Adverse Signaling of Scavenger Receptor Class B1 and PGC1s in Alcoholic Hepatosteatosis and Steatohepatitis and Protection by Betaine in Rat
2014; Elsevier BV; Volume: 184; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2014.03.005
ISSN1525-2191
AutoresRavi Varatharajalu, Mamatha Garige, Leslie C. Leckey, Jaime Arellanes‐Robledo, Karina Reyes‐Gordillo, Ruchi Shah, M Lakshman,
Tópico(s)Diet, Metabolism, and Disease
ResumoBecause scavenger receptor class B type 1 is the cholesterol uptake liver receptor, whereas peroxisome proliferator–activated receptor γ coactivator-1β (PGC-1β) and PGC-1α are critical for lipid synthesis and degradation, we investigated the roles of these signaling molecules in the actions of ethanol-polyunsaturated fatty acids and betaine on hepatosteatosis and steatohepatitis. Ethanol-polyunsaturated fatty acid treatment caused the following: i) hepatosteatosis, as evidenced by increased liver cholesterol and triglycerides, lipid score, and decreased serum adiponectin; ii) marked inhibition of scavenger receptor class B type 1 glycosylation, its plasma membrane localization, and its hepatic cholesterol uptake function; and iii) moderate steatohepatitis, as evidenced by histopathological characteristics, increased liver tumor necrosis factor α and IL-6, decreased glutathione, and elevated serum alanine aminotransferase. These actions of ethanol involved up-regulated PGC-1β, sterol regulatory element-binding proteins 1c and 2, acetyl-CoA carboxylase, and HMG-CoA reductase mRNAs/proteins and inactive non-phosphorylated AMP kinase; and down-regulated silence regulator gene 1 and PGC-1α mRNA/proteins and hepatic fatty acid oxidation. Betaine markedly blunted all these actions of ethanol on hepatosteatosis and steatohepatitis. Therefore, we conclude that ethanol-mediated impaired post-translational modification, trafficking, and function of scavenger receptor class B type 1 may account for alcoholic hyperlipidemia. Up-regulation of PGC-1β and lipid synthetic genes and down-regulation of silence regulator gene 1, PGC-1α, adiponectin, and lipid degradation genes account for alcoholic hepatosteatosis. Induction of proinflammatory cytokines and depletion of endogenous antioxidant, glutathione, account for alcoholic steatohepatitis. We suggest betaine as a potential therapeutic agent because it effectively protects against adverse actions of ethanol. Because scavenger receptor class B type 1 is the cholesterol uptake liver receptor, whereas peroxisome proliferator–activated receptor γ coactivator-1β (PGC-1β) and PGC-1α are critical for lipid synthesis and degradation, we investigated the roles of these signaling molecules in the actions of ethanol-polyunsaturated fatty acids and betaine on hepatosteatosis and steatohepatitis. Ethanol-polyunsaturated fatty acid treatment caused the following: i) hepatosteatosis, as evidenced by increased liver cholesterol and triglycerides, lipid score, and decreased serum adiponectin; ii) marked inhibition of scavenger receptor class B type 1 glycosylation, its plasma membrane localization, and its hepatic cholesterol uptake function; and iii) moderate steatohepatitis, as evidenced by histopathological characteristics, increased liver tumor necrosis factor α and IL-6, decreased glutathione, and elevated serum alanine aminotransferase. These actions of ethanol involved up-regulated PGC-1β, sterol regulatory element-binding proteins 1c and 2, acetyl-CoA carboxylase, and HMG-CoA reductase mRNAs/proteins and inactive non-phosphorylated AMP kinase; and down-regulated silence regulator gene 1 and PGC-1α mRNA/proteins and hepatic fatty acid oxidation. Betaine markedly blunted all these actions of ethanol on hepatosteatosis and steatohepatitis. Therefore, we conclude that ethanol-mediated impaired post-translational modification, trafficking, and function of scavenger receptor class B type 1 may account for alcoholic hyperlipidemia. Up-regulation of PGC-1β and lipid synthetic genes and down-regulation of silence regulator gene 1, PGC-1α, adiponectin, and lipid degradation genes account for alcoholic hepatosteatosis. Induction of proinflammatory cytokines and depletion of endogenous antioxidant, glutathione, account for alcoholic steatohepatitis. We suggest betaine as a potential therapeutic agent because it effectively protects against adverse actions of ethanol. Cholesterol uptake by the liver is crucial for maintaining cholesterol homeostasis in peripheral tissues of mammals, as exemplified by early onset of atherosclerosis in familial hypercholesterolemia.1Hobbs H.H. Brown M.S. Goldstein J.L. 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The effect of betaine in reversing alcoholic steatosis.Alcohol Clin Exp Res. 1997; 21: 1100-1102Crossref PubMed Scopus (82) Google Scholar and has been reported to restore the decreased liver glutathione (GSH) level in ethanol-treated guinea pigs.27Balkan J. Oztezcan S. Kucuk M. Cevikbas U. Kocak-Toker N. Uysal M. The effect of betaine treatment on triglyceride levels and oxidative stress in the liver of ethanol-treated guinea pigs.Exp Toxicol Pathol. 2004; 55: 505-509Crossref PubMed Scopus (59) Google Scholar Therefore, it is reasonable that betaine may prevent the deleterious effects of heavy alcohol and high omega-3 polyunsaturated fatty acids (ω-3 PUFAs) on SR-B1, plasma lipids, and hepatic lipid metabolizing pathway, and lipid homeostasis by altering hepatic GSH and reactive oxygen species (ROS). In view of the previous descriptions, we have explored the possible action of chronic alcohol/high PUFA fish oil diet and the protection by betaine on the following: i) hepatic SR-B1 expression, relative glycosylation rate, and its localization on the plasma membrane; ii) the expression of various lipogenic and lipid-oxidizing signaling pathway genes and activities, and hepatic lipid status versus serum adiponectin; and iii) liver injury status, as evidenced by depletion of hepatic endogenous antioxidant, GSH, proinflammatory cytokines, tumor necrosis factor α (TNFα) and IL-6, and serum alanine aminotransferase (ALT). Experimental animals were female Wistar rats (130 to 150 g) (Charles River Laboratories, Inc., Wilmington, MA) housed in groups of two per cage in plastic cages (40 × 24 × 18 cm), in a temperature-controlled room at 25°C with a 12-hour light-dark cycle. Experiments were performed according to the approved Institutional Animal Care and Use Committee protocol. After the first week of acclimation on a pelleted commercial diet (Purina Rodent Chow, number 500; TMI Nutrition, St. Louis, MO), the animals were randomly divided into the following three groups, and pair fed their respective high-fat diets (Dyets Inc., Bethlehem, PA). The high-fat control (HFC) group had a high menhaden fish oil liquid diet (35% of total calories came from ω-3 PUFA); the high-fat ethanol (HFE) group had 35% ethanol calories in place of carbohydrate; and in the HFEB group, HFE was supplemented with 10 g betaine/L of the diet for 8 weeks. This amounted to a consumption of 3.5 g betaine per kg body weight per day on the basis of daily consumption of approximately a 70 mL diet per rat per day. This dosage of betaine was chosen because of its beneficial effects in many alcoholic steatosis studies.28Graf D. Kurz A.K. Reinehr R. Fischer R. Kircheis G. Häussinger D. Prevention of bile acid-induced apoptosis by betaine in rat liver.Hepatology. 2002; 36: 829-839Crossref PubMed Scopus (81) Google Scholar, 29Barak A.J. Beckenhauer H.C. Junnila M. Tuma D.J. Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration.Alcohol Clin Exp Res. 1993; 17: 552-555Crossref PubMed Scopus (167) Google Scholar Liver cholesterol and triglycerides were determined using enzymatic reagents, as described by the manufacturer (Wako Pure Chemicals, Richmond, VA), adapted to a microtiter plate assay.30Garige M. Gong M. Varatharajalu R. Lakshman M.R. Quercetin up-regulates paraoxonase 1 gene expression via sterol regulatory element binding protein 2 that translocates from the endoplasmic reticulum to the nucleus where it specifically interacts with sterol responsive element-like sequence in paraoxonase 1 promoter in HuH7 liver cells.Metabolism. 2010; 59: 1372-1378Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 31Shireman R.B. Durieux J. Microplate methods for determination of serum cholesterol, high density lipoprotein cholesterol, triglyceride and apolipoproteins.Lipids. 1993; 28: 151-155Crossref PubMed Scopus (19) Google Scholar For histological examination, paraffin-embedded sections (5 μm thick) were processed by routine histological protocol for H&E staining. Commercially available enzyme-linked immunosorbent assay kits specific to rat liver TNFα, IL-6, and adiponectin (R&D Systems, Inc., Minneapolis, MN) were used to determine these parameters according to manufacturer's instructions, except that the plates were incubated overnight at 4°C to completely capture the respective target proteins. Standard curves for the respective rat recombinant cytokines were included in each assay; all samples from each group were assayed in triplicate. In the initial experiments, we determined that the optimal concentration of liver post-mitochondrial protein for the assay of IL-6 was 250 μg per well, whereas it was 100 μg per well for the assay of TNFα. To measure circulating adiponectin in plasma, we diluted each sample 1000 times in the diluent provided by the manufacturer. The ALT assay kit was used to measure plasma ALT activity, as per the manufacturer's instructions (Teco Diagnostic, Anaheim, CA). HDL was isolated from pooled HFC or HFE rat plasma, according to Gidez et al.32Gidez L.I. Miller G.J. Burstein M. Slagle S. Eder H.A. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure.J Lipid Res. 1982; 23: 1206-1223Abstract Full Text PDF PubMed Google Scholar Protein concentration was determined colorimetrically.33Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar HDL cholesterol content was measured according to Zlatkis and Zak.34Zlatkis A. Zak B. Study of a new cholesterol reagent.Anal Biochem. 1969; 29: 143-148Crossref PubMed Scopus (332) Google Scholar HDL labeling with [3H] cholesteryl oleate was performed according to Basu et al,35Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts.Proc Natl Acad Sci U S A. 1976; 73: 3178-3182Crossref PubMed Scopus (823) Google Scholar and the specific activity is expressed as dpm/mg HDL cholesterol. Hepatocytes were isolated from the HFC, HFE, and HFEB groups, and their cholesterol uptake capacity [3H] cholesteryl oleate–labeled HDL was determined, as we described.36Lakshman M.R. Muesing R.A. LaRosa J.C. Regulation of cholesterol biosynthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by chylomicron remnants in isolated hepatocytes and perfused liver.J Biol Chem. 1981; 256: 3037-3043PubMed Google Scholar, 37Marmillot P. Munoz J. Patel S. Garige M. Rosse R.B. Lakshman M.R. Long-term ethanol consumption impairs reverse cholesterol transport function of high-density lipoproteins by depleting high-density lipoprotein sphingomyelin both in rats and in humans.Metabolism. 2007; 56: 947-953Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar Similarly, the relative hepatic glycosylation rates of SR-B1 in various groups were determined as we described.12Ghosh P. Lakshman M.R. Chronic ethanol induced impairment of hepatic glycosylation machinery in rat is independent of dietary carbohydrate.Alcohol Clin Exp Res. 1997; 21: 76-81Crossref PubMed Scopus (12) Google Scholar Briefly, the hepatocytes (approximately 100 mg wet wt./mL; 10 mL final volume) from the respective groups were incubated for 120 minutes with either 100 μCi of [3H] ManNAc or 20 μCi of [35S]methionine, and the incorporations of [3H]ManNAc relative to that of [35S]methionine into immunoprecipitable SR-B1 were determined. The total RNA was isolated from each liver using the Tri-Reagent (Molecular Research Center, Cincinnati, OH), following the manufacturer's instructions, as we described.38Gong M. Castillo L. Redman R.S. Garige M. Hirsch K. Azuine M. Amdur R.L. Seth D. Haber P.S. Lakshman M.R. Down-regulation of liver Galbeta1, 4GlcNAc alpha2, 6-sialyltransferase gene by ethanol significantly correlates with alcoholic steatosis in humans.Metabolism. 2008; 57: 1663-1668Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar Isolated RNA was used immediately or stored at −80°C until use. PGC-1α, PGC-1β, SREBP1c, acetyl-CoA carboxylase (ACC), SIRT1, carnitine palmitoyltransferase (CPT)-1, and β-actin mRNAs were measured by real-time PCR using their corresponding primer pairs, as we described previously,39Luna L. Manual of Histologic Staining Methods of the Armed Forces Institutes of Pathology. ed 3. McGraw-Hill, New York1968Google Scholar using the housekeeping gene, β-actin (Ambion, Austin, TX), as the internal control. The relative expression level of the specific gene of interest was calculated by subtracting the CT of the β-actin gene from the CT of the gene of interest and raising 2 to the power of this difference. CT values are defined as the number of PCR cycles at which the fluorescence signal during the PCR reaches a fixed threshold. Immunohistochemistry of all liver sections was performed as we described,39Luna L. Manual of Histologic Staining Methods of the Armed Forces Institutes of Pathology. ed 3. McGraw-Hill, New York1968Google Scholar except that the sections were incubated overnight at 4°C with goat anti–SR-B1 (Novus Biologicals, Littleton, CO) at 1:500 dilution, and mouse anti-Na+/K+ATPase1, a plasma membrane marker, at 1:200 dilution, in 1% bovine serum albumin/PBS with Tween 20 (Abcam, Cambridge, MA). The liver sections were mounted with Prolong Gold Antifade mounting medium (Life Technologies, Carlsbad, CA). Confocal microscopy images were obtained with a Zeiss LSM 710 Zeiss Confocal microscope (Carl Zeiss Micro-Imaging, Inc., Thornwood, NY). Digital imaging software (Axio Vision Rel version 4.8.2; Carl Zeiss Micro-Imaging, Inc.) was used to generate the colocalization measurements using Pearson's correlation coefficient, describing the correlation of intensity distribution, and colocalization, which is denoted as the percentage of relative area of SR-B1 colocalized with Na+/K+ATPase1. The indicated proteins of interest were determined in liver extracts and subcellular fractions from various groups by using Western blot analyses, as we described,39Luna L. Manual of Histologic Staining Methods of the Armed Forces Institutes of Pathology. ed 3. McGraw-Hill, New York1968Google Scholar and the following primary antibodies were used: PGC-1α, ACC, phosphorylated (p)ACC, AMP kinase (AMPK), and pAMPK (Cell Signaling Technology, Inc., Danvers, MA), SREBP1c (Abcam), SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA), PGC-1β (Genway Biotech, San Diego, CA), and SR-B1 (Novus Biologicals). Lamin B1 (Abcam), β-actin (Sigma, St. Louis, MO), and calreticulin (Abcam) were used as the housekeeping nuclear, cytosolic, and endoplasmic reticulum proteins, respectively, to normalize the results. The specificity of each antibody was verified before use for the previously described analyses. Caspase 3 activity in liver lysate was measured with a colorimetric assay kit that relies on caspase-mediated cleavage of para-nitroanilide from a synthetic caspase-specific substrate peptide containing amino acid motif. The kit was used in accordance with the manufacturer's guidelines (R&D Systems Inc.). Liver mitochondria integrity was assessed by measuring its component enzyme, citrate synthase (CS) activity, in various groups, according to Srere et al,40Srere P.A. Bhaduri A. The citrate cleavage enzyme, III: citryl coenzyme a as a substrate and the stereospecificity of the enzyme.J Biol Chem. 1964; 239: 714-718PubMed Google Scholar using 5,5′-dithiobis-(2-nitrobenzoic acid) in a 96-well plate of 0.1-cm light path with a SpectroMAX 190 spectrophotometer (Molecular Devices Co, Sunnyvale, CA). Liver GSH was determined in samples from various groups using the GSH assay kit (Sigma) at 412 nm using a SpectroMAX 190 spectrophotometer. Fresh livers from experimental groups were immediately homogenized in ice-cold buffer containing 10 mmol/L HEPES (pH 7.4), 250 mmol/L mannitol, 75 mmol/L sucrose, 100 μmol/L EDTA, 500 μmol/L EGTA, and protease inhibitor cocktail (Sigma). Cell lysates were centrifuged at 1000 × g for 10 minutes to remove nuclear pellets. A supernatant fraction was centrifuged at 10,000 × g for 15 minutes to isolate the mitochondrial pellet. This pellet was washed twice in homogenizing buffer and then solubilized in the same buffer. The supernatant solution was centrifuged at 100,000 × g for 1 hour to collect the microsomal pellet. The protein estimation was performed with the Bradford method (Bio-Rad, Hercules, CA). All data are expressed as means ± SD using Microsoft Excel 2010 software (Microsoft Inc., Redmond, WA) and the significance of variance (analysis of variance), followed by the Tukey's test.41SAS InstituteSAS/STAT User's Guide, Version 6. SAS Institute, Cary, NC2000Google Scholar Histopathological analysis revealed a remarkable fat accumulation in the HFE group, but no visible hepatosteatosis was observed in other groups (Figure 1A). Liver cholesterol and triglycerides increased markedly by 258% (P < 0.01) (Figure 1B) and 186% (P < 0.01) (Figure 1C), respectively, in the HFE group, with a concomitant 260% (P < 0.01) increase in hepatic lipid score (Figure 1, D and E), which were significantly blunted in the HFEB groups. Plasma adiponectin level was significantly decreased by 37% in the HFE group compared with the HFC group (P < 0.01), whereas its level was markedly increased by 27% in the HFEB group, which was similar to the HFC group (Figure 2A). We next investigated the protective effect of betaine on hepatic proinflammatory cytokines: both IL-6 and TNFα showed an increasing trend in the HFE group compared with the HFC group (Figure 2, B and C); however, these differences were not statistically significant. Surprisingly, betaine significantly lowered both IL-6 and TNFα by 18% (Figure 2B) and 40% (Figure 2C), respectively, compared with the HFE group (P < 0.01). Concomitantly, compared with the HFC group, plasma ALT activity was significantly increased by 86% (P < 0.01) in the HFE group, which was significantly lowered by 70% (P < 0.01) in the HFEB group (Figure 2D). SR-B1 mRNA expression was markedly decreased by 58% (P < 0.05) in the HFE group compared with the HFC group, whereas it was increased by 56% (P < 0.05) in the HFEB group compared with the HFE group (Figure 3A). The mature 82-kDa SR-B1 species in plasma membrane was decreased markedly by 43% (P < 0.05) in the HFE group, which was restored nearly to the control level in the HFEB group (Figure 3B). There was a reciprocal increase in the 57-kDa unglycosylated SR-B1 species in HFE that was restored nearly to the control level in the HFEB group. In immunoprecipitated extracts of hepatocytes, the incorporation of labeled N-AcMnNH2 relative to labeled methionine incorporation into newly synthesized SR-B1 was decreased by 42% (P < 0.01) in the HFE group, which was restored nearly to the control level in the HFEB group (Figure 3C). Compared with HFC, the uptake of cholesterol by hepatocytes from the HFE group was significantly inhibited by 48% (P < 0.05), whereas it was stimulated to 154% (P < 0.001) in the HFEB group (Figure 3D). The intensity of the colocalized SR-B1 with ATPase in the plasma membrane was markedly decreased by 34% (P < 0.05) in the HFE group compared with the HFC group, which was completely reversed in the HFEB group (Figure 4). Liver mitochondrial cholesterol (Figure 5A) was markedly elevated by 61% (P < 0.01), accompanied by a significant 34% (P < 0.05) decrease in mitochondrial GSH (Figure 5B) in the HFE group, which was partially prevented in the HFEB group. Furthermore, the relative leakage of cytochrome c (Figure 5C) into the cytosolic fraction was increased by 30% (P < 0.05), accompanied by 75% (P < 0.01) elevation of caspase 3 activity in the HFE group (Figure 5D), which were partially blocked in the HFEB group (P < 0.05). Compared with the HFC group, liver CS activity in the HFE group (Figure 5E) was significantly decreased by 41% (P < 0.01), which was significantly restored in the HFEB group (P < 0.05). PGC-1β mRNA expression was significantly increased in the HFE group (Figure 6A) by 1.6-fold (P < 0.01) compared with the HFC group, which was partia
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