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Liver-specific ceramide reduction alleviates steatosis and insulin resistance in alcohol-fed mice

2020; Elsevier BV; Volume: 61; Issue: 7 Linguagem: Inglês

10.1194/jlr.ra119000446

1539-7262

Jason Correnti, Chelsea Lin, Jascha Brettschneider, Amy Kuriakose, Sookyoung Jeon, Eleonora Scorletti, Amanke Oranu, Dru McIver-Jenkins, Isabelle Kaneza, Delfin Buyco, Yedidya Saiman, Emma E. Furth, Josepmaría Argemí, Ramón Bataller, William L. Holland, Rotonya M. Carr,

Diet, Metabolism, and Disease

Alcohol's impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol's inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD. Alcohol's impairment of both hepatic lipid metabolism and insulin resistance (IR) are key drivers of alcoholic steatosis, the initial stage of alcoholic liver disease (ALD). Pharmacologic reduction of lipotoxic ceramide prevents alcoholic steatosis and glucose intolerance in mice, but potential off-target effects limit its strategic utility. Here, we employed a hepatic-specific acid ceramidase (ASAH) overexpression model to reduce hepatic ceramides in a Lieber-DeCarli model of experimental alcoholic steatosis. We examined effects of alcohol on hepatic lipid metabolism, body composition, energy homeostasis, and insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp. Our results demonstrate that hepatic ceramide reduction ameliorates the effects of alcohol on hepatic lipid droplet (LD) accumulation by promoting VLDL secretion and lipophagy, the latter of which involves ceramide cross-talk between the lysosomal and LD compartments. We additionally demonstrate that hepatic ceramide reduction prevents alcohol's inhibition of hepatic insulin signaling. These effects on the liver are associated with a reduction in oxidative stress markers and are relevant to humans, as we observe peri- LD ASAH expression in human ALD. Together, our results suggest a potential role for hepatic ceramide inhibition in preventing ALD. AKT serine/threonine kinase alcoholic liver disease alanine aminotransferase acid ceramidase N-acylsphingosine amidohydrolase 1 Derlin 3 doxycycline electron microscopy ethanol glucose infusion rate hepatitis C virus hepatic glucose production insulin resistance lysosomal-associated membrane protein 1 lipid droplet malondialdehyde perilipin 2 rate of endogenous glucose production rate of peripheral glucose disposal respiratory exchange ratio reverse tetracycline-controlled transactivator super-oxide dismutase 1 tetracycline response element The rise in cirrhosis-related mortality since 2009 in the United States is due largely to alcoholic liver disease (ALD) with those between the ages of 25 and 34 experiencing the most significant increase. Of these, women are most impacted (1Tapper E.B. Parikh N.D. Mortality due to cirrhosis and liver cancer in the United States, 1999–2016: observational study.BMJ. 2018; 362: k2817Crossref PubMed Scopus (415) Google Scholar). ALD progresses from alcoholic steatosis to steatohepatitis, fibrosis, and cirrhosis, and this progression is associated with insulin resistance (IR) (2Raynard B. Balian A. Fallik D. Capron F. Bedossa P. Chaput J.C. Naveau S. Risk factors of fibrosis in alcohol-induced liver disease.Hepatology. 2002; 35: 635-638Crossref PubMed Scopus (321) Google Scholar, 3Carr R.M. Correnti J. Insulin resistance in clinical and experimental alcoholic liver disease.Ann. N. Y. Acad. Sci. 2015; 1353: 1-20Crossref PubMed Scopus (31) Google Scholar), the key determinant of glucose intolerance in ALD patients (4Iversen J. Vilstrup H. Tygstrup N. Kinetics of glucose metabolism in relation to insulin concentrations in patients with alcoholic cirrhosis and in healthy persons.Gastroenterology. 1984; 87: 1138-1143Abstract Full Text PDF PubMed Scopus (62) Google Scholar). Because only a small percentage of patients with alcohol dependence or overuse are able to achieve abstinence (5O'Shea R.S. Dasarathy S. McCullough A.J. Alcoholic liver disease.Hepatology. 2010; 51: 307-328Crossref PubMed Scopus (956) Google Scholar, 6Watson C.G. Recidivism in "controlled drinker" alcoholics: a longitudinal study.J. Clin. Psychol. 1987; 43: 404-412Crossref PubMed Scopus (7) Google Scholar, 7National Institute on Alcohol Abuse and Alcoholism (US), . 2005. Alcohol alert number 66. National Institute on Alcohol Abuse and Alcoholism, Rockville, MD.Google Scholar), therapeutic strategies that target alcohol's hepatic and dysmetabolic effects at the stage of steatosis are needed to prevent the development of advanced disease. A key feature of alcoholic liver injury is the impairment of both hepatic lipid metabolism and hepatic insulin signaling. Indeed, we have demonstrated that the onset of lipid dysregulation and resultant alcoholic steatosis is temporally related with the onset of IR in alcohol-fed mice (8Carr R.M. Dhir R. Yin X. Agarwal B. Ahima R.S. Temporal effects of ethanol consumption on energy homeostasis, hepatic steatosis, and insulin sensitivity in mice.Alcohol. Clin. Exp. Res. 2013; 37: 1091-1099Crossref PubMed Scopus (41) Google Scholar). We have demonstrated further that these physiologic perturbations require the presence of the major hepatocellular lipid droplet (LD) protein, perilipin 2 (PLIN2) (9Carr R.M. Peralta G. Yin X. Ahima R.S. Absence of perilipin 2 prevents hepatic steatosis, glucose intolerance and ceramide accumulation in alcohol-fed mice.PLoS One. 2014; 9: e97118Crossref PubMed Scopus (75) Google Scholar), and the accumulation of hepatic ceramides (8Carr R.M. Dhir R. Yin X. Agarwal B. Ahima R.S. Temporal effects of ethanol consumption on energy homeostasis, hepatic steatosis, and insulin sensitivity in mice.Alcohol. Clin. Exp. Res. 2013; 37: 1091-1099Crossref PubMed Scopus (41) Google Scholar, 9Carr R.M. Peralta G. Yin X. Ahima R.S. Absence of perilipin 2 prevents hepatic steatosis, glucose intolerance and ceramide accumulation in alcohol-fed mice.PLoS One. 2014; 9: e97118Crossref PubMed Scopus (75) Google Scholar, 10Williams B. Correnti J. Oranu A. Lin A. Scott V. Annoh M. Beck J. Furth E. Mitchell V. Senkal C.E. et al.A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.FASEB J. 2018; 32: 130-142Crossref PubMed Scopus (19) Google Scholar), bioactive sphingolipids that can impair cell growth, promote apoptosis, and impair insulin signaling (3Carr R.M. Correnti J. Insulin resistance in clinical and experimental alcoholic liver disease.Ann. N. Y. Acad. Sci. 2015; 1353: 1-20Crossref PubMed Scopus (31) Google Scholar, 11Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2454) Google Scholar, 12Stith J.L. Velazquez F.N. Obeid L.M. Advances in determining signaling mechanisms of ceramide and role in disease.J. Lipid Res. 2019; 60: 913-918Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Hepatic ceramides are increased in ALD patients (13Longato L. Ripp K. Setshedi M. Dostalek M. Akhlaghi F. Branda M. Wands J.R. de la Monte S.M. Insulin resistance, ceramide accumulation, and endoplasmic reticulum stress in human chronic alcohol-related liver disease.Oxid. Med. Cell. Longev. 2012; 2012: 479348Crossref PubMed Scopus (87) Google Scholar), and we and others have demonstrated that long-chain hepatic ceramides are increased in ALD rodent models (8Carr R.M. Dhir R. Yin X. Agarwal B. Ahima R.S. Temporal effects of ethanol consumption on energy homeostasis, hepatic steatosis, and insulin sensitivity in mice.Alcohol. Clin. Exp. Res. 2013; 37: 1091-1099Crossref PubMed Scopus (41) Google Scholar, 9Carr R.M. Peralta G. Yin X. Ahima R.S. Absence of perilipin 2 prevents hepatic steatosis, glucose intolerance and ceramide accumulation in alcohol-fed mice.PLoS One. 2014; 9: e97118Crossref PubMed Scopus (75) Google Scholar, 13Longato L. Ripp K. Setshedi M. Dostalek M. Akhlaghi F. Branda M. Wands J.R. de la Monte S.M. Insulin resistance, ceramide accumulation, and endoplasmic reticulum stress in human chronic alcohol-related liver disease.Oxid. Med. Cell. Longev. 2012; 2012: 479348Crossref PubMed Scopus (87) Google Scholar, 14Deaciuc I.V. Nikolova-Karakashian M. Fortunato F. Lee E.Y. Hill D.B. McClain C.J. Apoptosis and dysregulated ceramide metabolism in a murine model of alcohol-enhanced lipopolysaccharide hepatotoxicity.Alcohol. Clin. Exp. Res. 2000; 24: 1557-1565Crossref PubMed Google Scholar, 15Liangpunsakul S. Sozio M.S. Shin E. Zhao Z. Xu Y. Ross R.A. Zeng Y. Crabb D.W. Inhibitory effect of ethanol on AMPK phosphorylation is mediated in part through elevated ceramide levels.Am. J. Physiol. Gastrointest. Liver Physiol. 2010; 298: G1004-G1012Crossref PubMed Scopus (69) Google Scholar, 16Zhao Z. Yu M. Crabb D. Xu Y. Liangpunsakul S. Ethanol-induced alterations in fatty acid-related lipids in serum and tissues in mice.Alcohol. Clin. Exp. Res. 2011; 35: 229-234Crossref PubMed Scopus (45) Google Scholar, 17Ramirez T. Longato L. Dostalek M. Tong M. Wands J.R. de la Monte S.M. Insulin resistance, ceramide accumulation and endoplasmic reticulum stress in experimental chronic alcohol-induced steatohepatitis.Alcohol Alcohol. 2013; 48: 39-52Crossref PubMed Scopus (57) Google Scholar). Ceramides are synthesized via three major pathways: 1) de novo synthesis resulting from the condensation of serine and palmitate in the ER by the pathway's rate limiting enzyme, serine palmitoyl transferase; 2) lysosomal salvage due to the reacylation of sphingosine derived from more complex sphingolipids; and 3) sphingomyelin hydrolysis. We recently established that ceramides are present in the LD fraction and regulate Plin2 gene transcription through the enzyme ceramide synthase (10Williams B. Correnti J. Oranu A. Lin A. Scott V. Annoh M. Beck J. Furth E. Mitchell V. Senkal C.E. et al.A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.FASEB J. 2018; 32: 130-142Crossref PubMed Scopus (19) Google Scholar), whose activity is required for both ER de novo and lysosomal ceramide synthesis. Lysosomal ceramides can be subsequently deacylated into the ceramide precursor, sphingosine, by ceramidases. There are currently no suitable experimental rodent models that replicate the progression from alcoholic steatosis to advanced chronic ALD. The Lieber-DeCarli chronic alcohol feeding model is a well-validated model of alcoholic steatosis in the setting of ongoing alcohol consumption but does not model advanced liver disease (18Lieber C.S. DeCarli L.M. The feeding of alcohol in liquid diets: two decades of applications and 1982 update.Alcohol. Clin. Exp. Res. 1982; 6: 523-531Crossref PubMed Scopus (603) Google Scholar, 19Guo F. Zheng K. Benede-Ubieto R. Cubero F.J. Nevzorova Y.A. The Lieber-DeCarli diet-a flagship model for experimental alcoholic liver disease.Alcohol. Clin. Exp. Res. 2018; 42: 1828-1840Crossref PubMed Scopus (44) Google Scholar). Using this model, we demonstrated previously that systemic pharmacologic reduction of ceramides prevents alcoholic steatosis and glucose intolerance in mice (10Williams B. Correnti J. Oranu A. Lin A. Scott V. Annoh M. Beck J. Furth E. Mitchell V. Senkal C.E. et al.A novel role for ceramide synthase 6 in mouse and human alcoholic steatosis.FASEB J. 2018; 32: 130-142Crossref PubMed Scopus (19) Google Scholar), but off-target effects of this strategy limit its utility. An inducible model of hepatic acid ceramidase (ASAH) overexpression and ceramide deacylation has been developed previously and used to demonstrate improved insulin sensitivity and hepatic steatosis in high-fat diet-fed mice (20Xia J.Y. Holland W.L. Kusminski C.M. Sun K. Sharma A.X. Pearson M.J. Sifuentes A.J. McDonald J.G. Gordillo R. Scherer P.E. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis.Cell Metab. 2015; 22: 266-278Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). We employed this model of hepatic-specific ASAH overexpression to reduce hepatic ceramides in mice fed alcohol chronically. Here we report that hepatic ceramide reduction via ASAH overexpression ameliorates alcoholic steatosis and improves hepatic insulin sensitivity; and that this enzyme is relevant in human patients with ALD. We additionally establish that there is inter-organelle cross-talk between the lysosomal and LD ceramide pools and that this cross-talk is mediated by lipophagy. Experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania. All efforts were made to minimize animal discomfort and animals were treated with humane care. Inducible, liver-specific ASAH-transgenic "ASAH+" mice were a generous gift of Drs. William Holland and Phillip Scherer while Dr. Holland was at the University of Texas Southwestern (20Xia J.Y. Holland W.L. Kusminski C.M. Sun K. Sharma A.X. Pearson M.J. Sifuentes A.J. McDonald J.G. Gordillo R. Scherer P.E. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis.Cell Metab. 2015; 22: 266-278Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). ASAH+ mice have liver-specific inducible expression of N-acylsphingosine amidohydrolase 1 (ASAH1), accomplished by three transgenes: Rosa26-loxP-stop-loxP-reverse tetracycline-controlled transactivator (rtTA), Albumin-Cre, and tetracycline response element (TRE)-ASAH1. Doxycycline (Dox) treatment activates rtTA binding to the TRE and drives transcription of the ceramidase ASAH1, specifically in hepatocytes. While all mice in our experiments are homozygous for the Rosa26-loxP-stop-loxP-rtTA and Albumin-Cre transgenes, only the ASAH+ mice have the TRE-ASAH1 transgene, and the ASAH1 transgene is only expressed in the presence of Dox (20Xia J.Y. Holland W.L. Kusminski C.M. Sun K. Sharma A.X. Pearson M.J. Sifuentes A.J. McDonald J.G. Gordillo R. Scherer P.E. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis.Cell Metab. 2015; 22: 266-278Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). For all experiments, ASAH+ and ASAH− littermates were used. Weight-matched 8- to 14-week-old female mice were used for the experiments, with weight distributed equally across the genotypes. We chose female mice to replicate the epidemiologic and experimental observations that females are more susceptible to the effects of alcohol than males (1Tapper E.B. Parikh N.D. Mortality due to cirrhosis and liver cancer in the United States, 1999–2016: observational study.BMJ. 2018; 362: k2817Crossref PubMed Scopus (415) Google Scholar, 21Wagnerberger S. Fiederlein L. Kanuri G. Stahl C. Millonig G. Mueller S. Bischoff S.C. Bergheim I. Sex-specific differences in the development of acute alcohol-induced liver steatosis in mice.Alcohol Alcohol. 2013; 48: 648-656Crossref PubMed Scopus (27) Google Scholar). To acclimate animals to the ethanol (Etoh) diet, Etoh concentration was increased gradually by 2 days of feeding each at 0, 5, 10, and 15% Etoh-derived calories. Mice were then pair-fed a Lieber-DeCarli 82, Shake and Pour liquid diet containing 28% Etoh-derived calories (Bioserv, Flemington, NJ; 36% fat, 13.5% carbohydrate, 1% protein). Dox mice were supplemented with 200 mg/l Dox hyclate (Sigma-Aldrich, St. Louis, MO). Food intake and body weight were measured twice a week. Blood glucose was measured using the Accu-Chek Nano glucose meter (Roche Diabetes Care, Inc., New York, NY). To assess insulin-stimulated hepatic AKT serine/threonine kinase (Akt) activation, animals were fasted for 6 h (8:00 AM to 2:00 PM), given 2 mU/g Novolin regular insulin (Novo Nordisc, Plainsboro, NJ) intraperitoneally, and livers were isolated by freeze clamping 20 min after injection. To estimate hepatic VLDL production, a VLDL kinetic experiment was performed. Mice were fasted for 4 h (7:00 AM to 11:00 AM) prior to injection of 1 g/kg poloxamer 407 (Sigma-Aldrich), which blocks the lipolysis of TGs, thereby enabling estimation of the rate of VLDL secretion (22Millar J.S. Cromley D.A. McCoy M.G. Rader D.J. Billheimer J.T. Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339.J. Lipid Res. 2005; 46: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Tail vein blood was collected at time 0, and 1, 2, and 4 h for TG measurement by colorimetric assay as described (23Carr R.M. Patel R.T. Rao V. Dhir R. Graham M.J. Crooke R.M. Ahima R.S. Reduction of TIP47 improves hepatic steatosis and glucose homeostasis in mice.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2012; 302: R996-R1003Crossref PubMed Scopus (53) Google Scholar). ApoB was measured by ELISA (Abcam, Cambridge, MA). To inhibit lipophagy, the lysosomal inhibitor leupeptin was administered. Leupeptin (40 mg/kg; Sigma-Aldrich) in PBS was given intraperitoneally to mice 2 h before euthanization and tissue harvest. For all experiments, mice were euthanized by CO2 inhalation, and tissue was collected for analysis. Mice were individually placed in the Oxymax laboratory animal monitoring system (Columbus Instruments, Columbus, OH). Following a 24 h acclimation period, oxygen consumption, carbon dioxide production, and locomotor activity were determined. Respiratory exchange ratio (RER) was calculated by dividing the volume of carbon dioxide produced by the volume of oxygen consumed (VCO2/VO2). Whole-body fat and lean mass determinations were performed using the EchoMRI-100 system (EchoMRI; Houston, TX). Clamp studies were performed at the University of Pennsylvania Diabetes Research Center Mouse Phenotyping, Physiology, and Metabolism Core. Indwelling jugular vein catheters were surgically implanted 5 days prior to the clamp study day as described previously (24Ayala J.E. Bracy D.P. Malabanan C. James F.D. Ansari T. Fueger P.T. McGuinness O.P. Wasserman D.H. Hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice.J. Vis. Exp. 2011; (doi:10.3791/3188.)Crossref PubMed Scopus (97) Google Scholar). Mice were fasted for 5 h prior to initiation of the clamp and acclimated to the plastic restrainers for tail sampling. A [3-3H] glucose infusion was primed (5-μCi) and continuously infused for a 120 min equilibration period (0.05 μCi/min). Baseline measurements were determined in blood samples collected at -10 and 0 min (relative to the start of the clamp) for analysis of glucose, [3-3H]glucose-specific activity, and basal insulin. The clamp was started at t = 0 min with a primed-continuous infusion of human insulin (16 mU/kg bolus followed by 2.5 mU/kg/min; Novolin regular insulin), and glucose (D20 mixed with [3-3H]glucose 0.03 μCi/μl) was infused at a variable glucose infusion rate (GIR) to maintain euglycemia. Blood samples were taken at t = 80–120 min for the measurement of [3-3H]glucose-specific activity and clamped insulin levels. After the final blood sample, animals were injected with a bolus of pentobarbital, and quadricep muscle and epididymal adipose tissue were collected and frozen in liquid nitrogen and stored in −20°C for subsequent analysis. The radioactivity of [3-3H]glucose, [14C]2DG, and [14C]2DG-6-phosphate were determined as described previously (25Ayala J.E. Bracy D.P. Julien B.M. Rottman J.N. Fueger P.T. Wasserman D.H. Chronic treatment with sildenafil improves energy balance and insulin action in high fat-fed conscious mice.Diabetes. 2007; 56: 1025-1033Crossref PubMed Scopus (186) Google Scholar). The glucose turnover rate [total rate of endogenous glucose production (Ra); milligrams per kilogram per minute) was calculated as the rate of tracer infusion (disintegrations per minute per minute) divided by the corrected plasma glucose specific activity (disintegrations per minute per milligram) per kilogram of body weight of the mouse. Glucose appearance (Ra) and disappearance [rate of peripheral glucose disposal (Rd)] rates were determined using steady-state equations, and endogenous glucose production (Ra) was determined by subtracting the GIR from total Ra. Tissue-specific glucose disposal (Rg; micromoles per 100 grams of tissue per minute) was calculated as described previously (25Ayala J.E. Bracy D.P. Julien B.M. Rottman J.N. Fueger P.T. Wasserman D.H. Chronic treatment with sildenafil improves energy balance and insulin action in high fat-fed conscious mice.Diabetes. 2007; 56: 1025-1033Crossref PubMed Scopus (186) Google Scholar). TGs (Stanbio, St. Boerne, TX), cholesterol (Wako, Mountain View, CA), β-hydroxybutyrate (Stanbio), alanine aminotransferase (ALT; Stanbio), and NEFAs (Wako) were measured using enzymatic colorimetric assays. Liver TGs were measured in Etoh:KOH lipid extracts. For liver ceramide analysis, liver was homogenized in RIPA buffer and quantitated using the Pierce™ BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Samples were analyzed by mass spectrometry for ceramide content at the metabolomics cores at Stony Brook University School of Medicine and at the Medical University of South Carolina. Whole and fractionated liver samples were normalized to protein and equal serum volumes were measured. Isolation of LDs, lysosomes, and ER for ceramide analysis was performed as described (26Brettschneider J. Correnti J.M. Lin C. Williams B. Oranu A. Kuriakose A. McIver-Jenkins D. Haba A. Kaneza I. Jeon S. et al.Rapid lipid droplet isolation protocol using a well-established organelle isolation kit.J. Vis. Exp. 2019; (doi:10.3791/59290.)Crossref PubMed Scopus (2) Google Scholar). Liver lysates for Western blot were generated by homogenization in RIPA buffer and protein content was assessed by BCA assay (Thermo Fisher Scientific). Protein (50 μg) was separated by SDS-PAGE, transferred, and probed with antibodies specific for PLIN2 (1:1,000; ab108323, Abcam), lysosomal-associated membrane protein 1 (LAMP1) (1:1;000; 3243, Cell Signaling Technology, Danvers, MA), microtubule-associated protein light chain 3 (LC3B) (1:1,000; nb100-2220, Novus Biologicals, Centennial, CO), or GAPDH (1:2,000; MAB374, MilliporeSigma, Burlington, MA). All blots were visualized by chemiluminescence using secondaries conjugated to HRP (1:5,000; sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA), except GAPDH, which was visualized by infrared secondary (1:15,000; 926-68070, LiCor, Lincoln, NE) scanned on a LiCor Odyssey (LiCor). Quantitation was done using LiCor software and on scans of radiographs in ImageJ software (National Institutes of Health, https://imagej.nih.gov/ij/). RNA was extracted using the PureLink™ RNA mini kit (Life Technologies, Carlsbad, CA). DNase I-treated (Life Technologies) RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit, and mRNA expression was measured by real-time PCR (Applied Biosystems) using TaqMan primers from Life Technologies. ASAH1 mRNA was quantified using SYBR Green (Applied Biosystems) with PCR primers described previously (20Xia J.Y. Holland W.L. Kusminski C.M. Sun K. Sharma A.X. Pearson M.J. Sifuentes A.J. McDonald J.G. Gordillo R. Scherer P.E. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis.Cell Metab. 2015; 22: 266-278Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Relative mRNA expression was normalized to GAPDH, 18s rRNA, or 36B4. Total RNA quantity and quality were assayed with an Agilent 2100 bioanalyzer instrument using the RNA 6000 Nano kit (Agilent Technologies). Libraries were prepared at Next Generation Sequencing Core at the University of Pennsylvania using TruSeq Stranded mRNA HT Sample Prep Kit (Illumina) as per the standard protocol in the kit's sample preparation guide. Libraries were assayed for size using a DNA 1000 kit of Agilent 2100 Bioanalyzer (Agilent Technologies) and quantified using the KAPA Library quantification kit for Illumina platforms (KAPA Biosystems). One hundred base pair single-read sequencing of multiplexed samples was performed on an Illumina HiSeq 4000 sequencer. Illumina's bcl2fastq version 2.20.0.422 software was used to convert bcl to fastq files. The Molecular Profiling Facility at the University of Pennsylvania performed data analysis. Raw sequence files (fastq) were mapped using salmon (https://combine-lab.github.io/salmon/) against the mouse transcripts described in GENCODE (version M19, built on the mouse genome GRCm38.p6, https://www.gencodegenes.org). Transcript counts were summarized to the gene level using tximport (https://bioconductor.org/packages/release/bioc/html/tximport.html) and normalized and tested for differential expression using DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html). Normalized data were visualized with principal components analysis to assess global relationships among the samples using Partek Genomics Suite (Partek, Inc., St. Louis, MO). A false discovery rate-corrected P-value was calculated by DESeq2 using the Benjamini-Hochberg method. Institutional review board-exempt status was obtained to use archived human liver tissue. The protocol conforms to the ethics outlined in the 1975 Declaration of Helsinki. Most samples were fixed in 10% neutral buffered formalin for 24 h and then transferred to 70% Etoh until paraffin embedding. Frozen tissue was thawed, fixed overnight in 10% NBF, transferred to 70% Etoh, and then paraffin embedded for sectioning. Paraffin sections were stained with hematoxylin and eosin. Oil Red O staining on liver sections frozen in cryoprotectant media was performed by the Molecular Pathology and Imaging Core at the University of Pennsylvania. Immunohistochemistry on mouse and de-identified human liver samples was performed using a BOND instrument (Leica Microsystems, Mannheim, Germany) with heat-epitope retrieval for 20 min in ER1 solution. Electron microscopy (EM) sample preparation, immunogold labeling, and imaging were performed by the EM Resource Lab at the University of Pennsylvania. For human RNaseq studies, human liver samples were obtained from the Human Biorepository Core from the National Institutes of Health-funded international InTeam consortium and from Cliniques Universitaires Saint-Luc (Brussels, Belgium), as described previously (27Argemi J. Latasa M.U. Atkinson S.R. Blokhin I.O. Massey V. Gue J.P. Cabezas J. Lozano J.J. Van Booven D. Bell A. et al.Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.Nat. Commun. 2019; 10: 3126Crossref PubMed Scopus (78) Google Scholar). All participants gave written informed consent and the research protocols were approved by the local Ethics Committees and by the central Institutional Review Board of the University of North Carolina at Chapel Hill. A total of 90 patients were included. Patients were grouped into seven categories: nonobese, high alcohol intake, early alcoholic steatohepatitis ("Early", N = 12); nonsevere alcoholic hepatitis ("Nsev AH", N = 11); severe alcoholic steatohepatitis ("Sev AH", N = 29); nondiseased ("Normal", N = 10); NAFLD without alcohol abuse ("NAFLD", N = 9); chronic hepatitis C virus (HCV) (HCV, N = 10); and compensated HCV cirrhosis ("Comp HCV Cirr", N = 9). Patients with malignancies were excluded. RNA purity and quality assessment, library preparation, sequencing, and bioinformatic analyses have been described previously (27Argemi J. Latasa M.U. Atkinson S.R. Blokhin I.O. Massey V. Gue J.P. Cabezas J. Lozano J.J. Van Booven D. Bell A. et al.Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis.Nat. Commun. 2019; 10: 3126Crossref PubMed Scopus (78) Google Scholar). Total RNA libraries were built using TruSeq Stranded Total RNA Ribo-Zero GOLD (Illumina) and sequenced using the Illumina HiSeq2000 platform. Sequencing was paired end (2 × 100 bp) and multiplexed. Limma package (2Raynard B. Balian A. Fallik D. Capron F. Bedossa P. Chaput J.C. Naveau S. Risk factors of fibrosis in alcohol-induced liver disease.Hepatology. 2002; 35: 635-638Crossref PubMed Scopus (321) Google Scholar) was used for cyclic loss normalization followed by log transformation of the counts per million and mean-variance adjustment using the voom function (28Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. Smyth G.K. limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (15626) Google Scholar). Statistical analysis was performed using t-test or ANOVA with post hoc Newman-Keuls multiple comparison test (GraphPad Prism, La Jolla, CA). P < 0.05 was considered significant in all cases. ASAH is a lysosomal enzyme that hydrolyzes ceramide to sphingosine and a free FA (20Xia J.Y. Holland W.L. Kusminski C.M. Sun K. Sharma A.X. Pearson M.J. Sifuentes A.J. McDonald J.G. Gordillo R. Scherer P.E. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis.Cell Metab. 2015; 22: 266-278Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). To achieve inducible liver-specific ceramide reduction, ASAH-overexpressing (ASAH+