Leptin Deficiency Contributes to the Pathogenesis of Alcoholic Fatty Liver Disease in Mice
2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2012.06.013
ISSN1525-2191
AutoresXiaobing Tan, Xiuhua Sun, Qiong Li, Yantao Zhao, Wei Zhong, Xinguo Sun, Jia Wang, Craig J. McClain, Zhanxiang Zhou,
Tópico(s)Alcohol Consumption and Health Effects
ResumoWhite adipose tissue (WAT) secretes adipokines, which critically regulate lipid metabolism. The present study investigated the effects of alcohol on adipokines and the mechanistic link between adipokine dysregulation and alcoholic fatty liver disease. Mice were fed alcohol for 2, 4, or 8 weeks to document changes in adipokines over time. Alcohol exposure reduced WAT mass and body weight in association with hepatic lipid accumulation. The plasma adiponectin concentration was increased at 2 weeks, but declined to normal at 4 and 8 weeks. Alcohol exposure suppressed leptin gene expression in WAT and reduced the plasma leptin concentration at all times measured. There is a highly positive correlation between plasma leptin concentration and WAT mass or body weight. To determine whether leptin deficiency mediates alcohol-induced hepatic lipid dyshomeostasis, mice were fed alcohol for 8 weeks with or without leptin administration for the last 2 weeks. Leptin administration normalized the plasma leptin concentration and reversed alcoholic fatty liver. Alcohol-perturbed genes involved in fatty acid β-oxidation, very low-density lipoprotein secretion, and transcriptional regulation were attenuated by leptin. Leptin also normalized alcohol-reduced phosphorylation levels of signal transducer Stat3 and adenosine monophosphate–activated protein kinase. These data demonstrated for the first time that leptin deficiency in association with WAT mass reduction contributes to the pathogenesis of alcoholic fatty liver disease. White adipose tissue (WAT) secretes adipokines, which critically regulate lipid metabolism. The present study investigated the effects of alcohol on adipokines and the mechanistic link between adipokine dysregulation and alcoholic fatty liver disease. Mice were fed alcohol for 2, 4, or 8 weeks to document changes in adipokines over time. Alcohol exposure reduced WAT mass and body weight in association with hepatic lipid accumulation. The plasma adiponectin concentration was increased at 2 weeks, but declined to normal at 4 and 8 weeks. Alcohol exposure suppressed leptin gene expression in WAT and reduced the plasma leptin concentration at all times measured. There is a highly positive correlation between plasma leptin concentration and WAT mass or body weight. To determine whether leptin deficiency mediates alcohol-induced hepatic lipid dyshomeostasis, mice were fed alcohol for 8 weeks with or without leptin administration for the last 2 weeks. Leptin administration normalized the plasma leptin concentration and reversed alcoholic fatty liver. Alcohol-perturbed genes involved in fatty acid β-oxidation, very low-density lipoprotein secretion, and transcriptional regulation were attenuated by leptin. Leptin also normalized alcohol-reduced phosphorylation levels of signal transducer Stat3 and adenosine monophosphate–activated protein kinase. These data demonstrated for the first time that leptin deficiency in association with WAT mass reduction contributes to the pathogenesis of alcoholic fatty liver disease. Alcoholic liver disease is traditionally described as three progressive pathologic conditions: steatosis (fatty liver), hepatitis, and cirrhosis.1Hall P. Pathological spectrum of alcoholic liver disease Alcoholic Liver Disease. ed 2. Edward Arnold, London1995: 41-88Google Scholar Alcoholic fatty liver is characterized by lipid droplet accumulation in the cytoplasm of hepatocytes, and is one of the earliest pathologic alterations in the liver. Accumulation of lipids in the hepatocytes makes the liver susceptible to inflammatory mediators or other toxic agents, leading to further progression to hepatitis and eventually to fibrosis. Alcohol consumption may affect multiple pathways of hepatic lipid metabolism including de novo lipogenesis, fatty acid oxidation, lipid uptake, and lipid export in the form of very low-density lipoproteins.2Lakshman M.R. Some novel insights into the pathogenesis of alcoholic steatosis.Alcohol. 2004; 34: 45-48Crossref PubMed Scopus (42) Google Scholar, 3Gao B. Bataller R. Alcoholic liver disease: pathogenesis and new therapeutic targets.Gastroenterology. 2011; 141: 1572-1585Abstract Full Text Full Text PDF PubMed Scopus (1387) Google Scholar However, recent studies have suggested that extrahepatic factors such as adiponectin critically modulate hepatic lipid metabolism.4Rogers C.Q. Ajmo J.M. You M. Adiponectin and alcoholic fatty liver disease.IUBMB Life. 2008; 60: 790-797Crossref PubMed Scopus (109) Google Scholar, 5You M. Rogers C.Q. Adiponectin: a key adipokine in alcoholic fatty liver.Exp Biol Med (Maywood). 2009; 234: 850-859Crossref PubMed Scopus (95) Google Scholar White adipose tissue (WAT) is a major organ for body fat storage, and also functions as an endocrine organ.6Galic S. Oakhill J.S. Steinberg G.R. Adipose tissue as an endocrine organ.Mol Cell Endocrinol. 2010; 316: 129-139Crossref PubMed Scopus (1261) Google Scholar The hormones secreted by WAT are adipokines, and two of the most important adipokines related to energy homeostasis are adiponectin and leptin. Both adiponectin and leptin critically modulate hepatic lipid homeostasis toward reduction of lipid content in the liver. Adiponectin signaling in the liver leads to activation of the adenosine monophosphate-activated protein kinase (AMPK) pathway via adiponectin receptor.4Rogers C.Q. Ajmo J.M. You M. Adiponectin and alcoholic fatty liver disease.IUBMB Life. 2008; 60: 790-797Crossref PubMed Scopus (109) Google Scholar, 5You M. Rogers C.Q. Adiponectin: a key adipokine in alcoholic fatty liver.Exp Biol Med (Maywood). 2009; 234: 850-859Crossref PubMed Scopus (95) Google Scholar, 6Galic S. Oakhill J.S. Steinberg G.R. Adipose tissue as an endocrine organ.Mol Cell Endocrinol. 2010; 316: 129-139Crossref PubMed Scopus (1261) Google Scholar, 7Marra F. Bertolani C. Adipokines in liver disease.Hepatology. 2009; 50: 957-969Crossref PubMed Scopus (397) Google Scholar AMPK activation negatively regulates the hepatic lipid level by stimulating fatty acid oxidation and suppressing fatty acid influx and de novo lipogenesis. Leptin critically regulates whole-body energy homeostasis by inhibiting energy intake and stimulating energy expenditure. Leptin signaling in the liver via leptin receptor b (LepRb) activates AMPK and signal transducer Stat3 pathways.6Galic S. Oakhill J.S. Steinberg G.R. Adipose tissue as an endocrine organ.Mol Cell Endocrinol. 2010; 316: 129-139Crossref PubMed Scopus (1261) Google Scholar, 7Marra F. Bertolani C. Adipokines in liver disease.Hepatology. 2009; 50: 957-969Crossref PubMed Scopus (397) Google Scholar Mice lacking functional leptin develop not only obesity but also fatty liver. Therefore, adipose tissues via adipokine secretion significantly affect lipid homeostasis in the liver. Alcohol exposure has been shown to affect adipose mass and adipokine secretion in both humans and animals. Patients with alcoholism have lower body mass index (BMI) and fat mass (FM) but higher liver fat levels.8Addolorato G. Capristo E. Greco A.V. Stefanini G.F. Gasbarrini G. Energy expenditure, substrate oxidation, and body composition in subjects with chronic alcoholism: new findings from metabolic assessment.Alcohol Clin Exp Res. 1997; 21: 962-967PubMed Google Scholar, 9Addolorato G. Capristo E. Greco A.V. Stefanini G.F. Gasbarrini G. Influence of chronic alcohol abuse on body weight and energy metabolism: is excess ethanol consumption a risk factor for obesity or malnutrition?.J Intern Med. 1998; 244: 387-395Crossref PubMed Scopus (112) Google Scholar, 10Addolorato G. Capristo E. Marini M. Santini P. Scognamiglio U. Attilia M.L. Messineo D. Sasso G.F. Gasbarrini G. Ceccanti M. Body composition changes induced by chronic ethanol abuse: evaluation by dual energy X-ray absorptiometry.Am J Gastroenterol. 2000; 95: 2323-2327Crossref PubMed Google Scholar, 11Greco A.V. Mingrone G. Favuzzi A. Capristo E. Gniuli D. Addolorato G. Brunani A. Cavagnin F. Gasbarrini G. Serum leptin levels in post-hepatitis liver cirrhosis.J Hepatol. 2000; 33: 38-42Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Studies in humans have also found that the serum leptin concentration was reduced by either chronic alcohol consumption or acute alcohol abuse.11Greco A.V. Mingrone G. Favuzzi A. Capristo E. Gniuli D. Addolorato G. Brunani A. Cavagnin F. Gasbarrini G. Serum leptin levels in post-hepatitis liver cirrhosis.J Hepatol. 2000; 33: 38-42Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 12Santolaria F. Pérez-Cejas A. Alemán M.R. González-Reimers E. Milena A. de la Vega M.J. Martínez-Riera A. Gómez-Rodríguez M.A. Low serum leptin levels and malnutrition in chronic alcohol misusers hospitalized by somatic complications.Alcohol Alcohol. 2003; 38: 60-66Crossref PubMed Scopus (47) Google Scholar, 13Calissendorff J. Brismar K. Röjdmark S. Is decreased leptin secretion after alcohol ingestion catecholamine-mediated?.Alcohol Alcohol. 2004; 39: 281-286Crossref PubMed Scopus (16) Google Scholar, 14Kalousová M. Zima T. Popov P. Spacek P. Braun M. Soukupová J. Pelinkova K. Kientsch-Engel R. Advanced glycation end-products in patients with chronic alcohol misuse.Alcohol Alcohol. 2004; 39: 316-320Crossref PubMed Scopus (46) Google Scholar Chronic alcohol exposure in rodents reduced adipose tissue weight,15Kang L. Chen X. Sebastian B.M. Pratt B.T. Bederman I.R. Alexander J.C. Previs S.F. Nagy L.E. Chronic ethanol and triglyceride turnover in white adipose tissue in rats: inhibition of the anti-lipolytic action of insulin after chronic ethanol contributes to increased triglyceride degradation.J Biol Chem. 2007; 282: 28465-28473Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 16Kang X. Zhong W. Liu J. Song Z. McClain C.J. Kang Y.J. Zhou Z. Zinc supplementation reverses alcohol-induced steatosis in mice through reactivating hepatocyte nuclear factor-4alpha and peroxisome proliferator-activated receptor-alpha.Hepatology. 2009; 50: 1241-1250Crossref PubMed Scopus (143) Google Scholar, 17Zhong W. Zhao Y. Tang Y. Wei X. Shi X. Sun W. Sun X. Yin X. Sun X. Kim S. McClain C.J. Zhang X. Zhou Z. Chronic alcohol exposure stimulates adipose tissue lipolysis in mice: role of reverse triglyceride transport in the pathogenesis of alcoholic steatosis.Am J Pathol. 2012; 180: 998-1007Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar and serum adiponectin18Xu A. Wang Y. Keshaw H. Xu L.Y. Lam K.S. Cooper G.J. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice.J Clin Invest. 2003; 112: 91-100Crossref PubMed Scopus (1150) Google Scholar, 19You M. Considine R.V. Leone T.C. Kelly D.P. Crabb D.W. Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice.Hepatology. 2005; 42: 568-577Crossref PubMed Scopus (226) Google Scholar, 20Chen X. Sebastian B.M. Nagy L.E. Chronic ethanol feeding to rats decreases adiponectin secretion by subcutaneous adipocytes.Am J Physiol Endocrinol Metab. 2007; 292: E621-E628Crossref PubMed Scopus (73) Google Scholar, 21Esfandiari F. You M. Villanueva J.A. Wong D.H. French S.W. Halsted C.H. S-adenosylmethionine attenuates hepatic lipid synthesis in micropigs fed ethanol with a folate-deficient diet.Alcohol Clin Exp Res. 2007; 31: 1231-1239Crossref PubMed Scopus (81) Google Scholar, 22Song Z. Zhou Z. Deaciuc I. Chen T. McClain C.J. Inhibition of adiponectin production by homocysteine: potential mechanism for alcoholic liver disease.Hepatology. 2008; 47: 867-879Crossref PubMed Scopus (111) Google Scholar, 23Shen Z. Liang X. Rogers C.Q. Rideout D. You M. Involvement of adiponectin-SIRT1-AMPK signaling in the protective action of rosiglitazone against alcoholic fatty liver in mice.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G364-G374Crossref PubMed Scopus (178) Google Scholar and leptin24Otaka M. Konishi N. Odashima M. Jin M. Wada I. Matsuhashi T. Ohba R. Watanabe S. Effect of alcohol consumption on leptin level in serum, adipose tissue, and gastric mucosa.Dig Dis Sci. 2007; 52: 3066-3069Crossref PubMed Scopus (26) Google Scholar, 25Maddalozzo G.F. Turner R.T. Edwards C.H. Howe K.S. Widrick J.J. Rosen C.J. Iwaniec U.T. Alcohol alters whole body composition, inhibits bone formation, and increases bone marrow adiposity in rats.Osteoporos Int. 2009; 20: 1529-1538Crossref PubMed Scopus (77) Google Scholar concentrations in association with the development of fatty liver. Administration of exogenous adiponectin or stimulation of endogenous adiponectin production attenuated alcoholic fatty liver in mice.18Xu A. Wang Y. Keshaw H. Xu L.Y. Lam K.S. Cooper G.J. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice.J Clin Invest. 2003; 112: 91-100Crossref PubMed Scopus (1150) Google Scholar, 19You M. Considine R.V. Leone T.C. Kelly D.P. Crabb D.W. Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice.Hepatology. 2005; 42: 568-577Crossref PubMed Scopus (226) Google Scholar, 23Shen Z. Liang X. Rogers C.Q. Rideout D. You M. Involvement of adiponectin-SIRT1-AMPK signaling in the protective action of rosiglitazone against alcoholic fatty liver in mice.Am J Physiol Gastrointest Liver Physiol. 2010; 298: G364-G374Crossref PubMed Scopus (178) Google Scholar, 26Ajmo J.M. Liang X. Rogers C.Q. Pennock B. You M. Resveratrol alleviates alcoholic fatty liver in mice.Am J Physiol Gastrointest Liver Physiol. 2008; 295: G833-G842Crossref PubMed Scopus (331) Google Scholar These studies indicate that adipokines critically regulate lipid homeostasis at the adipose tissue–liver axis. The present study reports that chronic alcohol exposure caused leptin deficiency, and leptin administration for 2 weeks reversed alcohol-induced fatty liver in mice previously exposed to alcohol for 6 weeks. Male C57BL/6 mice were obtained from Harlan Laboratories, Inc. (Indianapolis, IN). All of the mice were treated according to the experimental procedures approved by the Institutional Animal Care and Use Committee. At age 4 months, mice were pair-fed a modified Lieber-DeCarli alcohol or isocaloric maltose dextrin control liquid diet for up to 8 weeks in a stepwise procedure. Caloric content of the alcohol diet was 16% protein, 12% carbohydrate, 34% fat, and 38% ethanol (Sigma-Aldrich Corp., St. Louis, MO). The ethanol content (w/v) in the diet was 4.8% (34% of total calories) for the first 2 weeks, and was increased by 0.2% every 2 weeks, reaching 5.4% (38% of total calories) for the last 2 weeks. On the basis of our observations, this stepwise feeding protocol improves the performance of the mouse model of alcohol feeding, as indicated by reduced mortality. Two sets of animal experiments were performed. Experiment 1 was performed to determine adipokine production in association with development of alcoholic liver disease, and mice were subjected to alcohol exposure for 2, 4, or 8 weeks. Experiment 2 was performed to determine the role of leptin in alcoholic liver disease because leptin deficiency was detected in experiment 1. Mice were subjected to alcohol exposure for 8 weeks, with leptin (purity >95%; ProSpec, East Brunswick, NJ) administration at 0.5 mg/d per kilogram body weight or saline solution as vehicle, via subcutaneous osmotic minipump (ALZET Osmotic Pump; DURECT Corp., Cupertino, CA) for the last 2 weeks. Blood glucose concentration was measured using a OneTouch Ultra2 Blood Glucose Meter (LifeScan, Inc., Milpitas, CA), and ketone bodies using PTS PANELS Ketone Test Strips (Polymer Technology Systems, Inc., Indianapolis, IN). Plasma alanine aminotransferase activity and triglyceride and cholesterol concentrations were determined using Infinity Reagents (Thermo Fisher Scientific, Inc., Middletown, VA). Plasma free fatty acids (FFAs) were quantified using an FFA Quantification Kit (BioVision, Inc., Milpitas, CA). Plasma leptin and adiponectin concentrations were measured using commercial mouse enzyme-linked immunosorbent assay kits (Millipore Corp., Billerica, MA). Liver tissues were fixed in 10% formalin and embedded in paraffin. Tissues were cut into 5-μm sections and processed for H&E staining and Sirus Red staining. Quantitative assay of lipids was conducted by measuring triglyceride, cholesterol, and FFA concentrations in the liver. Hepatic lipids are extracted by homogenizing liver tissue in chloroform using 1% Triton X-100. The organic extracts were air dried, vacuumed, and dissolved in 1% Triton X-100. Triglyceride, cholesterol, and FFA concentrations in extracts were determined using commercial kits (see Blood Parameters). The total RNA was isolated from liver or WAT using the TRIzol method (Life Technologies Corp., Grand Island, NY), and reverse transcription was conducted using the TaqMan Reverse Transcription Reagents Kit (Applied Biosystems, Inc., Foster City, CA). The forward and reverse primers (Table 1) were designed using Primer Express Software v3.0.1 (Applied Biosystems), and quantitative RT-PCR (RT-qPCR) analysis with SYBR Green PCR master mix (Qiagen, Inc., Valencia, CA) was performed using a PRISM 7500 Sequence Detection System (Applied Biosystems). Data were normalized to β-actin expression, and are given as fold changes, setting the values of pair-fed mice at 1.Table 1Primer Sequences for Real-Time RT-PCRGeneGeneBank accession no.Sequences (Forward/Reverse)ACADLNM_0073815′-TCTTTTCCTCGGAGCATGACA-3′5′-GACCTCTCTACTCACTTCTCCAG-3′ACOX1NM_0157295′-TCCAGACTTCCAACATGAGGA-3′5′-CTGGGCGTAGGTGCCAATTA-3′ApoBNM_0096935′-TTGGCAAACTGCATAGCATCC-3′5′-TCAAATTGGGACTCTCCTTTAGC-3′C/EBP-αNM_0076785′-CAAGAACAGCAACGAGTACCG-3′5′-GTCACTGGTCAACTCCAGCAC-3′FABP1NM_0173995′-ATGAACTTCTCCGGCAAGTACC-3′5′-CTGACACCCCCTTGATGTCC-3′FATP2NM_0119785′-TCCTCCAAGATGTGCGGTACT-3′5′-TAGGTGAGCGTCTCGTCTCG-3′FATP5NM_0095125′-CTACGCTGGCTGCATATAGATG-3′5′-CCACAAAGGTCTCTGGAGGAT-3′HNF-1αNM_0093275′-GACCTGACCGAGTTGCCTAAT-3′5′-CCGGCTCTTTCAGAATGGGT-3′HNF-4αNM_0082615′-GCCTTCTGCGAACTCCTTCTG-3′5′-GGGACGATGTAGTCATTGCCT-3′CPT1aNM_0134955′-CTCCGCCTGAGCCATGAAG-3′5′-CACCAGTGATGATGCCATTCT-3′LeptinNM_0084935′-GAGACCCCTGTGTCGGTTC-3′5′-CTGCGTGTGTGAAATGTCATTG-3′LepR-bNM_1461465′-TGGTCCCAGCAGCTATGGT-3′5′-ACCCAGAGAAGTTAGCACTGT-3′MTTPNM_0086425′-TCAAGAGAGGCTTGGCTAGCTT-3′5′-GCCTGGTAGGTCACTTTACAATCC-3′PPAR-αNM_0111445′-AGAGCCCCATCTGTCCTCTC-3′5′-ACTGGTAGTCTGCAAAACCAAA-3′PPAR-γNM_0111465′-TCGCTGATGCACTGCCTATG-3′5′-GAGAGGTCCACAGAGCTGATT-3′β-ActinNM_0073935′-GGCTGTATTCCCCTCCATCG-3′5′-CCAGTTGGTAACAATGCCATGT-3′ Open table in a new tab Whole protein lysates of liver were extracted using 10% Nonidet P-40 lysis buffer supplemented with 1% protease inhibitor cocktail and 1% phenylmethylsulfonyl fluoride. Aliquots containing 60 μg proteins were loaded onto 10% SDS-PAGE, transblotted onto polyvinylidene difluoride membrane, blocked using 5% nonfat dry milk in Tris-buffered saline solution with 0.1% Tween-20, and incubated with rabbit anti-pStat3, Stat3, pAMPK, AMPK (Cell Signaling Technology, Inc., Danvers, MA), PPAR-α (peroxisome proliferator-activated receptor-α), HNF-1α (hepatocyte nuclear factor-1α), C/EBP-α (CCAAT/enhancer binding protein), or β-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The membrane was then incubated using horseradish peroxidase–conjugated anti-rabbit IgG. The bound complexes were detected via chemiluminescence. The immunoblot bands were quantified via densitometry analysis, and the ratio to β-actin was calculated and is given as fold changes, setting the values of pair-fed mice at 1. All data are given as mean ± SD. The results were analyzed using the one-sample t-test for two groups, and one-way analysis of variance with Turkey post hoc comparison for more than two groups. Correlation coefficients, analyzed using Prime 3, were used to determine linear association between plasma leptin concentration and WAT mass or body weight. In all statistical tests, P < 0.05 was considered significant. Alcohol exposure for 2, 4, and 8 weeks caused liver damage that was time-dependent (Figure 1A). Lipid droplet accumulation in the liver is the major pathologic change at 2 weeks. At 4 and 8 weeks, the number and size of the lipid droplets in the hepatocytes gradually increased, and inflammatory cell infiltration was frequently found. In contrast, alcohol exposure significantly reduced epididymal and subcutaneous WAT mass at all three times measured (Figure 1B). In comparison with a gradual increase in WAT weight in pair-fed mice, alcohol-fed mice maintained a low WAT weight. The body weight of alcohol-fed mice was lower than that of pair-fed mice at all three times measured (data not shown). To determine whether alcohol-induced lipodystrophy leads to alterations in adipokine secretion, plasma adiponectin and leptin levels were measured. Plasma adiponectin levels in pair-fed mice did not change at any times measured (Figure 2A). Alcohol exposure for 2 weeks increased the plasma adiponectin concentration, but it declined to normal at 4 and 8 weeks. The plasma leptin concentration in pair-fed mice showed a significant increase at 4 and 8 weeks in comparison with that at 0 and 2 weeks (Figure 2A). Alcohol exposure reduced the plasma leptin concentration by 40%, 20%, and 59% at 2, 4, and 8 weeks, respectively, compared with that at 0 week. RT-qPCR analysis demonstrated that alcohol exposure for 8 weeks dramatically down-regulated leptin gene expression in both epididymal and subcutaneous WATs (Figure 2B). Correlation analysis showed highly positive correlation coefficients between plasma leptin concentration and epididymal and subcutaneous WAT mass and body weight (Figure 2C). To determine the role of leptin deficiency in the pathogenesis of alcoholic fatty liver, leptin was administered to alcohol-fed mice for the last 2 weeks of the study. Leptin administration normalized the alcohol-decreased plasma leptin concentration and stimulated hepatic LepRb gene expression (Figure 3A), although alcohol alone also up-regulated hepatic LepRb (Figure 3B). Effects of leptin administration on routine parameters are given in Table 2. Alcohol exposure with or without leptin administration reduced body weight, whereas liver weight was reduced only by alcohol plus leptin. As a consequence, the liver–body weight ratio was increased by alcohol exposure, which was partially reversed by leptin. Epididymal WAT weight and its ratio to body weight were reduced by alcohol, and the reduction was promoted by leptin. Alcohol exposure significantly elevated the plasma alanine aminotransferase concentration, which was normalized by leptin. Alcohol exposure decreased blood glucose and cholesterol concentrations, and increased the plasma ketone body concentration; these alterations were not affected by leptin. However, leptin administration normalized alcohol-reduced plasma FFA concentrations.Table 2Effects of Chronic Alcohol Exposure and Leptin Administration on Body Weight, Liver Weight, Epididymal WAT Weight, and Blood MetabolitesMeasurementsPFAFAF/LeptinFood intake (g/d per mouse)10.36 ± 0.8311.02 ± 0.7610.67 ± 1.47Alcohol intake (g/d per mouse)NA0.59 ± 0.040.56 ± 0.07Body weight (g)29.77 ± 1.7624.05 ± 1.40⁎P < 0.05 versus PF (ANOVA).22.60 ± 0.52⁎P < 0.05 versus PF (ANOVA).Liver weight (g)1.25 ± 0.081.30 ± 0.071.09 ± 0.05⁎P < 0.05 versus PF (ANOVA).Epididymal WAT weight (g)1.28 ± 0.210.42 ± 0.04⁎P < 0.05 versus PF (ANOVA).0.18 ± 0.04⁎P < 0.05 versus PF (ANOVA).†P < 0.05 versus AF (ANOVA).Liver–body weight ratio4.17 ± 0.365.41 ± 0.14⁎P < 0.05 versus PF (ANOVA).4.83 ± 0.14⁎P < 0.05 versus PF (ANOVA).Epididymal WAT–body weight ratio4.27 ± 0.591.74 ± 0.12⁎P < 0.05 versus PF (ANOVA).0.78 ± 0.18⁎P < 0.05 versus PF (ANOVA).†P < 0.05 versus AF (ANOVA).Plasma ALT (U/mL)24.36 ± 7.1965.82 ± 9.60⁎P < 0.05 versus PF (ANOVA).31.50 ± 11.18†P < 0.05 versus AF (ANOVA).Blood glucose (mg/dL)277.0 ± 45.4221.0 ± 19.1⁎P < 0.05 versus PF (ANOVA).204.7 ± 26.1⁎P < 0.05 versus PF (ANOVA).Blood ketone bodies (mg/dL)4.90 ± 1.597.08 ± 1.65⁎P < 0.05 versus PF (ANOVA).8.34 ± 1.25⁎P < 0.05 versus PF (ANOVA).Plasma triglycerides (mg/dL)75.45 ± 14.1276.98 ± 21.3257.83 ± 20.40Plasma cholesterol (mg/dL)91.29 ± 17.8859.81 ± 11.24⁎P < 0.05 versus PF (ANOVA).47.85 ± 9.66⁎P < 0.05 versus PF (ANOVA).Plasma free fatty acids (mmol/L)0.80 ± 0.150.43 ± 0.21⁎P < 0.05 versus PF (ANOVA).0.72 ± 0.21†P < 0.05 versus AF (ANOVA).Mice were pair-fed alcohol or isocaloric maltose dextran for 8 weeks in the presence or absence of external leptin for the last 2 weeks.Data are given as mean ± SD (n = 6–10).AF, alcohol-fed; ALT, alanine aminotransferase; ANOVA, analysis of variance; PF, pair-fed; WAT, white adipose tissue. P < 0.05 versus PF (ANOVA).† P < 0.05 versus AF (ANOVA). Open table in a new tab Mice were pair-fed alcohol or isocaloric maltose dextran for 8 weeks in the presence or absence of external leptin for the last 2 weeks. Data are given as mean ± SD (n = 6–10). AF, alcohol-fed; ALT, alanine aminotransferase; ANOVA, analysis of variance; PF, pair-fed; WAT, white adipose tissue. Alcohol exposure for 8 weeks caused lipid droplet accumulation in the liver, which was attenuated by leptin (Figure 4A). Quantitative assay of hepatic lipids demonstrated that alcohol exposure significantly increased the concentrations of triglycerides, cholesterol, and FFAs in the liver (Figure 4B). Leptin administration normalized the concentrations of hepatic triglycerides and FFAs, but did not affect the alcohol-elevated cholesterol concentration. To exclude the possibility that leptin may affect liver fibrogenesis, the hepatic collagen level was assessed using Sirius Red staining. Neither alcohol feeding alone nor alcohol feeding plus leptin administration caused significant collagen accumulation in the liver (see Supplemental Figure S1 at http://ajp.amjpathol.org). The relative mRNA levels of hepatic genes related to lipid metabolism are given in Table 3. Among three fatty acid transport genes, FABP1 was down-regulated by alcohol regardless of leptin administration, whereas FATP2 was up-regulated by leptin. Alcohol exposure down-regulated all three genes related to fatty acid β-oxidation, CPT1a, ACADL, and ACOX1, which was normalized by leptin. Leptin administration also normalized alcohol-down-regulated very low-density lipoprotein assembly genes MTTP and ApoB. Among the five transcription factors related to lipid metabolism regulation, alcohol exposure down-regulated C/EBP-α, HNF-1α, PPAR-α, and PPAR-γ, and leptin normalized the expression levels of the first three but not PPAR-γ. The HNF-4α gene was not affected by either alcohol or leptin. Immunoblot analysis demonstrated that leptin administration reversed alcohol-reduced protein levels of PPAR-α and HNF-1α (Figure 5A). However, the protein level of C/EBP-α was not affected by alcohol regardless of leptin administration. Immunoblot analysis also showed that the phosphorylation levels of Stat3 and AMPK, the major molecules mediating leptin signaling, were reduced after alcohol exposure, but were normalized by leptin administration (Figure 5B).Table 3Effects of Leptin Administration on Hepatic Genes Related to Lipid MetabolismMeasurementsPFAFAF/LeptinFatty acid transport FABP11.006 ± 0.130.41 ± 0.13⁎P < 0.05 versus PF (ANOVA).0.33 ± 0.09⁎P < 0.05 versus PF (ANOVA). FATP21.005 ± 0.120.98 ± 0.291.50 ± 0.29⁎P < 0.05 versus PF (ANOVA).†P < 0.05 versus AF (ANOVA). FATP51.003 ± 0.100.98 ± 0.101.23 ± 0.17Fatty acid β-oxidation CPT1a1.001 ± 0.050.53 ± 0.02⁎P < 0.05 versus PF (ANOVA).1.06 ± 0.27†P < 0.05 versus AF (ANOVA). ACADL1.009 ± 0.160.70 ± 0.15⁎P < 0.05 versus PF (ANOVA).1.007 ± 0.11†P < 0.05 versus AF (ANOVA). ACOX11.001 ± 0.050.58 ± 0.14⁎P < 0.05 versus PF (ANOVA).0.86 ± 0.26†P < 0.05 versus AF (ANOVA).VLDL assembly MTTP1.04 ± 0.350.64 ± 0.09⁎P < 0.05 versus PF (ANOVA).0.71 ± 0.11 ApoB1.003 ± 0.100.76 ± 0.02⁎P < 0.05 versus PF (ANOVA).1.17 ± 0.20†P < 0.05 versus AF (ANOVA).Transcription factors C/EBP-α1.004 ± 0.110.67 ± 0.02⁎P < 0.05 versus PF (ANOVA).0.93 ± 0.08†P < 0.05 versus AF (ANOVA). HNF-1α1.008 ± 0.150.56 ± 0.12⁎P < 0.05 versus PF (ANOVA).0.84 ± 0.12†P < 0.05 versus AF (ANOVA). HNF-4α1.01 ± 0.211.07 ± 0.151.27 ± 0.27 PPAR-α1.004 ± 0.110.62 ± 0.09⁎P < 0.05 versus PF (ANOVA).0.93 ± 0.05†P < 0.05 versus AF (ANOVA). PPAR-γ1.007 ± 0.140.27 ± 0.16⁎P < 0.05 versus PF (ANOVA).0.16 ± 0.06⁎P < 0.05 versus PF (ANOVA).Mice were pair-fed alcohol on isocaloric maltose dextran for 8 weeks in the presence or absence of external leptin for the last 2 weeks.Gene expression was analyzed using quantitative RT-PCR.Data are given as mean ± SD (n = 4).AF, alcohol-fed; ANOVA, analysis of variance; PF, pair-fed; VLDL, very low-density lipoprotein. P < 0.05 versus PF (ANOVA).† P < 0.05 versus AF (ANOVA). Open table in a new tab Mice were pair-fed alcohol on isocaloric maltose dextran for 8 weeks in the presence or absence of external leptin for the last 2 weeks. Gene expression was analyzed using quantitative RT-PCR. Data are given as mean ± SD (n = 4). AF, alcohol-fed; ANOVA, analysis of variance; PF, pair-fed; VLDL, very low-density lipoprotein. Alcohol consumption has been shown to modulate plasma adipokine levels, in particular adiponectin. Alcohol exposure in mice decreased the serum adiponectin concentration in a time-dependent manner, whereas administration with mouse recombinant adiponectin attenuated alcohol-induced fatty liver and inflammation.18Xu A. Wang Y. Keshaw H. Xu L.Y. Lam K.S. Cooper G.J. The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice.J Clin Invest. 2003; 112: 91-100Crossref PubMed Scopus (1150) Google Scholar The decrease in plasma adiponectin concent
Referência(s)