP ⬍ 0.05 compared with maltosedextrin control of the same time point after PHx. ALDH, aldehyde dehydrogenase; Alda-1, ALDH2 inducer.
cient for the liver to recover from injury, thereby contributing to the chronicity of disease. The finding that chronic ethanol exposure impairs hepatic regeneration after PHx (e.g., Ref. 20) is in line with this concept. Indeed, several studies have demonstrated that mice deficient in proliferation progress to experimental fibrosis and cirrhosis more rapidly, the most notable being telomerase-deficient mice (29). Previous studies have shown that acute ethanol exposure will impair hepatic regeneration if administered 1 h before or 1 h after PHx surgery in rodents (19, 44). However, such a protocol is complicated by the direct and indirect effects of ethanol and/or its metabolites. Here, the effect of ethanol was moved more distally (24 h prior) from the surgery (Fig. 1A), which removes concerns about ethanol and its metabolites, per se, on the observed effects. Previous work by this group using this exposure model has shown that ethanol preexposure enhances lipopolysaccharide-induced liver injury (1), analogous
to findings after chronic ethanol exposure (6). Based on the previous similarities between this acute exposure model and chronic ethanol exposure (1, 6), it was initially hypothesized that acute ethanol exposure would prevent hepatic regeneration, analogous to findings after chronic exposure. Surprisingly, acute ethanol preexposure enhanced hepatic regeneration under these conditions and accelerated the gain of liver mass (Fig. 1B) at later time points (⬎48 h). This gain in liver mass is likely the result of the increased entry and progression through the cell cycle observed in the livers from alcoholpreexposed mice (Fig. 1, C and D). Koteish et al. (20) reported that chronic ethanol exposure downregulates cyclin D1 and upregulates p21 expression, thereby suppressing liver regeneration. However, the effect of acute ethanol preexposure observed did not correlate with any apparent changes in expression of these regulators of the cell cycle (Fig. 2). The effect of ethanol under these conditions
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Fig. 7. Role of ALDH2 in enhanced regeneration after acute ethanol preexposure. A: representative immunoblots (upper, left), densitometric analysis (top, right) at the time of PHx (t ⫽ 0). Total ALDH activity for all time points (bottom, left), mitochondrial ALDH2 (bottom, right) activity at the time of PHx (t ⫽ 0) are also shown. B: representative photomicrographs (⫻200) of PCNA immunostaining of livers from mice 24 h after PHx. aP ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx; cP ⬍ 0.05 compared with ethanol exposure in the absence of antisense oligonucleotides (ASO).
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does not appear to be mediated via changes in expression of these genes. Mitogen-activated protein kinases play important roles in regulating the proliferation and cell death, depending on the cellular context. Previous studies have shown that inhibition the phosphorylation of JNK2 and p38 could promote
liver regeneration (24, 35). In this study, acute ethanol preexposure also decreased phosphorylation of JNK2 and p38 after PHx, which may play a role in promoting liver regeneration after ethanol exposure. However, the changes caused by ethanol preexposure were rather small relative to the effects on
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Fig. 8. ALDH2 activation protects against 4-hydroxynonenal toxicity in mouse hepatocytes. A: mitochondrial membrane potential (TMRM fluorescence), cell membrane permeability (TOTO-3), and nuclear area (Hoechst) were determined. aP ⬍ 0.05 compared with control, bP ⬍ 0.05 compared with Alda-1 alone. B: representative photomicrographs (⫻20) depicting nuclear fluorescence (Hoechst; blue), mitochondrial membrane potential (TMRM; red), and cell membrane permeability (TOTO-3; green). C: individual values for TOTO-3 fluorescence as a function of TMRM fluorescence for individual valid objects (cells).
proliferation (Fig. 3), suggesting that other mechanisms may contribute to the observed effects. Role of energy supply in hepatic regeneration. Hepatic regeneration is a highly complex response, requiring coordination of several processes at the organismal level. Not surprisingly, the metabolic demand on a regenerating liver is very high and requires energy to, not only construct new liver cells, but also maintain hepatic function. Changes in this energy supply could, in principle, alter the regenerative response, even under conditions in which expression of key genes involved in regeneration (e.g., Cyclin D1) are unaltered. Therefore, the effect of acute ethanol preexposure and PHx on indices of hepatic glucose and lipid metabolism were determined (Fig. 4). As has been observed by several groups (e.g., Refs. 31 and 37), it was found here that PHx robustly and transiently
increased hepatic lipids in control animals (Fig. 4). This effect is mediated, in part, by elevated lipogenesis (Fig. 5) and impaired mitochondrial -oxidation of nonesterified fatty acids (NEFA) (31). Acute ethanol preexposure almost completely prevented the increase in hepatic NEFA, triglycerides, and phospholipids caused by PHx (Fig. 4). This effect did not correlate with a decrease in lipid synthesis; indeed, Fas expression was higher in the ethanol vs. maltose-dextrin groups (Fig. 5). It is therefore likely that these effects represent more efficient consumption of these pools (e.g., via mitochondria; see below). Likewise, ethanol preexposure caused hepatic glycogen levels to deplete more rapidly after PHx (Fig. 4A and RESULTS), suggesting elevated consumption of glycogen reserves. This effect is despite the elevated GLUT4 expression after PHx in ethanol-preexposed animals (Fig. 5), which should
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favor glucose uptake over glycogenolysis for aerobic metabolism. Taken together, these indices of lipid and carbohydrate metabolism suggest that livers preexposed to acute ethanol are more efficiently able to generate energy from lipid and carbohydrate supplies. Mitochondria are key to maintaining cellular energy supply under basal and stressful conditions. Previous work has shown that mitochondria are transiently uncoupled after PHx and have diminished function. For example, Rehman et al. (27) demonstrated that preventing mitochondrial uncoupling is sufficient to enhance liver regeneration after PHx in mouse liver. Guerrieri et al. (9) showed that mitochondria isolated from rat livers during the early phase after PHx (⬍24 h) show impaired ATP synthesis, elevated lipid peroxides, and increased oxidative damage. It has therefore been hypothesized that oxidative stress caused by PHx (34) impairs mitochondrial function and contributes to altered energy metabolism observed after PHx (40). Furthermore, altering oxidative stress could affect hepatic regeneration by changing the energy generation by mitochondria (4, 14). Here, as has been observed by others, PHx caused lipid peroxidation (Fig. 6). Furthermore, aconitase activity was inhibited (Fig. 6), which is indicative of general oxidative damage to the mitochondria (7). Acute ethanol preexposure blunted both the lipid peroxidation and the aconitase inhibition caused by PHx (Fig. 6). Taken together, these data suggest that ethanol preexposure protected hepatic mitochondria from oxidative damage caused by PHx. This antioxidant effect most likely prevented mitochondrial dysfunction, which allowed the cells to more efficiently prepare for entry into the cell cycle. ALDH2 plays a key role in enhancing regeneration after PHx in mouse liver. Alcohol exposure is well known to induce expression and/or activity of enzymes responsible for its metabolism. ALDH2 is a key mitochondrial enzyme responsible for acetaldehyde oxidation in ethanol metabolism. However, other aldehydes, including lipid aldehydes (4-HNE) (14, 30) are substrates for this enzyme; induction/activation of this enzyme could therefore protect mitochondria from oxidative stress mediated by lipid aldehydes (Fig. 6, bottom, left). In this study, acute ethanol preexposure increased the activity of ALDHs and ALDH2 (Fig. 7A). The increase in ALDH2 activity caused by acute ethanol preexposure is most likely mediated by the increase in PKC-ε, a known activator of ALDH2 (5), as ASO knockdown of PKC-ε prevented this effect (Fig. 7). Previous work by this group has indicated that acute ethanol exposure likely activates PKC-ε by increasing diacylglycerol levels in the liver (25). Taken together, these data suggest that the transient oxidative stress caused by PHx is partially prevented by alcohol preexposure by activating ALDH2; this protective effect is likely responsible for mediating the more rapid entry into the cell cycle observed in the livers from ethanol-preexposed animals (Fig. 1). Indeed, an ALDH2 inducer (Alda-1) (5) mimicked the ethanol effect (Fig. 7B, bottom, left), and an ALDH2 inhibitor (Daidzin) (21) prevented the ethanol effect (Fig. 7B, bottom, right). This hypothesis is further supported by the fact that, in primary hepatocytes, ethanol preexposure in vivo or Alda-1 in vitro conferred protection against mito- and cytotoxicity caused by 4-HNE (Fig. 8). Summary and conclusions. Taken together, this study identifies a new role of mitochondrial ALDH2 in promoting hepatic regeneration in the mouse liver. Recently, a similar protective
mechanism of ALDH2 induction was observed in experimental cardiac ischemia/reperfusion in rats (5). The proposed mechanism by which ALDH2 enhances hepatic regeneration under these conditions is via protecting against mitochondrial dysfunction caused by oxidative stress early after PHx (Fig. 6, bottom, left). This protective effect against mitochondrial damage allows the cells to more efficiently generate the energy reserves required to enter the cell cycle. ALDH2 activity may also explain, at least in part, the differences between the effects of acute and chronic alcohol exposure on hepatic regeneration after PHx; specifically, whereas acute ethanol activates ALDH2 (Fig. 7), chronic alcohol exposure actually inhibits activity (11). The clinical implications of these data are that they suggest that targeting ALDH2 may be a useful strategy for improving hepatic regeneration. For example, activating ALDH (e.g., with Alda-1) may improve hepatic regeneration after hepatic resection surgery. These data further suggest that, in addition to altered alcohol metabolism, loss-of-function single nucleotide polymorphisms of ALDH2 may increase the risk of developing liver disease by impairing hepatic regeneration. This latter point may be of concern, not only for alcoholic liver disease, but also for other liver diseases (18). ACKNOWLEDGMENTS X. Ding is currently affiliated with the Department of Transplantation, Xiangya hospital, Central South University, Changsha, Hunan, China. GRANTS This work was supported, in part, by a grant from the National Institute of Alcohol Abuse and Alcoholism (AA003624). J. Beier was supported by a postdoctoral (T32) fellowship from National Institute of Environmental Health Science (ES011564). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: X.D. and G.E.A. conception and design of research; X.D., J.I.B., K.J.B., J.D.J., H.Z., and G.E.A. performed experiments; X.D., J.I.B., K.J.B., J.D.J., H.Z., and G.E.A. analyzed data; X.D., J.I.B., K.J.B., and G.E.A. interpreted results of experiments; X.D., K.J.B., and G.E.A. prepared figures; X.D. and G.E.A. drafted manuscript; X.D., J.I.B., K.J.B., and G.E.A. edited and revised manuscript; X.D., J.I.B., K.J.B., J.D.J., H.Z., and G.E.A. approved final version of manuscript. REFERENCES 1. Beier JI, Luyendyk JP, Guo L, von Montfort C, Staunton DE, Arteel GE. Fibrin accumulation plays a critical role in the sensitization to lipopolysaccharide-induced liver injury caused by ethanol in mice. Hepatology 49: 1545–1553, 2009. 2. Bergheim I, Guo L, Davis MA, Lambert JC, Beier JI, Duveau I, Luyendyk JP, Roth RA, Arteel GE. Metformin prevents alcohol-induced liver injury in the mouse: Critical role of plasminogen activator inhibitor-1. Gastroenterology 130: 2099 –2112, 2006. 3. Berry MN, Friend DS. High yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study. J Cell Biol 43: 506 –520, 1969. 4. Beyer TA, Werner S. The cytoprotective Nrf2 transcription factor controls insulin receptor signaling in the regenerating liver. Cell Cycle 7: 874 –879, 2008. 5. Chen CH, Budas GR, Churchill EN, Disatnik MH, Hurley TD, Mochly-Rosen D. Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart. Science 321: 1493–1495, 2008. 6. Enomoto N, Ikejima K, Bradford BU, Rivera CA, Kono H, Brenner DA, Thurman RG. Alcohol causes both tolerance and sensitization of rat
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Acute ethanol preexposure promotes liver regeneration after partial hepatectomy in mice by activating ALDH2 Xiang Ding,1,2 Juliane I. Beier,1,2 Keegan J. Baldauf,1,2 Jenny D. Jokinen,1,2 Hai Zhong,1,2 and Gavin E. Arteel1,2 1
Department of Pharmacology and Toxicology, University of Louisville Health Sciences Center, Louisville; 2University of Louisville Alcohol Research Center, Louisville, Kentucky Submitted 18 March 2013; accepted in final form 23 October 2013
Ding X, Beier JI, Baldauf KJ, Jokinen JD, Zhong H, Arteel GE. Acute ethanol preexposure promotes liver regeneration after partial hepatectomy in mice by activating ALDH2. Am J Physiol Gastrointest Liver Physiol 306: G37–G47, 2014. First published October 31, 2013; doi:10.1152/ajpgi.00085.2013.—It is known that chronic ethanol significantly impairs liver regeneration. However, the effect of acute ethanol exposure on liver regeneration remains largely unknown. To address this question, C57Bl6/J mice were exposed to acute ethanol (6 g/kg intragastrically) for 3 days, and partial hepatectomy (PHx) was performed 24 h after the last dose. Surprisingly, acute ethanol preexposure promoted liver regeneration. This effect of ethanol did not correlate with changes in expression of cell cycle regulatory genes (e.g., cyclin D1, p21, and p27) but did correlate with protection against the effect of PHx on indices of impaired lipid and carbohydrate metabolism. Ethanol preexposure protected against inhibition of the oxidant-sensitive mitochondrial enzyme, aconitase. The activity of aldehyde dehydrogenase 2 (ALDH2) was significantly increased by ethanol preexposure. The effect of ethanol was blocked by inhibiting (Daidzin) and was mimicked by activating (Alda-1) ALDH2. Lipid peroxides are also substrates for ALDH2; indeed, alcohol preexposure blunted the increase in lipid peroxidation (4OH-nonenal adducts) caused by PHx. Taken together, these data suggest that acute preoperative ethanol exposure “preconditions” the liver to respond more rapidly to regenerate after PHx by activating mitochondrial ALDH2, which prevents oxidative stress in this compartment. aldehyde dehydrogenase 2; alcohol; liver regeneration; oxidative stress THE LIVER HAS TREMENDOUS REGENERATIVE capacity that distinguishes it from other vital organs (e.g., the brain, heart, and lungs) that are far less able to replace functional tissue. As the main detoxifying organ in the body, the liver is prone to toxic injury. Due to its regenerative properties, however, the liver is able to restore to full size and ensure survival. In experimental models (e.g., mice), full regeneration occurs within 7–10 days (36). Although hepatocytes rarely proliferate in the healthy adult liver, virtually all surviving hepatocytes replicate at least once after 70% partial hepatectomy (PHx). In addition to hepatocyte proliferation, there is a tightly coordinated response to complement the regenerative process (34). Perturbations of this complex and synchronized response can impact normal tissue recovery from injury or damage. Indeed, it is now clear that impaired regeneration and/or restitution is critical to the chronicity of numerous hepatic diseases. Studies have shown that chronic ethanol (EtOH)
Address for reprint requests and other correspondence: G. E. Arteel, Dept. of Pharmacology and Toxicology, 505 S Hancock St., CTRB, Rm 506, Univ. of Louisville Health Sciences Center, Louisville, KY, 40292 (e-mail: [email protected]). http://www.ajpgi.org
exposure delays the induction of hepatocyte DNA synthesis in response to PHx (20). This effect of chronic ethanol may contribute to impaired regeneration and restitution from damage associated with alcoholic liver disease. In contrast to chronic exposure, little is known about the effect of acute ethanol administration on liver regeneration. Under some conditions, acute and chronic ethanol exposure mediate similar responses in the organ. For example, both acute and chronic ethanol exposure enhance lipopolysaccharide-induced liver damage (1, 6). Should acute ethanol exposure also impair the hepatic regenerative response, this model could be employed as a screening tool for subsequent chronic studies. The purpose of the present study was a priori to test the hypothesis that acute alcohol exposure will impair hepatic regeneration after PHx, analogous to findings with chronic ethanol exposure (20). Surprisingly, it was found that acute ethanol exposure enhanced hepatic regeneration after PHx. The purpose of this study was then to determine the mechanisms behind this effect. MATERIALS AND METHODS
Animals and treatments. Male C57BL/6J mice, 6 – 8 weeks old, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a pathogen-free barrier facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and procedures were approved by the University of Louisville Institutional Animal Care and Use Committee. Food and tap water were allowed ad libitum. Animals received ethanol (6 g/kg intragastrically) or isocaloric/isovolumetric maltose-dextrin solution for 3 days (Fig. 1A) (1). With this dose of alcohol, peak blood levels are ⬃300 mg/dl, and mice are sluggish but conscious; the animals regain normal behavior after ⬃6 h of alcohol dosing (2). Some maltosedextrin-exposed mice were injected with a specific aldehyde dehydrogenase 2 (ALDH2) agonist: Alda-1 (20 mg/kg ip; EMD Chemicals, Gibbstown, NJ), or vehicle (50% DMSO and 50% PEG) for 3 days before PHx, and the last dose of Alda-1 was given during surgery. Some ethanol-exposed mice were given 7-(-D-Glucopyranosyloxy)-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (75 mg/kg ip, Daidzin; Sigma, St. Louis, MO) or vehicle (50% DMSO and 50% PEG) during PHx surgery to inhibit ALDH2 activity. Seventy percent PHxs were performed 24 h after the last ethanol dose under isofluorane anesthesia (8) with minor modifications. Mice were anesthetized with ketamine/xylazine (100/15 mg/kg intramuscularly) at select time points up to 96 h after PHx (see timeline in Fig. 1A) and samples collected (1). Hepatocytes were isolated from some ethanol- or maltose-dextran-exposed mice 24 h after the last administration (see below). Biochemical analyses and histology. The capacity of liver regeneration was assessed by liver weight/body weight ratio; cell cycle progression (per 1,000 hepatocytes) was estimated using specific proliferating chain nuclear antigen (PCNA) staining patterns and cell morphology as described previously (41, 42). Briefly, G0 cells do not
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Fig. 1. Effect of acute ethanol (EtOH) preexposure on liver regeneration after partial hepatectomy (PHx). A: experimental timeline. B: plasma transaminases (left) and liver weight/body weight ratios (right) at the time of death. ALT, alanine aminotransferase; AST, aspartate aminotransferase. C: representative photomicrographs (⫻200) depicting proliferating chain nuclear antigen (PCNA) immunostaining 12 h and 24 h after PHx in maltose-dextrin (MD)-exposed (left) and ethanolexposed (right) groups. D: cell cycle progression (per 1,000 hepatocytes) at different time points. aP ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
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stain for PCNA; G1 cells are lightly stained in the nucleus, whereas S-phase cell nuclei are darkly stained; G2 cells have a cytoplasmic staining pattern with or without nuclear staining; M cells have diffuse cytoplasmic and deep blue chromosomal staining and a clear presence of mitotic bodies. Frozen sections of liver (10 m) were stained with oil red O (2). Hepatic lipids were determined in extracted liver samples (2). Hepatic glycogen reserves were determined by periodic acid (0.5%) Schiff (PAS) staining (12) and by anthrone reagent (32). Mitochondrial protein for the ALDH2 and aconitase activity assays were extracted from liver, as described by Wiekowski et al. (43). Enzymatic activity of ALDHs and ALDH2 was determined spectrophotometrically by monitoring the reduction of NAD⫹ to NADH at 340 nm (15). To determine the role of PKC-ε on activating ALDH2, the effect of knocking down this enzyme with antisense oligonucleotides (ASO) against PKC-ε was determined, as described previously by Kaiser et al. (17). Briefly, mice received the PKC-ε ASO or vehicle saline at a dose of 25 mg/kg ip twice a week before ethanol or maltose dextrin administration (see above). Mice were killed 24 h after the last
ethanol gavage. Mitochondrial aconitase activity was determined by monitoring the reduction of NAD⫹ to NADH at 340 nm (26). Plasma levels of aminotransferases (alanine aminotransferase and aspartate aminotransferase, ALT and AST, respectively) were determined using standard kits (Thermotrace, Melbourne, Australia). Adducts of 4-hydroxynonenal (4-HNE) were detected by immunohistochemistry using a rabbit polyclonal 4-HNE antibody (Alpha Diagnostic, San Antonio, TX) (10). RNA isolation and real-time RT-PCR. RNA extraction and realtime RT-PCR was performed as described previously (2). The PCR primers and probes PCK1, Fas, and G6P were designed using Primer 3 (Whitehead Institute for Biomedical Research, Cambridge, MA). Primers were designed to cross exons to ensure that only cDNA and not genomic DNA was amplified. Other PCR primers and probes (e.g., cyclin D1, p21, and p27) were purchased premade (Applied Biosystems, Carlsbad, CA). The comparative CT method was used to determine fold differences between samples and the calibrator gene (-actin), and purity of PCR products was verified by gel electropho-
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resis. The comparative CT method determines the amount of target, normalized to an endogenous reference (-actin) and relative to a calibrator (2⫺⌬⌬Ct). Immunoblots. Liver samples were homogenized in RIPA buffer [20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (wt/vol) Triton X-100], containing protease and phosphatase inhibitor cocktails (Sigma). Samples were loaded onto SDS-polyacrylamide gels of 12% (wt/vol) acrylamide followed by electrophoresis and Western blotting onto nitrocellulose membranes (Hybond P; GE Healthcare, Piscataway, NJ). Primary antibodies against phosphorylated and total extracellular-signal-regulated kinases1/2 (ERK1/ 2), p38, and c-Jun NH2-terminal kinases (JNK1/2) were from Cell Signaling (Billerica, MA). Primary antibodies against PKC-ε and ALDH2 (Santa Cruz Biotechnology, Santa Cruz, CA) were used. Bands were visualized using an ECL kit (Pierce, Rockford, IL) and Hyperfilm (GE Healthcare). Densitometric analysis was performed using UN-SCAN-IT Gel (Silk Scientific, Orem, UT) software. Hepatocyte isolation and culture. Hepatocytes were isolated and separated from liver by collagenase perfusion (3, 33), with minor modifications. Isolated hepatocytes were cultured (10,000 cells/well) overnight on collagen-coated 96-well plates optimized for fluorescent microscopy (NUNC, Rochester, NY). Cells were then exposed to 4-HNE (0 –1 mM) for up to 24 h. Some cells were exposed to Alda-1 (1.5 M) and/or Daidzin (60 M) 30 min before 4-HNE addition. After the predetermined incubation period with 4-HNE, the media was removed, and media containing Hoechst 33342 (1.5 M; nuclear fluorescence), TMRM (20 nM; mitochondrial membrane potential), and TOTO-3 (1 M; cell membrane permeability) dyes was added to the wells. Following a 1-h incubation with the dyes, the plate was placed into the Cellomics Array Scan VTI HCS reader and analyzed, analogous to what has been previously described by O’Brien et al. (22). The first of either 800 valid objects (defined by Hoechst 33342 staining) or five microscope fields were analyzed. The average of each 96-well plate was determined, as well as the individual values for all three fluorophores for each valid object. Statistical analyses. ANOVA with Bonferroni’s post hoc test or the Mann-Whitney Rank Sum test was used for the determination of statistical significance among treatment groups, as appropriate. Results are reported as means ⫾ SE (n ⫽ 4 – 6). RESULTS
Effect of acute ethanol preexposure on indices of liver damage after PHx. As observed previously (1), plasma levels of AST and ALT were within normal ranges in mice exposed to maltose-dextrin in the absence of surgery (Fig. 1B, left); acute ethanol exposure in the absence of PHx did not significantly alter AST and ALT levels compared with maltose-dextrinexposed mice. Preoperative acute ethanol exposure did not significantly alter the increase in plasma AST and ALT caused by PHx. Acute ethanol preexposure promotes liver regeneration after PHx. As is well known for this paradigm, PHx rapidly induces a regenerative response in the remnant liver. A crude index of this response is the increase in liver weight/body weight ratio (Fig. 1B, right), which increased rapidly in maltose-dextrinexposed mice after surgery. Acute ethanol preexposure did not significantly affect the liver/body weight ratio in sham-operated animals or in animals killed immediately after PHx (Fig. 1B). However, acute ethanol administration significantly increased liver mass gain compared with maltose-dextrin controls at later time points (⬎48 h). The effect of ethanol on hepatocyte proliferation after PHx was further analyzed by PCNA immunostaining (Fig. 1C). In maltose-dextrin controls, the amount of PCNA-positive cells
G39
increased dramatically 24 h after PHx and returned to G0 by the 96-h time point. Acute ethanol preexposure dramatically enhanced the rate of entry into the cell cycle (Fig. 1D). Indeed, ethanol preexposure increased the number of hepatocytes in G1 phase (88 ⫾ 24 per 1,000 hepatocytes) 12 h after PHx, a time point at which few hepatocytes in the control PHx group had entered G1 (3 ⫾ 1 per 1,000 hepatocytes). The rate of progression through the cell cycle was also apparently enhanced by acute ethanol preexposure. For example, although the number of cells in G1 was ⬃18% lower in ethanol-exposed mice compared with control mice (688 ⫾ 63 vs. 843 ⫾ 26, respectively) 24 h after PHx, the number of cells in S phase was ⬃2.6-fold higher (233 ⫾ 60 vs. 87 ⫾ 27, respectively) in the ethanol group. This effect greatly decreased the G1/S ratio in the ethanol group (3 vs. 10), which is indicative of an accelerated G1/S transition. The G1/S ratio was similarly affected by ethanol preexposure 48 h after PHx. Acute ethanol preexposure did not alter the expression of key cell cycle-regulating genes. The rate of hepatocyte division is regulated, not only at the level of entry into the cell cycle, but also at key cell cycle checkpoints (e.g., G1/S transition). As the PCNA results (see Fig. 1D) suggested, an accelerated progression through the G1/S transition in ethanol-exposed mice and the expression of key regulators (cyclin D1, p21 and p27) of the G1/S transition were determined (Fig. 2). PHx surgery increased the expression of all three genes, but with different temporal patterns. Acute ethanol exposure did not significantly change hepatic expression of any of these genes after PHx. Effect of acute ethanol preexposure on the activation of MAPKs caused by PHx. The mitogenic effects of growth factors during hepatic regeneration are mediated, at least in part, by SAPK (e.g., p38, JNK1/2, and ERK1/2). The phosphorylation status of these proteins was therefore assessed by immunoblot (Fig. 3). The phosphorylation of p38, JNK1/2, and ERK1/2 was significantly increased by PHx and peaked 24 h after surgery in the maltose-dextrin group. Although the increase of ERK1/2 and JNK1 phosphorylation caused by PHx was not significantly different between the maltose-dextrin- and ethanol-exposed mice, the increase of p38 and JNK2 phosphorylation was significantly attenuated in ethanol-exposed mice at the 24-h time point after PHx by ⬃10% and 30%, respectively (Fig. 3). Acute ethanol preexposure alters hepatic lipid and glycogen metabolism after PHx. Ethanol is well known to alter hepatic lipid and carbohydrate metabolism. As changes in these pools can potentially impact hepatic regeneration (13), the effect of acute ethanol and PHx on indices of these pools was determined. Figure 4 shows representative photomicrographs depicting lipid and glycogen accumulation in the liver (Oil red O stain and PAS stain, respectively) and quantitative analysis of the pools of hepatic lipids. As observed previously (1), lipid and glycogen levels were within normal ranges in mice exposed to maltose-dextrin. PHx robustly and transiently increased Oil red O staining (Fig. 4A) and hepatic lipid pools in maltose-dextrin-treated animals (Fig. 4B). Acute ethanol exposure did not significantly alter most lipid pools before PHx, except cholesterol, which was approximately twofold higher than maltose-dextrin-treated controls (Fig. 4B). However, the increase in free fatty acids, phospholipids, and triglycerides caused by PHx was almost completely attenuated by acute alcohol preexposure (Fig. 4B).
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00085.2013 • www.ajpgi.org
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Fold of Control
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alone decreased PCK1 expression by ⬃50% before PHx (P ⬍ 0.05) but did not significantly affect the expression of this gene at other time points. PHx significantly decreased Fas gene expression in the maltose-dextrin group at the 12-h time point after PHx; this effect was significantly attenuated in acute ethanol-exposed mice. GLUT4 expression was also suppressed in the maltose-dextrin group 12–24 h after PHx; in contrast, acute ethanol exposure significantly increased the expression of this gene at these time points after PHx. Acute ethanol preexposure attenuated oxidative stress caused by PHx. PHx is known to cause oxidative stress, which delays liver regeneration (9). As a general index of oxidative stress, the effect of PHx and ethanol preexposure on the accumulation of 4-HNE protein adducts was determined immunohistochemically (Fig. 6). Ethanol preexposure did not affect the amount of adducts in livers before PHx (0 h). PHx dramatically increased the number of 4-HNE adducts in the liver lobule, an effect that peaked 24 h after surgery. Ethanol preexposure decreased the amount of 4-HNE adducts as early as 6 h after PHx (not shown), but the effect was most dramatic at the 24-h time point (Fig. 6).
a
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Fig. 2. Effect of ethanol preexposure on the expression of key cell cycle control genes after PHx. Animal treatments and methods are as described in MATERIALS AND METHODS. Syclin D1, p21, and p27 gene expression at different time points after PHx, as determined by real-time RT-PCR are shown. Data are means ⫾ SE (n ⫽ 4 – 6); aP ⬍ 0.05 compared with t ⫽ 0 h.
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Hepatic PAS staining (Fig. 4A) and glycogen content decreased with time after PHx. Acute ethanol exposure alone did not significantly alter hepatic glycogen levels before PHx although relatively diminished PAS staining could be seen at some hepatic perivascular areas of ethanol-exposed mice (Fig. 4A). Acute ethanol exposure increased the rate of glycogen depletion after PHx. For example, 12 h after PHx, hepatic glycogen values in the maltose-dextrin group were approximately twofold higher compared with ethanol-treated group (13 ⫾ 2 vs. 6. ⫾ 1 g/mg tissue weight, respectively; P ⬍ 0.05). Effect of acute ethanol preexposure on the expression of genes associated with hepatic lipid and carbohydrate metabolism after PHx. One level of regulation of carbohydrate and lipid metabolism is via expression of rate-limiting enzymes in the processes. Therefore, the mRNA expression of genes critical for lipid synthesis (Fas) and carbohydrate metabolism [phosphoenolpyruvate carboxykinase-1 (PCK1), glucose-6 phosphatase (G6P), and insulin-responsive glucose transporter 4 (GLUT4)] was quantified (Fig. 5). G6P expression was not significantly different between ethanol- and maltose-dextrinexposed groups at any time point after PHx. Ethanol exposure
48
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Fig. 3. Effect of ethanol preexposure on MAPK activation after PHx. Animal treatments and methods are as described in MATERIALS AND METHODS. A: representative immunoblots 24 h after PHx. B: densitometric analysis of immunoblotting at all time points. Quantitative data are means ⫾ SE (n ⫽ 4 – 6); a P ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00085.2013 • www.ajpgi.org
ALDH2 ACTIVATION AND LIVER REGENERATION
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A
Fig. 4. Effect of preoperative ethanol exposure and PHx on lipid and glycogen content in liver. A: representative photomicrographs (⫻200) depicting Oil Red O (lipids; columns 1 and 3) and periodic acid Schiff (PAS) (glycogen; columns 2 and 4) staining from mouse liver after PHx. B: biochemical determination of lipid pools in liver at various times after PHx. aP ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
B
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Preexposure to acute ethanol robustly prevented lipid accumulation in the liver after PHx (Fig. 4) but had little effect on the expression of key synthesis genes (Fig. 5). Another mechanism by which lipid metabolism can be impaired is via oxidative stress damage to the mitochondria (28). Aconitase is
an mitochondrial enzyme known to be sensitive to oxidative stress (38). The effect of PHx and acute ethanol preexposure on aconitase activity was therefore determined (Fig. 6, bottom, right). There was no significant difference in mitochondrial aconitase activity in livers from ethanol-exposed mice and
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00085.2013 • www.ajpgi.org
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Fold of control
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Fig. 5. Effect of acute ethanol preexposure on expression of key lipid and glucose metabolism genes. Real-time RT-PCR was performed as described in MATERIALS AND METHODS. Data are means ⫾ SE (n ⫽ 4 – 6) and are expressed as fold of control. aP ⬍ 0.05 compared with maltose-dextrin control of the same time-point after PHx; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx.
maltose-dextrin controls before PHx. In maltose-dextrin controls, aconitase activity was dramatically inhibited by PHx; indeed, as early as 6 h after PHx, aconitase activity was ⬃20% of baseline in this group. Although PHx also inhibited aconitase activity in livers from ethanol-preexposed mice, this inhibition was blunted by ethanol preexposure at early time points. Indeed, 6 h after PHx, aconitase activity was approximately threefold higher in the ethanol group compared with the maltose-dextrin controls. However, 24 h after PHx, the effect of ethanol on this variable was absent. Effect of acute ethanol preexposure on hepatic PKC- expression and mitochondrial ALDH2 activity and total ALDHs activity. In addition to acetaldehyde, lipid aldehydes (e.g., 4-HNE) are also substrates for some ALDHs (e.g., ALDH2; Fig. 6, bottom, left), which may protect the mitochondria against oxidative damage caused by these compounds (23, 39). The effect of ethanol preexposure and PHx on ALDH levels and activity was therefore determined (Fig. 7A). Ethanol exposure did not significantly alter protein levels of ALDH2, as determined by Western blot. However, the activity of ALDH2 was approximately twofold higher in mitochondria from alcohol-exposed mice before PHx (Fig. 7A). As other ALDH isozymes may also play an antioxidant role in the mitochondrion, the total enzyme activity of ALDHs in regenerating liver was also measured (Fig. 7A). Total activity of all ALDHs was significantly suppressed in the livers of maltose-dextrin-exposed mice 12 h and 24 h after PHx. Acute ethanol preexposure significantly blunted this decrease in total ALDH activity caused by PHx. Protein levels of PKC-ε, an upstream activator of ALDH2, was also elevated by ethanol preexposure under these conditions (Fig. 7A). Because previous studies have shown that PKC-ε is activated by acute alcohol exposure (e.g.,
Ref. 17), and that PKC-ε is a known activator of ALDH2 (5), the effect of knocking down PKC-ε with antisense ASO on ALDH2 activity under these conditions was determined. As was observed previously (17), PKC-ε mRNA levels were approximately fourfold lower in knockdown than in control or ethanol-exposure mice given scrambled control ASO. Knockdown of PKC-ε almost completely prevented the increase in ALDH2 activity caused by ethanol (Fig. 7A, bottom, right). ALDH2 mediates the proliferative effect of acute ethanol preexposure on liver regeneration. Increased hepatic regeneration caused by alcohol preexposure correlated with an increase in ALDH2 activity (Fig. 7A) and protection against oxidative stress (Fig. 6). Therefore, the specific role of ALDH2 on hepatic regeneration under these conditions was determined (Fig. 7B) with Alda-1, a specific activator of ALDH2 (5), and Daidzin, an ALDH2 inhibitor (21). Alda-1 administration to maltose-dextrin-treated mice (Fig. 7B, bottom, left) increased hepatic proliferation to an extent similar to ethanol preexposure 24 h after PHx (Fig. 7B, top, right; see also Fig. 1C). Indeed, analogous to ethanol pretreatment, the number of cells in S-phase was ⬃2.5-fold higher in Alda-1-treated mice compared with controls, and the G1/S ratio was also decreased approximately threefold. In contrast, inhibiting ALDH2 with Daidzin (Fig. 7B, bottom, right) completely blocked the increase in PCNA incorporation caused by ethanol preexposure; the distribution pattern of cell cycle phases was similar to maltose-dextrin-treated mice under these conditions. ALDH2 activation protects hepatocytes from 4-HNE cytotoxicity. The in vivo work summarized supported the hypothesis that ALDH2 activation by acute ethanol preexposure enhanced liver regeneration after PHx via protecting against mitochondrial oxidative stress, thereby metabolically allowing the cells to enter the cell cycle more rapidly (see Fig. 6, bottom, left). To further test this hypothesis, the effect of ALDH2 activation on cytotoxicity caused by 4-HNE exposure in primary hepatocytes was determined (Fig. 8). In naïve cells, exposure of cells to 4-HNE for 24 h dose dependently decreased polarized mitochondria (TMRM; Fig. 8A) and concomitantly increased membrane permeability (TOTO-3). The decrease in the latter value at higher 4-HNE concentrations (⬎100 M) is likely due to cell losses before high-content screening analysis (data not shown). These effects of 4-HNE were not coupled with a decrease in mitochondrial size, as would be expected by apoptotic killing. Activation of ALDH2 by Alda-1 significantly blunted the toxicity of 4-HNE (Fig. 8A) and this protective effect of Alda-1 was significantly attenuated by Daidzin coadministration. Ethanol preexposure in vivo conferred similar protection against 4-HNE toxicity in isolated hepatocytes. As summarized in Fig. 8C, ethanol preexposure prevented the loss of TMRM fluorescence and cytotoxicity (TOTO-3 fluorescence) caused by 125 and 250 M 4-HNE. DISCUSSION
Acute ethanol preexposure enhances liver regeneration after PHx. Chronic ethanol exposure induces multiple effects on liver cell proliferation. For example, chronic ethanol exposure increases the basal number of proliferating cells in the liver (16), which may contribute to hepatomegaly caused by ethanol. However, this increase in basal proliferation may be insuffi-
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00085.2013 • www.ajpgi.org
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Fig. 6. Effect of acute ethanol preexposure on 4-hydroxynonenal (4-HNE) accumulation after PHx. Animal treatments and methods are as described in MATERIALS AND METHODS. Top: representative photomicrographs (⫻200) depicting immunohistochemical detection of 4-HNE protein adducts (brown) against a hematoxylin counterstain are shown. Bottom, left: working hypothesis. Bottom, right: mitochondrial aconitase activity at select times after PHx. aP ⬍ 0.05 compared with maltosedextrin control of the same time-point after PHx; bP ⬍ 0.05 compared with maltosedextrin control of the same time point after PHx. ALDH, aldehyde dehydrogenase; Alda-1, ALDH2 inducer.
cient for the liver to recover from injury, thereby contributing to the chronicity of disease. The finding that chronic ethanol exposure impairs hepatic regeneration after PHx (e.g., Ref. 20) is in line with this concept. Indeed, several studies have demonstrated that mice deficient in proliferation progress to experimental fibrosis and cirrhosis more rapidly, the most notable being telomerase-deficient mice (29). Previous studies have shown that acute ethanol exposure will impair hepatic regeneration if administered 1 h before or 1 h after PHx surgery in rodents (19, 44). However, such a protocol is complicated by the direct and indirect effects of ethanol and/or its metabolites. Here, the effect of ethanol was moved more distally (24 h prior) from the surgery (Fig. 1A), which removes concerns about ethanol and its metabolites, per se, on the observed effects. Previous work by this group using this exposure model has shown that ethanol preexposure enhances lipopolysaccharide-induced liver injury (1), analogous
to findings after chronic ethanol exposure (6). Based on the previous similarities between this acute exposure model and chronic ethanol exposure (1, 6), it was initially hypothesized that acute ethanol exposure would prevent hepatic regeneration, analogous to findings after chronic exposure. Surprisingly, acute ethanol preexposure enhanced hepatic regeneration under these conditions and accelerated the gain of liver mass (Fig. 1B) at later time points (⬎48 h). This gain in liver mass is likely the result of the increased entry and progression through the cell cycle observed in the livers from alcoholpreexposed mice (Fig. 1, C and D). Koteish et al. (20) reported that chronic ethanol exposure downregulates cyclin D1 and upregulates p21 expression, thereby suppressing liver regeneration. However, the effect of acute ethanol preexposure observed did not correlate with any apparent changes in expression of these regulators of the cell cycle (Fig. 2). The effect of ethanol under these conditions
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A
ALDH2 Fold of Control
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Fig. 7. Role of ALDH2 in enhanced regeneration after acute ethanol preexposure. A: representative immunoblots (upper, left), densitometric analysis (top, right) at the time of PHx (t ⫽ 0). Total ALDH activity for all time points (bottom, left), mitochondrial ALDH2 (bottom, right) activity at the time of PHx (t ⫽ 0) are also shown. B: representative photomicrographs (⫻200) of PCNA immunostaining of livers from mice 24 h after PHx. aP ⬍ 0.05 compared with t ⫽ 0 h; bP ⬍ 0.05 compared with maltose-dextrin control of the same time point after PHx; cP ⬍ 0.05 compared with ethanol exposure in the absence of antisense oligonucleotides (ASO).
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does not appear to be mediated via changes in expression of these genes. Mitogen-activated protein kinases play important roles in regulating the proliferation and cell death, depending on the cellular context. Previous studies have shown that inhibition the phosphorylation of JNK2 and p38 could promote
liver regeneration (24, 35). In this study, acute ethanol preexposure also decreased phosphorylation of JNK2 and p38 after PHx, which may play a role in promoting liver regeneration after ethanol exposure. However, the changes caused by ethanol preexposure were rather small relative to the effects on
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00085.2013 • www.ajpgi.org
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Fig. 8. ALDH2 activation protects against 4-hydroxynonenal toxicity in mouse hepatocytes. A: mitochondrial membrane potential (TMRM fluorescence), cell membrane permeability (TOTO-3), and nuclear area (Hoechst) were determined. aP ⬍ 0.05 compared with control, bP ⬍ 0.05 compared with Alda-1 alone. B: representative photomicrographs (⫻20) depicting nuclear fluorescence (Hoechst; blue), mitochondrial membrane potential (TMRM; red), and cell membrane permeability (TOTO-3; green). C: individual values for TOTO-3 fluorescence as a function of TMRM fluorescence for individual valid objects (cells).
proliferation (Fig. 3), suggesting that other mechanisms may contribute to the observed effects. Role of energy supply in hepatic regeneration. Hepatic regeneration is a highly complex response, requiring coordination of several processes at the organismal level. Not surprisingly, the metabolic demand on a regenerating liver is very high and requires energy to, not only construct new liver cells, but also maintain hepatic function. Changes in this energy supply could, in principle, alter the regenerative response, even under conditions in which expression of key genes involved in regeneration (e.g., Cyclin D1) are unaltered. Therefore, the effect of acute ethanol preexposure and PHx on indices of hepatic glucose and lipid metabolism were determined (Fig. 4). As has been observed by several groups (e.g., Refs. 31 and 37), it was found here that PHx robustly and transiently
increased hepatic lipids in control animals (Fig. 4). This effect is mediated, in part, by elevated lipogenesis (Fig. 5) and impaired mitochondrial -oxidation of nonesterified fatty acids (NEFA) (31). Acute ethanol preexposure almost completely prevented the increase in hepatic NEFA, triglycerides, and phospholipids caused by PHx (Fig. 4). This effect did not correlate with a decrease in lipid synthesis; indeed, Fas expression was higher in the ethanol vs. maltose-dextrin groups (Fig. 5). It is therefore likely that these effects represent more efficient consumption of these pools (e.g., via mitochondria; see below). Likewise, ethanol preexposure caused hepatic glycogen levels to deplete more rapidly after PHx (Fig. 4A and RESULTS), suggesting elevated consumption of glycogen reserves. This effect is despite the elevated GLUT4 expression after PHx in ethanol-preexposed animals (Fig. 5), which should
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favor glucose uptake over glycogenolysis for aerobic metabolism. Taken together, these indices of lipid and carbohydrate metabolism suggest that livers preexposed to acute ethanol are more efficiently able to generate energy from lipid and carbohydrate supplies. Mitochondria are key to maintaining cellular energy supply under basal and stressful conditions. Previous work has shown that mitochondria are transiently uncoupled after PHx and have diminished function. For example, Rehman et al. (27) demonstrated that preventing mitochondrial uncoupling is sufficient to enhance liver regeneration after PHx in mouse liver. Guerrieri et al. (9) showed that mitochondria isolated from rat livers during the early phase after PHx (⬍24 h) show impaired ATP synthesis, elevated lipid peroxides, and increased oxidative damage. It has therefore been hypothesized that oxidative stress caused by PHx (34) impairs mitochondrial function and contributes to altered energy metabolism observed after PHx (40). Furthermore, altering oxidative stress could affect hepatic regeneration by changing the energy generation by mitochondria (4, 14). Here, as has been observed by others, PHx caused lipid peroxidation (Fig. 6). Furthermore, aconitase activity was inhibited (Fig. 6), which is indicative of general oxidative damage to the mitochondria (7). Acute ethanol preexposure blunted both the lipid peroxidation and the aconitase inhibition caused by PHx (Fig. 6). Taken together, these data suggest that ethanol preexposure protected hepatic mitochondria from oxidative damage caused by PHx. This antioxidant effect most likely prevented mitochondrial dysfunction, which allowed the cells to more efficiently prepare for entry into the cell cycle. ALDH2 plays a key role in enhancing regeneration after PHx in mouse liver. Alcohol exposure is well known to induce expression and/or activity of enzymes responsible for its metabolism. ALDH2 is a key mitochondrial enzyme responsible for acetaldehyde oxidation in ethanol metabolism. However, other aldehydes, including lipid aldehydes (4-HNE) (14, 30) are substrates for this enzyme; induction/activation of this enzyme could therefore protect mitochondria from oxidative stress mediated by lipid aldehydes (Fig. 6, bottom, left). In this study, acute ethanol preexposure increased the activity of ALDHs and ALDH2 (Fig. 7A). The increase in ALDH2 activity caused by acute ethanol preexposure is most likely mediated by the increase in PKC-ε, a known activator of ALDH2 (5), as ASO knockdown of PKC-ε prevented this effect (Fig. 7). Previous work by this group has indicated that acute ethanol exposure likely activates PKC-ε by increasing diacylglycerol levels in the liver (25). Taken together, these data suggest that the transient oxidative stress caused by PHx is partially prevented by alcohol preexposure by activating ALDH2; this protective effect is likely responsible for mediating the more rapid entry into the cell cycle observed in the livers from ethanol-preexposed animals (Fig. 1). Indeed, an ALDH2 inducer (Alda-1) (5) mimicked the ethanol effect (Fig. 7B, bottom, left), and an ALDH2 inhibitor (Daidzin) (21) prevented the ethanol effect (Fig. 7B, bottom, right). This hypothesis is further supported by the fact that, in primary hepatocytes, ethanol preexposure in vivo or Alda-1 in vitro conferred protection against mito- and cytotoxicity caused by 4-HNE (Fig. 8). Summary and conclusions. Taken together, this study identifies a new role of mitochondrial ALDH2 in promoting hepatic regeneration in the mouse liver. Recently, a similar protective
mechanism of ALDH2 induction was observed in experimental cardiac ischemia/reperfusion in rats (5). The proposed mechanism by which ALDH2 enhances hepatic regeneration under these conditions is via protecting against mitochondrial dysfunction caused by oxidative stress early after PHx (Fig. 6, bottom, left). This protective effect against mitochondrial damage allows the cells to more efficiently generate the energy reserves required to enter the cell cycle. ALDH2 activity may also explain, at least in part, the differences between the effects of acute and chronic alcohol exposure on hepatic regeneration after PHx; specifically, whereas acute ethanol activates ALDH2 (Fig. 7), chronic alcohol exposure actually inhibits activity (11). The clinical implications of these data are that they suggest that targeting ALDH2 may be a useful strategy for improving hepatic regeneration. For example, activating ALDH (e.g., with Alda-1) may improve hepatic regeneration after hepatic resection surgery. These data further suggest that, in addition to altered alcohol metabolism, loss-of-function single nucleotide polymorphisms of ALDH2 may increase the risk of developing liver disease by impairing hepatic regeneration. This latter point may be of concern, not only for alcoholic liver disease, but also for other liver diseases (18). ACKNOWLEDGMENTS X. Ding is currently affiliated with the Department of Transplantation, Xiangya hospital, Central South University, Changsha, Hunan, China. GRANTS This work was supported, in part, by a grant from the National Institute of Alcohol Abuse and Alcoholism (AA003624). J. Beier was supported by a postdoctoral (T32) fellowship from National Institute of Environmental Health Science (ES011564). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: X.D. and G.E.A. conception and design of research; X.D., J.I.B., K.J.B., J.D.J., H.Z., and G.E.A. performed experiments; X.D., J.I.B., K.J.B., J.D.J., H.Z., and G.E.A. analyzed data; X.D., J.I.B., K.J.B., and G.E.A. interpreted results of experiments; X.D., K.J.B., and G.E.A. prepared figures; X.D. and G.E.A. drafted manuscript; X.D., J.I.B., K.J.B., and G.E.A. edited and revised manuscript; X.D., J.I.B., K.J.B., J.D.J., H.Z., and G.E.A. approved final version of manuscript. REFERENCES 1. Beier JI, Luyendyk JP, Guo L, von Montfort C, Staunton DE, Arteel GE. Fibrin accumulation plays a critical role in the sensitization to lipopolysaccharide-induced liver injury caused by ethanol in mice. Hepatology 49: 1545–1553, 2009. 2. Bergheim I, Guo L, Davis MA, Lambert JC, Beier JI, Duveau I, Luyendyk JP, Roth RA, Arteel GE. Metformin prevents alcohol-induced liver injury in the mouse: Critical role of plasminogen activator inhibitor-1. Gastroenterology 130: 2099 –2112, 2006. 3. Berry MN, Friend DS. High yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study. J Cell Biol 43: 506 –520, 1969. 4. Beyer TA, Werner S. The cytoprotective Nrf2 transcription factor controls insulin receptor signaling in the regenerating liver. Cell Cycle 7: 874 –879, 2008. 5. Chen CH, Budas GR, Churchill EN, Disatnik MH, Hurley TD, Mochly-Rosen D. Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart. Science 321: 1493–1495, 2008. 6. Enomoto N, Ikejima K, Bradford BU, Rivera CA, Kono H, Brenner DA, Thurman RG. Alcohol causes both tolerance and sensitization of rat
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