Acute and Chronic Ethanol Administration ... - Wiley Online Library

ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 39, No. 12 December 2015

Acute and Chronic Ethanol Administration Differentially Modulate Hepatic Autophagy and Transcription Factor EB Paul G. Thomes, Casey S. Trambly, Howard S. Fox, Dean J. Tuma, and Terrence M. Donohue Jr.

Background: Chronic ethanol (EtOH) consumption decelerates the catabolism of long-lived proteins, indicating that it slows hepatic macroautophagy (hereafter called autophagy) a crucial lysosomal catabolic pathway in most eukaryotic cells. Autophagy and lysosome biogenesis are linked. Both are regulated by the transcription factor EB (TFEB). Here, we tested whether TFEB can be used as a singular indicator of autophagic activity, by quantifying its nuclear content in livers of mice subjected to acute and chronic EtOH administration. We correlated nuclear TFEB to specific indices of autophagy. Methods: In acute experiments, we gavaged GFP-LC3tg mice with a single dose of EtOH or with phosphate buffered saline (PBS). We fed mice chronically by feeding them control or EtOH liquid diets. Results: Compared with PBS-gavaged controls, livers of EtOH-gavaged mice exhibited greater autophagosome (AV) numbers, a higher incidence of AV-lysosome co-localization, and elevated levels of free GFP, all indicating enhanced autophagy, which correlated with a higher nuclear content of TFEB. Compared with pair-fed controls, livers of EtOH-fed mice exhibited higher AV numbers, but had lower lysosome numbers, lower AV-lysosome co-localization, higher P62/SQSTM1 levels, and lower free GFP levels. The latter findings correlated with lower nuclear TFEB levels in EtOH-fed mice. Thus, enhanced autophagy after acute EtOH gavage correlated with a higher nuclear TFEB content. Conversely, chronic EtOH feeding inhibited hepatic autophagy, associated with a lower nuclear TFEB content. Conclusions: Our findings suggest that the effect of acute EtOH gavage on hepatic autophagy differs significantly from that after chronic EtOH feeding. Each regimen distinctly affects TFEB localization, which in turn, regulates hepatic autophagy and lysosome biogenesis. Key Words: Autophagosome, Lysosome, Transcription Factor EB, Proteopathy, Steatosis.

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N LIVERS OF heavy drinkers and experimental alcoholfed animals, hepatic proteopathy (protein accumulation) and steatosis (lipid accumulation) reflect disrupted protein and lipid metabolism and both are early signs of alcoholic liver injury. Breakdown of most cellular macromolecules and organelles occurs in lysosomes (Donohue et al., 1989, 1994; Singh et al., 2009). We have reported that chronic ethanol

From the Liver Study Unit (PGT, CST, DJT, TMD), Department of Veterans Affairs, VA Nebraska-Western Iowa Health Care System (NWIHCS), Omaha, Nebraska; Department of Internal Medicine (PGT, CST, DJT, TMD), College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska; Department of Pharmacology and Experimental Neuroscience (HSF), College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska; Department of Biochemistry and Molecular Biology (DJT, TMD), College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska; Department of Pathology and Microbiology (TMD), College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska; and The Center for Environmental Health and Toxicology (TMD), College of Public Health, University of Nebraska Medical Center, Omaha, Nebraska. Received for publication July 5, 2015; accepted September 10, 2015. Reprint requests: Terrence M. Donohue Jr, PhD, Liver Study Unit, Research Service (151), Omaha Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, NE 68501; Tel: 402-995-3556; Fax: 402-449-0604; E-mail: [email protected] Copyright © 2015 by the Research Society on Alcoholism. DOI: 10.1111/acer.12904 2354

(EtOH) administration inhibits lysosome function, caused in part by a significant disruption of lysosome biogenesis (Kharbanda et al., 1995, 1996, 1997). Lysosomes have a key function in the catabolic phase of macroautophagy (autophagy), which begins with the formation of autophagosomes (AVs) that envelop and sequester both particulate and soluble cytoplasmic components. AVs are then trafficked to and fuse with lysosomes, forming autolysosomes that degrade the AV contents, generating low molecular weight substrates that are either recycled for biosynthesis or further broken down to generate energy. Recent reports on the status of hepatic autophagy after alcohol exposure are diverse. Ding and colleagues (2010) showed that acute (binge) EtOH administration (using 4 equally divided gavages in 20-minute intervals, followed by euthanasia 16 hours later) enhances autophagy. They later showed that FOXO3a, a transcription factor that regulates several cellular processes including apoptosis, oxidant stress, and glucose metabolism, also partially regulates acute EtOH-induced autophagy (Ni et al., 2013). In contrast, Wu and colleagues (2012) used a longer EtOH binge regimen (i.e., twice daily for 4 days) and reported decreased liver autophagy. After 4 weeks of chronic EtOH feeding to mice, Lin and colleagues (2013) reported enhanced autophagy. A recent immuno-electron microscopy study described that livers of 10-wk EtOH-fed rats had elevated AVs that sequesAlcohol Clin Exp Res, Vol 39, No 12, 2015: pp 2354–2363

ETHANOL AND HEPATIC AUTOPHAGY

tered greater numbers of lipid droplets and mitochondria, with a higher incidence of AV-cathepsin co-localization. They too concluded that livers of EtOH-fed rats had enhanced autophagy (Eid et al., 2013). Thus, the foregoing summary of earlier and more recent findings clearly indicates conflicting results from various laboratories, underscoring our belief that this area of investigation is still unsettled and thus prompted the present investigation. A recent advance in autophagy research has been the identification of the transcription factor EB (TFEB). This protein activates transcription of the coordinated lysosomal expression and regulation (CLEAR) genes that encode proteins with functional roles in autophagy and lysosome biogenesis (Settembre et al., 2011). Transcription of CLEAR genes by TFEB enhances AV formation, lysosome biogenesis, and autophagic flux, thereby accelerating autophagy (Settembre and Ballabio, 2011; Settembre et al., 2011). Here, we hypothesized that the nuclear localization of TFEB is a singular indicator of autophagic activity and serves as a reliable marker to quantify autophagy and lysosome biogenesis. Thus, in addition to measuring standard autophagy markers, as described in the guidelines (Klionsky et al., 2008), we sought to correlate autophagic activity with the hepatic nuclear content of TFEB. Using a rodent model of acute and chronic EtOH administration, we quantified TFEB and autophagy markers in primary hepatocytes and in whole livers and their subcellular fractions from mice subjected to each EtOH feeding regimen. Here, we report that acute and chronic EtOH treatments affected liver autophagy in distinctly opposite ways and that these effects were closely associated with the nuclear localization of TFEB. MATERIALS AND METHODS Reagents Anti-LC3, anti-pERK1/2, and anti-ERK1/2 were from Cell Signaling Technology Inc. (Danvers MA). Anti-P62/SQSTM1 was purchased from Medical and Biological Laboratories Ltd (Nayoga, Japan). Anti-GFP was from Rockland Inc. (Gilbertsville, PA). AntiTFEB was from MyBioSource Inc. (San Diego, CA). Anti-b-actin was from Calbiochem (La Jolla, CA). We purchased protease inhibitor cocktail, the proteasome substrate, N-succinyl-L-leucyl-L-leucylL-valyl-L-tyrosyl-7-amino-4-methyl-coumarin (N-Suc-LLVY-AM C), and other specialized reagents from Sigma (St. Louis, MO). Animal Treatments The animal studies subcommittee (IACUC) of the VA Nebraska, Western Iowa Health Care System, approved the animal protocol described here. We followed the 8th edition of the Guidelines for the Use and Care of Laboratory Animals, published by the National Institutes of Health. We used mice (C57BL/6 background) transgenic for the fusion protein GFP-LC3, a precursor form of the AV marker protein, GFP-LC3-II. In acute studies, male and female mice were given a single dose of EtOH (6 g/kg body weight) by gastric intubation (gavage), as described previously (Donohue et al., 2012; Thomes et al., 2012). Three or 12 hours postgavage, we anesthetized each animal and collected blood from the brachial vessels. Following exsanguination and pneumothorax, the liver of each animal was removed and a portion was homogenized and subsequently

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subjected to subcellular and nuclear fractionation (Donohue et al., 1994; McMullen et al., 2005). We confirmed the relative purity of nuclear fractions by Western blot, which revealed that they were ≥95% free of the cytosolic protein, alcohol dehydrogenase. Similarly, we used antibody against the cyclic AMP response element binding protein (anti-CREB) to detect nuclear contamination of cytosolic fractions and detected no CREB protein band in these fractions (data not shown). We also sacrificed mice to prepare hepatocytes by perfusing their livers in situ with collagenase (Thomes et al., 2012). Hepatocytes obtained from these animals were pelleted by centrifugation, then further centrifuged in Percoll to yield ≥85% viable cells, which we plated onto collagen IV-precoated chambered cover glasses (Thermo Scientific, Rockford, IL). We incubated the cells overnight in Williams Medium E containing 5% (v/v) fetal bovine serum, after which we processed the slides for confocal microscopy. For chronic EtOH studies, we used female mice. We initially fed them Purina chow and then acclimated them to the liquid control diet (Dyets Inc., Bethlehem, PA) for 3 days. We then weightmatched and paired the animals, giving the EtOH-fed mice increasing doses of EtOH in the diet. We subjected mice to a 10-day adaptation period during which the EtOH concentration (as percent of total calories) was gradually increased from 3.6% on day 1, to 7.2% on day 4, to 10.2% on day 6, to 21.6% on day 8, and finally to 29.2% (5.2% EtOH by volume) on day 10 and thereafter. Every day, we gave each control mouse the same volume of diet that was consumed the previous day by its weight-matched EtOH-fed mouse. To minimize nutritional inequalities during overnight consumption, we fed mice ad lib with one-fourth the average daily volume of their respective diets 90 minutes before euthanasia. Serum Analyses We measured serum EtOH levels by head space chromatography (Donohue et al., 2007). The clinical laboratory at the Omaha Veterans Affairs Medical Center performed automated measurements of alanine transaminase and aspartate transaminase activities. Microscopy After overnight incubation of hepatocytes on collagen-coated cover glass, we examined lysosomes after staining with the acidophilic dye, Lyso-tracker redÒ (Invitrogen, Grand Island, NY). We previously showed that lysosome detection using this dye is as reliable as immunofluorescent detection using the lysosomal membrane marker, lysosome-associated membrane protein-1 [LAMP-1] (Thomes et al., 2013), and its staining of lysosomes provides an accurate measure of their numbers (Klionsky et al., 2008). Endogenously produced green puncta, corresponding to GFP-containing AVs, were also detected in the cytoplasm and quantified. GFP-LC3 was also readily detected in hepatocyte nuclei as previously reported (Martinez-Lopez et al., 2013), as diffuse, nonpunctate material. We digitally acquired microscope images using a Zeiss confocal fluorescent microscope (LSM 510 Meta confocal laser scanning microscope; Carl Zeiss, Pleasanton, CA) and individually quantified green AVs, red lysosomes, and their co-localization (orange to yellow puncta) in multiple images using NIH ImageJ analyzer software (Bethesda, MD), as described before (Thomes et al., 2013). Proteasome Activity We measured the chymotrypsin-like activity of the 20S proteasome in crude liver homogenates using our published protocol (Osna et al., 2007). Enzyme activity is expressed as nmoles AMC generated per hour after its hydrolysis from the N-Suc-LLVYAMC substrate.

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Hepatic Triglycerides From snap-frozen livers, we organically extracted total lipids using method of Folch and colleagues (1957). We filtered the extracts through Whatman #1 filter paper and then dried the filtrates by vacuum centrifugation. Dried lipid pellets were reconstituted in 86.3% (v/v) EtOH containing 0.73 N KOH. Triglycerides were saponified at 65°C for 20 minutes and then spectrophotometrically quantified for glycerol content using the Thermo DMA reagent (Thermo Electron Inc., Middletown, VA) according to the manufacturer’s instructions. Detection of Proteins on Western Blots From individual samples, we separated proteins on 12% polyacrylamide mini-gels by SDS-PAGE and transferred them onto nitrocellulose membranes. We incubated membranes with the primary antibodies listed above usually overnight at 4°C. After washing, we incubated the membranes for 45 to 60 minutes with secondary antibodies that were conjugated to either horseradish peroxidase or infrared dyes. Proteins were detected using either enhanced chemiluminescence (ECL) (Pierce ECL supersignal; Thermo Scientific) or by the OdysseyTM infrared imaging system (Licor, Inc., Lutz, FL). We quantified protein band densities using Quantity One software (Bio-Rad, Hercules, CA) or by the Licor analysis software. We normalized protein load by calculating the densitometric ratio of the protein of interest to that of b-actin or, in the case of heavy membrane fractions (i.e., 16,451 9 g pellets 9 10 minutes, representing lysosomes and mitochondria), to LAMP-1. Because there was interexperimental variation in the densitometric measurements, we expressed the values from each EtOH-treated mouse as the percent of its control, after which the mean percent of control was calculated from all experiments. We calculated TFEB nuclear to cytosolic ratio by dividing TFEB content in the nucleoplasm by that in the cytosolic fraction after normalizing each TFEB band density by that of its loading control, b-actin. We must emphasize that the nuclear quantity of b-actin is comparable to that in the cytosol. Alcohol administration does not affect b-actin content in either cellular compartment (Donohue et al., 2012).

control mice. AV-lysosome co-localization in cells from EtOH-gavaged mice was 1.8-fold higher than in PBS controls, indicating an EtOH-induced acceleration of autophagic flux (Fig. 1). These findings were confirmed on Western blots as we observed 2.3-fold higher levels of the AV marker, LC3II (Fig. 2A,B), and 1.7-fold higher levels of free GFP (derived from GFP-LC3 hydrolysis) (Fig. 2C,D) in livers of EtOHgavaged mice. Enhanced autophagy in EtOH-gavaged mice was associated with a higher hepatic nuclear content of TFEB (expressed as a 1.6-fold higher nuclear/cytosolic ratio) than in PBS-gavaged control animals (Fig. 3A,B). There was no gender-related difference in TFEB content, as male and female EtOH-treated mice showed similar nuclear TFEB levels (males PBS = 100 18 (n = 4): males, EtOH-gavaged = 166 12 (n = 4); females PBS = 100 4 (n = 3); females EtOH-gavaved = 147 15, (n = 3) p = 0.35. The higher nuclear TFEB content in EtOH-treated mice was inversely associated with 2.6- and 1.8-fold lower ratios of phosphorylated ERK1/2 (pERK1/2) to the total content of ERK1/2, to suggest that acute EtOH administration decreased the combined activity of ERK1/2 (Fig. 3C,D). Chronic EtOH Feeding Enhanced Hepatic AV Content, But Lowered Autophagic Flux, and Nuclear TFEB Content Compared with their pair-fed controls, mice subjected to chronic EtOH administration exhibited liver enlargement, A PBS-gavaged Lys

AVs

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Other Methods We performed RNA isolation and real-time polymerase chain reaction as described earlier (Thomes et al., 2013). We used the enzymatic recycling assay (Griffith, 1980) to quantify liver glutathione (GSH) levels. We spectrophotometrically measured liver malondialdehyde (MDA) levels as thiobarbituric acid-reactive substances, using authentic MDA as a standard (Janero, 1990).

13 ± 3a

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Statistical Analysis Data are expressed as mean values SEM. We determined statistical significance between 2 groups by Student’s t-test and among multiple groups by 1-way analysis of variance, using a Neuman– Keuls post hoc analysis. For chronic EtOH studies, we performed paired analyses. A probability (p) value ≤0.05 was considered statistically significant.

RESULTS Acute EtOH Gavage Enhanced AV Content, AV-Lysosome Fusion, Autophagic Flux, and Nuclear TFEB Content Confocal microscopy of hepatocytes revealed 1.7fold more AVs in cells from EtOH-gavaged mice than in hepatocytes from phosphate buffered saline (PBS)-gavaged

17 ± 1a

22 ± 3 b

7 7 ± 1b 7.7

Fig. 1. Acute ethanol (EtOH) gavage increased autophagosome (AV) numbers and AV-lysosome fusion events. Hepatocytes from phosphate buffered saline- and EtOH-gavaged GFP-LC3 mice (each treated 3 hours prior to death) were loaded with LysoTracker red. Lysosomes are red dots (puncta). AVs are green puncta. Nuclei are shown as much larger structures with more diffuse (not punctate) green color. Presumed AV-lysosome fusion events are orange or yellow puncta, owing to co-localization of red (lysosomes) and green (AVs). Data below each panel are mean values ( SE) from 15 to 20 individual cells per group and are the number of puncta per nucleus. Values bearing different letter superscripts are significantly different from each other. Values bearing the same letter superscripts are not significantly different. Similar results were obtained from 3 sets of individual acute EtOH experiments.


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Fig. 2. Acute ethanol (EtOH) gavage enhanced autophagic flux. (A) Representative Western blot of LC3-I and LC3-II in livers of acutely treated mice. (B) Densitometric ratios of LC3-II/actin (phosphate buffered saline [PBS]- vs. EtOH-gavaged; p = 0.01). (C) Representative Western blot showing GFPLC3-I, GFP-LC3-II, and free GFP. (D) Densitometric ratio of free GFP/actin (PBS- vs. EtOH-gavaged; p = 0.01). N = 7 PBS- and EtOH-gavaged mice. Bars with different letters are significantly different. Bars with the same letter are not significantly different.

protein accumulation (proteopathy), fatty liver (steatosis), and mild liver toxicity (Table 1). We detected oxidant stress in livers of EtOH-fed animals, as judged by a 26% decline in reduced GSH and a 58% rise in MDA, compared with pairfed controls. The ratio of the lipid peroxide, MDA to the antioxidant, GSH, was 2-fold higher in livers of EtOH-fed mice than in pair-fed control mice. Hepatocytes from EtOH-fed mice exhibited 60% higher numbers of AV puncta, but 25% lower lysosome numbers than controls (Fig. 4). Interestingly, there was a 6-fold reduction in the frequency of AV-lysosome co-localization events in cells from EtOH-fed mice compared with those from pairfed controls (Fig. 4). We also confirmed higher AV numbers in EtOH-fed mice by immunoblot analyses of the AV marker, LC3-II. The protein was elevated 1.7-fold in heavy membrane subcellular fractions compared with the same fractions from pair-fed control mice (Fig. 5A,B), using LAMP-1 as a loading control. Liver postnuclear fractions from EtOH-fed mice also contained higher LC3-II levels (LC3-II/b-actin; control = 1 0.04, EtOH = 1.34 0.07; p = 0.008, N = 4 to 6 pairs). AV (LC3-II) enhancement did not reflect de novo LC3 biogenesis, as the content of LC3B mRNA was equal in livers of both control and EtOH-fed mice (control relative quantity [RQ] = 1 0.8; EtOH-fed RQ = 0.9 0.7; p = 0.56, N = 7 pairs). Instead, LC3-II enhancement likely reflects its intracellular accumulation because its lysosomal degradation was decelerated. Additionally, part of the rise in LC3-II was also due to its reduced catabolism by the 20S

proteasome. The specific activity of the 20S enzyme was 27% lower in livers of EtOH-fed mice than controls (pair-fed controls = 15 0.5; EtOH-fed = 11 0.4 nmoles AMC per hour per mg protein p = 50% decline in hepatic GSH (Donohue et al., 2012), likely accelerated autophagy by increasing intracellular oxidants to levels that blocked the activity of the autophagy suppressor, MTORC1. Additionally, a previously reported EtOH-elicited rise in plasma glucagon (Donohue et al., 1998; Forman et al., 1988; Simanowski et al., 1989), a proautophagy hormone that represses MTORC1 signaling by activating AMP-activated protein kinase (AMPK) (Kimball et al., 2004) likely contributed to this induction. Our findings are also consistent with our earlier study, which demonstrated that acute EtOH gavage induced the transcription factor, early growth response-1, which co-activates LC3B gene transcription (Chen et al., 2008). All the foregoing signs of autophagic enhancement were associated with higher levels of nuclear TFEB, suggesting that its elevated nuclear content reflects greater transcriptional activity (Sardiello et al., 2009). Further, higher nuclear TFEB in livers of EtOH-gavaged mice correlated inversely with lower levels of the phosphorylated extracellular and signal-regulated kinases 1 and 2 (ERK1/2). By phosphorylating TFEB, ERK1/2 blocks TFEB translocation into the nucleus to enhance transcription of CLEAR genes and accelerate autophagy (Settembre et al., 2011). Thus, a single EtOH gavage caused a burst of oxidant stress that began with the swift


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Table 1. Physiological Parameters in Control- and Ethanol-Fed GFP-LC3 Female Mice Parameter Starting weight (g) Daily volume of ethanol diet consumed (ml) Final weight (g) Absolute liver weight (g) Relative liver weight (g/100 g body wt) Liver proteins (mg/100 g body wt) Liver triglycerides (mg/100 g body wt) Serum ethanol (mM) Serum ALT (Units/l) Serum AST (Units/l) Liver glutathione (nmole GSH/g liver) Liver malondialdehyde (MDA) (nmoles MDA/g liver) (nmoles MDA/nmoles GSH)

Ethanol-fed mice

Pair-fed control mice

20 0.6 16 0.6

21 0.5a –

22 0.2a 1.34 0.06a 6.1 0.1a

24 0.6b 1.12 0.03b 4.6 0.2b

937 90a

671 30b

709 119a

241 21b

a

22 141 259 5,435

4 20a 23a 280a

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87 ± 10a

51 ± 13a

18 ± 5 a

B EtOH-fed Lys

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Background* 60 10b 176 26b 7,156 500b

632 74a

401 34b

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0.06 0.004

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38 ± 7b

138 ± 20 b

3 ± 1b

b

ALT, alanine transaminase activity; AST aspartate transaminase activity in the serum; GSH, glutathione; MDA, malondialdehyde. *Serum ethanol concentrations in ethanol-fed mice are adjusted values after subtracting background ethanol concentrations (mean = 4 1 mM) detected in the sera of pair-fed control mice. Data are compiled from 5 separate chronic ethanol feeding studies and are mean values ( SEM) from 14 to 28 samples per group. Female mice were pair-fed the Lieber-DeCarli control and ethanol liquid diets for 35 to 62 days. Values with different superscript letters are statistically different, p < 0.05.

increase in EtOH oxidation to bring about elevated TFEB nuclear localization and an activation of autophagy. Figure 7A depicts the major factors involved in autophagic acceleration after acute EtOH gavage. While acute EtOH gavage to rodents is similar to binge drinking by humans, chronic EtOH feeding is clinically relevant to liver injury that occurs after years of heavy drinking. Here, we demonstrated that continuous EtOH administration disrupted autophagy and lysosome biogenesis, resulting in steatosis and proteopathy, both of which contribute to liver enlargement (Table 1). Compared with pair-fed controls, we detected lower lysosome numbers in hepatocytes of EtOH-fed mice (Fig. 4). These findings are consistent with our previous reports of faulty lysosome biogenesis in hepatocytes of rats fed EtOH for 5 to 7 weeks (Haorah et al., 2003; Kharbanda et al., 1996). Higher AV numbers in hepatocytes of alcohol-fed mice likely resulted from a striking reduction in autophagic flux, as judged by decreased catabolic rates of P62/SQSTM1 and GFP-LC3, causing P62/SQSTM1 protein accumulation and yielding lower intracellular levels of GFP (Fig. 5). Our findings are consistent with our earlier report, showing that after EtOH-oxidizing VL-17A cells are exposed for 24 hours to 50 mM EtOH, there are lower lysosome numbers and a reduced incidence of AV-lysosome colocalization (Thomes et al., 2013). We reported no such findings in Hep G2 cells, because they do not oxidize EtOH (Thomes et al., 2013). These results imply that prolonged EtOH oxidation during chronic alcohol consumption is the

Fig. 4. Chronic ethanol (EtOH) feeding increased autophagosome (AV) numbers but decreased lysosome numbers and AV-lysosome fusion events in mouse hepatocytes. Micrographs of hepatocytes from pair-fed and EtOH-fed GFP-LC3 mice were quantified for lysosomes (left panels), AVs (middle panels) and their co-localization (right panels) as described in Fig. 1. Numbers below each panel are mean values ( SE) from 15 to 20 individual cells per group and are expressed as puncta per nucleus. Values bearing different letter superscripts are significantly different from each other. Values bearing the same letter superscripts are not significantly different. Similar results were obtained from 3 sets of individual chronic EtOH feeding experiments.

probable cause of the autophagic deficiencies reported here. Most significant is that these deficiencies were closely linked with lower levels of nuclear TFEB, which correlated inversely with higher ERK1/2 phosphorylation levels, compared with controls (Fig. 6). Thus, animals subjected to chronic EtOH feeding exhibited elevated ERK1/2 phosphorylation, which likely increased TFEB phosphorylation, preventing its entry into the nucleus. It is also noteworthy that these findings were not unique to liver. We observed 2-fold lower TFEB content in nuclear fractions from brains of EtOH-fed mice (p ≤ 0.05) than in brains of pair-fed controls (data not shown). Others have reported differential effects of acute and chronic alcohol treatment on ERK1/2 activity in monocytes (Mandrekar et al., 2009) and in livers of patients with alcoholic liver disease (Nguyen and Gao, 2002) in whom ERK1/2 is also activated. Others report the association of ERK1/2 activation with exposure to arachidonic acid generated in Kuppfer cells of EtOH-fed rats (Cubero and Nieto, 2012). To account for the differences in hepatic autophagy between the acute and chronic EtOH feeding models, we present Fig. 7B in which we propose that chronic EtOH oxidation in vivo creates a nearly continuous condition of oxidant stress during which generation of reactive species (e.g., MDA, acetaldehyde, and superoxide) is sustained at higher levels than in pair-fed control mice. This condition is exacerbated by a chronic decline in hepatic GSH. One could argue that this stressful condition is similar to that produced by

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Fig. 5. Chronic ethanol (EtOH) feeding caused increased autophagosomes (AVs) but lower degradation of autophagosomes (AVs) cargo. (A) Representative Western blot of LC3. (B) Densitometric ratio of LC3-II/LAMP-1 (pair-fed vs. EtOH-fed; p = 0.03). (C) Representative Western blot of SQSTM1/ P62. (D) Densitometric ratio of P62/SQSTM1/LAMP-1 in the liver mitochondria/lysosome fraction (pair-fed vs. EtOH-fed; p = 0.03). (E) Representative Western blot showing GFP-LC3-I, GFP-LC3-II, and free GFP. (F) Densitometric ratio of GFP/actin (pair-fed vs. EtOH-fed; p = 0.002) in the postnuclear supernatants. N = 7 pairs of EtOH -fed and pair-fed mice. Bars with different letters are significantly different. Bars with the same letter are not significantly different.

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Fig. 6. Chronic ethanol (EtOH) feeding decreased transcription factor EB (TFEB) nuclear content. (A) Representative Western blot of TFEB and loading control (actin) in liver cytosol (Cyto) and nuclear (Nuc) fractions and (B) densitometric ratio of Nuc TFEB/Cyto TFEB (pair-fed vs. EtOH-fed; p = 0.007). (C) Representative Western blot of pERK1/2 and ERK1/2. (D) Densitometric ratio of pERK1/ERK1 (pair-fed vs. EtOH-fed; p = 0.007), pERK2/ERK2 (pair-fed vs. EtOH-fed; p = 0.03) in postnuclear fractions of liver homogenates. N = 9 pairs of pair-fed and EtOH-fed mice. Bars with different letters are significantly different. Bars with the same letter are not significantly different.


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Fig. 7. Transcription factor EB (TFEB) and autophagy regulation after acute and chronic ethanol (EtOH) administration. (A) Acute EtOH administration elicits a swift increase in alcohol metabolism (SIAM) giving rise to enhanced oxidant production (EtOH metabolites) and oxidants from depolarized mitochondria. Such oxidants and elevated glucagon (acting via activated AMPK) block MTORC1 signaling, thereby lowering TFEB phosphorylation (via ERK1/2), allowing TFEB nuclear entry and enhancing transcription of CLEAR genes. The net result is autophagy acceleration. (B) Chronic EtOH feeding gradually accelerates EtOH metabolism, forming oxidants and reactive species (RS) and EtOH metabolites, which deplete intracellular glutathione (GSH), causing fatty liver (steatosis) and oxidant stress, to inhibit MTOR activity. However, hepatic leucine levels also rise during chronic EtOH feeding. Leucine activates MTORC1, increasing ERK1/2 phosphorylation to phosphorylate TFEB, preventing its nuclear entry. The latter slows expression of key autophagy genes and those involved in lysosome biogenesis. Cytoplasmic autophagy is also impaired by accumulated fats, which inhibit lysosome acidification and their fusion with AVs. EtOH metabolism generates acetaldehyde, which is closely associated with microtubule acetylation, to impair organelle trafficking, which may occur during autophagy but is not depicted in B. Short vertical arrows (↓↑) denote an increase or decrease in content; longer arrows denote an activation or a subsequent pathway step; crossbars (┤) denote inhibition. The relative degree of autophagic activity is also denoted by the thickness of arrows or by their relative intensites (i.e., darkness).

acute EtOH gavage, such that MTORC1 activity would be inhibited, thereby allowing accelerated autophagy. However, additional factors contribute to autophagic deceleration

during chronic EtOH feeding. One is EtOHinduced steatosis. Others report that steatosis that develops in animals after consuming excessive dietary fat, disrupts hepatic AV-lysosome fusion, increases lysosomal pH, and lowers expression of the major lysosomal cathepsins, B and L (Inami et al., 2011; Koga et al., 2010). These findings are consistent with our earlier reports in livers of rats subjected to chronic EtOH administration (Kharbanda et al., 1995, 1997) and our more recent investigations with EtOH-exposed VL-17A cells (Thomes et al., 2013). Another factor is the reported rise in intrahepatic leucine (Baraona et al., 1977; Bernal et al., 1993), which rather distinctly activates MTORC1 activity (Jewell et al., 2013) to partially or fully restore autophagic suppression. Still, another cause of AV suppression during chronic EtOH exposure is the well-documented ability of EtOH metabolites to disrupt hepatic protein trafficking. Autophagy requires the action of microtubules, which direct AV-lysosome fusion. Both microtubule content and function are disrupted by EtOH metabolites (Baraona et al., 1977; Smith et al., 1989; Tuma and Sorrell, 1981, 1988). Recent findings indicate a strong association between EtOH oxidation (i.e., acetaldehyde generation) and protein hyperacetylation, which reportedly destabilizes microtubules (Fernandez et al., 2012; Shepard et al., 2010, 2012). Here, we observed a 6-fold decline in AVlysosome co-localization in hepatocytes of EtOH-fed mice, a change that was greater than both the rise in AVs and the decline in lysosomes (Fig. 4). Thus we propose that in addition to defective lysosome biogenesis, AV-lysosome fusion is disrupted because of faulty microtubule assembly. Direct proof of this awaits further investigation. Here, for the first time, we have identified differential responses of TFEB to acute and chronic EtOH treatment. Our findings correlated closely with hepatic autophagic activity, suggesting that nuclear TFEB content is an indicator of the state of autophagy in liver after acute and chronic EtOH administration. Moreover, TFEB is a prospective therapeutic target that likely responds to physiological or pharmacological agents to alleviate EtOH-induced and other forms of liver pathology.

ACKNOWLEDGMENT Dr. Carol Casey provided valuable technical expertise in liver perfusion and hepatocyte isolation.

RESOURCES, FACILITIES, AND GRANT SUPPORT Financial support for this project was provided by institutional funds from the Section of Gastroenterology and Hepatology, UNMC Department of Internal Medicine, and a Dean’s Reviewed Research Grant from the University of Nebraska Medical Center. The data presented are results of work supported with resources and use of facilities at the Nebraska-Western Iowa Health Care System of the

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