Amelioration of diet-induced steatohepatitis in mice following combined therapy with ASO-Fsp27 and fenofibrate
2017; Elsevier BV; Volume: 58; Issue: 11 Linguagem: Inglês
10.1194/jlr.m077941
ISSN1539-7262
AutoresAnanthi Rajamoorthi, N. Arias, Jeannine Basta, Richard Lee, Ángel Baldán,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoNonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. NAFLD progresses from benign steatosis to steatohepatitis (NASH) to cirrhosis and is linked to hepatocellular carcinoma. No targeted treatment is currently approved for NAFLD/NASH. We previously showed that fat-specific protein 27 (FSP27), a lipid droplet-associated protein that controls triglyceride turnover in the hepatocyte, is required for fasting- and diet-induced triglyceride accumulation in the liver. However, silencing Fsp27 with antisense oligonucleotides (ASOs) did not improve hepatosteatosis in genetic nor nutritional mouse models of obesity. Herein, we tested the therapeutic potential of ASO-Fsp27 when used in combination with the PPARα agonist fenofibrate. C57BL/6 mice were fed a high-trans-fat, high-cholesterol, high-fructose diet for eight weeks to establish NASH, then kept on diet for six additional weeks while dosed with ASOs and fenofibrate, alone or in combination. Data show that ASO-Fsp27 and fenofibrate synergize to promote resistance to diet-induced obesity and hypertriglyceridemia and to reverse hepatic steatosis, inflammation, oxidative stress, and fibrosis. This multifactorial improvement of liver disease noted when combining both drugs suggests that a course of treatment that includes both reduced FSP27 activity and activation of PPARα could provide therapeutic benefit to patients with NAFLD/NASH. Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease. NAFLD progresses from benign steatosis to steatohepatitis (NASH) to cirrhosis and is linked to hepatocellular carcinoma. No targeted treatment is currently approved for NAFLD/NASH. We previously showed that fat-specific protein 27 (FSP27), a lipid droplet-associated protein that controls triglyceride turnover in the hepatocyte, is required for fasting- and diet-induced triglyceride accumulation in the liver. However, silencing Fsp27 with antisense oligonucleotides (ASOs) did not improve hepatosteatosis in genetic nor nutritional mouse models of obesity. Herein, we tested the therapeutic potential of ASO-Fsp27 when used in combination with the PPARα agonist fenofibrate. C57BL/6 mice were fed a high-trans-fat, high-cholesterol, high-fructose diet for eight weeks to establish NASH, then kept on diet for six additional weeks while dosed with ASOs and fenofibrate, alone or in combination. Data show that ASO-Fsp27 and fenofibrate synergize to promote resistance to diet-induced obesity and hypertriglyceridemia and to reverse hepatic steatosis, inflammation, oxidative stress, and fibrosis. This multifactorial improvement of liver disease noted when combining both drugs suggests that a course of treatment that includes both reduced FSP27 activity and activation of PPARα could provide therapeutic benefit to patients with NAFLD/NASH. Nonalcoholic fatty liver disease (NAFLD) is behind most cases of chronic liver disease in adults and children in Western societies (reviewed in Ref. 1Fazel Y. Koenig A.B. Sayiner M. Goodman Z.D. Younossi Z.M. Epidemiology and natural history of non-alcoholic fatty liver disease.Metabolism. 2016; 65: 1017-1025Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 2Lomonaco R. Sunny N.E. Bril F. Cusi K. Nonalcoholic fatty liver disease: current issues and novel treatment approaches.Drugs. 2013; 73: 1-14Crossref PubMed Scopus (121) Google Scholar). NAFLD is a spectrum of liver disorders in the absence of significant alcohol intake that ranges from benign steatosis to steatohepatitis (NASH) to cirrhosis. NASH is characterized by hepatocyte ballooning and triglyceride (TAG) accumulation, inflammation, oxidative stress, and collagen deposition. Up to 25% of NASH patients develop hepatocellular carcinoma (1Fazel Y. Koenig A.B. Sayiner M. Goodman Z.D. Younossi Z.M. Epidemiology and natural history of non-alcoholic fatty liver disease.Metabolism. 2016; 65: 1017-1025Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 2Lomonaco R. Sunny N.E. Bril F. Cusi K. Nonalcoholic fatty liver disease: current issues and novel treatment approaches.Drugs. 2013; 73: 1-14Crossref PubMed Scopus (121) Google Scholar). Liver failure from NAFLD and its complications are the third most common cause for liver transplantation and the twelfth leading cause of death in the United States. Together with dyslipidemia, central obesity, hypertension, and insulin resistance, NAFLD is a component of the metabolic syndrome, which confers increased risk for CVD (2Lomonaco R. Sunny N.E. Bril F. Cusi K. Nonalcoholic fatty liver disease: current issues and novel treatment approaches.Drugs. 2013; 73: 1-14Crossref PubMed Scopus (121) Google Scholar). Several epidemiological studies suggest that up to 30% of Americans have some degree of NAFLD, but in obese, diabetic patients, these statistics climb to up to 80% (1Fazel Y. Koenig A.B. Sayiner M. Goodman Z.D. Younossi Z.M. Epidemiology and natural history of non-alcoholic fatty liver disease.Metabolism. 2016; 65: 1017-1025Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 2Lomonaco R. Sunny N.E. Bril F. Cusi K. Nonalcoholic fatty liver disease: current issues and novel treatment approaches.Drugs. 2013; 73: 1-14Crossref PubMed Scopus (121) Google Scholar). Most projections suggest that the prevalence of NAFLD will continue to increase over the next few decades. Despite the growing threat of NAFLD, there remains a substantial lack of therapeutic tools to manage these patients. Fat-specific protein 27 (FSP27), also known as CIDEC (cell death-inducing DFFA-like effector C) in humans, is a member of the CIDE family of lipid droplet-associated proteins that localizes to the surface of lipid droplet (LD) contact sites and promotes the formation of large, unilocular LDs in adipocytes by mediating the directional net transfer of lipids from small to large LDs. The three CIDE genes have distinct tissue distribution: CIDEA is abundant in brown adipose tissue, CIDEB in liver, and CIDEC in both white and brown adipose tissue (3Xu L. Zhou L. Li P. CIDE proteins and lipid metabolism.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1094-1098Crossref PubMed Scopus (127) Google Scholar, 4Yonezawa T. Kurata R. Kimura M. Inoko H. Which CIDE are you on? Apoptosis and energy metabolism.Mol. Biosyst. 2011; 7: 91-100Crossref PubMed Google Scholar). Overexpression of Fsp27 promotes the accumulation of larger LDs in several cell lines (5Yu S. Matsusue K. Kashireddy P. Cao W.Q. Yeldandi V. Yeldandi A.V. Rao M.S. Gonzalez F.J. Reddy J.K. Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor gamma1 (PPARgamma1) overexpression.J. Biol. Chem. 2003; 278: 498-505Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 6Danesch U. Hoeck W. Ringold G.M. Cloning and transcriptional regulation of a novel adipocyte-specific gene, FSP27. CAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression.J. Biol. Chem. 1992; 267: 7185-7193Abstract Full Text PDF PubMed Google Scholar, 7Keller P. Petrie J.T. De Rose P. Gerin I. Wright W.S. Chiang S.H. Nielsen A.R. Fischer C.P. Pedersen B.K. MacDougald O.A. Fat-specific protein 27 regulates storage of triacylglycerol.J. Biol. Chem. 2008; 283: 14355-14365Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 8Gong J. Sun Z. Wu L. Xu W. Schieber N. Xu D. Shui G. Yang H. Parton R.G. Li P. Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites.J. Cell Biol. 2011; 195: 953-963Crossref PubMed Scopus (233) Google Scholar). Conversely, shRNA-mediated knockdown of Fsp27 decreases LD size (9Puri V. Konda S. Ranjit S. Aouadi M. Chawla A. Chouinard M. Chakladar A. Czech M.P. Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage.J. Biol. Chem. 2007; 282: 34213-34218Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Interestingly, FSP27 is detected in fatty, but not in normal, livers (3Xu L. Zhou L. Li P. CIDE proteins and lipid metabolism.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1094-1098Crossref PubMed Scopus (127) Google Scholar, 10Langhi C. Baldan A. CIDEC/FSP27 is regulated by peroxisome proliferator-activated receptor alpha and plays a critical role in fasting- and diet-induced hepatosteatosis.Hepatology. 2015; 61: 1227-1238Crossref PubMed Scopus (54) Google Scholar), and we showed that FSP27 promotes TAG accumulation in the hepatocyte under conditions of fasting and diet-induced hepatosteatosis and is under the transcriptional control of PPARα (10Langhi C. Baldan A. CIDEC/FSP27 is regulated by peroxisome proliferator-activated receptor alpha and plays a critical role in fasting- and diet-induced hepatosteatosis.Hepatology. 2015; 61: 1227-1238Crossref PubMed Scopus (54) Google Scholar). Chow-fed Fsp27−/− mice are lean, show small LDs in adipose tissue, and have lower plasma glucose and leptin, resulting in enhanced insulin sensitivity and resistance to diet-induced obesity (11Nishino N. Tamori Y. Tateya S. Kawaguchi T. Shibakusa T. Mizunoya W. Inoue K. Kitazawa R. Kitazawa S. Matsuki Y. et al.FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets.J. Clin. Invest. 2008; 118: 2808-2821PubMed Google Scholar, 12Toh S.Y. Gong J. Du G. Li J.Z. Yang S. Ye J. Yao H. Zhang Y. Xue B. Li Q. et al.Up-regulation of mitochondrial activity and acquirement of brown adipose tissue-like property in the white adipose tissue of fsp27 deficient mice.PLoS One. 2008; 3: e2890Crossref PubMed Scopus (215) Google Scholar). Finally, a partially lipodystrophic patient was identified who carries a homozygous nonsense mutation in FSP27 (13Rubio-Cabezas O. Puri V. Murano I. Saudek V. Semple R.K. Dash S. Hyden C.S. Bottomley W. Vigouroux C. Magre J. et al.Partial lipodystrophy and insulin resistant diabetes in a patient with a homozygous nonsense mutation in CIDEC.EMBO Mol. Med. 2009; 1: 280-287Crossref PubMed Scopus (206) Google Scholar). The fact that hepatic FSP27 is induced under pathologic conditions and correlates with lipid accumulation (3Xu L. Zhou L. Li P. CIDE proteins and lipid metabolism.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1094-1098Crossref PubMed Scopus (127) Google Scholar, 5Yu S. Matsusue K. Kashireddy P. Cao W.Q. Yeldandi V. Yeldandi A.V. Rao M.S. Gonzalez F.J. Reddy J.K. Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor gamma1 (PPARgamma1) overexpression.J. Biol. Chem. 2003; 278: 498-505Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 10Langhi C. Baldan A. CIDEC/FSP27 is regulated by peroxisome proliferator-activated receptor alpha and plays a critical role in fasting- and diet-induced hepatosteatosis.Hepatology. 2015; 61: 1227-1238Crossref PubMed Scopus (54) Google Scholar, 14Aibara D. Matsusue K. Matsuo K. Takiguchi S. Gonzalez F.J. Yamano S. Expression of hepatic fat-specific protein 27 depends on the specific etiology of fatty liver.Biol. Pharm. Bull. 2013; 36: 1766-1772Crossref PubMed Scopus (13) Google Scholar, 15Matsusue K. Kusakabe T. Noguchi T. Takiguchi S. Suzuki T. Yamano S. Gonzalez F.J. Hepatic steatosis in leptin-deficient mice is promoted by the PPARgamma target gene Fsp27.Cell Metab. 2008; 7: 302-311Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) suggests that FSP27 might be a therapeutic target for NAFLD/NASH patients. Consistent with that proposal, hepatocyte-specific Fsp27−/− mice are protected from diet-induced hepatosteatosis (16Wang W. Xu M.J. Cai Y. Zhou Z. Cao H. Mukhopadhyay P. Pacher P. Zheng S. Gonzalez F.J. Gao B. Inflammation is independent of steatosis in a murine model of steatohepatitis.Hepatology. 2017; 66: 108-123Crossref PubMed Scopus (44) Google Scholar), and a recent study showed that hepatic TAG contents were reduced in mice fed a high-fat diet (HFD) following a single infusion of an adenoviral-encoded shRNA against Fsp27 (10Langhi C. Baldan A. CIDEC/FSP27 is regulated by peroxisome proliferator-activated receptor alpha and plays a critical role in fasting- and diet-induced hepatosteatosis.Hepatology. 2015; 61: 1227-1238Crossref PubMed Scopus (54) Google Scholar, 16Wang W. Xu M.J. Cai Y. Zhou Z. Cao H. Mukhopadhyay P. Pacher P. Zheng S. Gonzalez F.J. Gao B. Inflammation is independent of steatosis in a murine model of steatohepatitis.Hepatology. 2017; 66: 108-123Crossref PubMed Scopus (44) Google Scholar). Interestingly, this latter effect was enhanced by concomitant gavage with the synthetic PPARα agonist Wy14643 (10Langhi C. Baldan A. CIDEC/FSP27 is regulated by peroxisome proliferator-activated receptor alpha and plays a critical role in fasting- and diet-induced hepatosteatosis.Hepatology. 2015; 61: 1227-1238Crossref PubMed Scopus (54) Google Scholar). To test the therapeutic potential of FSP27-based therapies on the regression of preexistent NASH, herein we defined the consequences of long-term therapeutic silencing of Fsp27 using generation 2.0 antisense oligonucleotides (ASOs), alone or in combination with the PPARα agonist fenofibrate, on the progression of diet-induced steatohepatitis. We hypothesized that PPARα-mediated increase in fatty acid oxidation is more efficient in promoting the hepatic clearance of lipids when used in combination with agents that prevent the storage of TAG in lipid droplets. C57BL/6 mice were dosed with ASOs, fenofibrate, or both after an 8-week feeding regimen with a high-trans-fat, high-cholesterol, high-fructose NASH diet, which was recently shown to induce hepatic steatosis, hepatocyte ballooning, inflammation, and fibrosis in mice (17Savard C. Tartaglione E.V. Kuver R. Haigh W.G. Farrell G.C. Subramanian S. Chait A. Yeh M.M. Quinn L.S. Ioannou G.N. Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis.Hepatology. 2013; 57: 81-92Crossref PubMed Scopus (191) Google Scholar, 18Neuschwander-Tetri B.A. Ford D.A. Acharya S. Gilkey G. Basaranoglu M. Tetri L.H. Brunt E.M. Dietary trans-fatty acid induced NASH is normalized following loss of trans-fatty acids from hepatic lipid pools.Lipids. 2012; 47: 941-950Crossref PubMed Scopus (40) Google Scholar, 19Tetri L.H. Basaranoglu M. Brunt E.M. Yerian L.M. Neuschwander-Tetri B.A. Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent.Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G987-G995Crossref PubMed Scopus (334) Google Scholar), better modeling the histopathological features noted in the livers of NASH patients. Our data show that silencing Fsp27 in the presence of fenofibrate results in the synergistic reduction of body weight, visceral adiposity, hepatic TAG accumulation, and liver inflammation, oxidative stress, and fibrosis, as well as changes in transcriptional programs regulating lipolysis, fatty acid utilization, and de novo lipogenesis in both liver and white adipose tissue (WAT). Importantly, we also report a major reduction in plasma VLDL-TAG upon silencing of Fsp27. Collectively, these data highlight the therapeutic potential of combination therapies that target both lipid storage and oxidation to manage NAFLD/NASH and reduce cardiovascular risk in patients. Chimeric 2′ methoxyethyl control (5′-CCTTCCCTGAAGGTTCCTCC) and anti-Fsp27 (5′-CAGACTCTAATACCATTCAC) oligonucleotides were synthesized and purified, as has been described (20Langhi C. Arias N. Rajamoorthi A. Basta J. Lee R.G. Baldan A. Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance.J. Lipid Res. 2017; 58: 81-91Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), suspended in saline, and stored at −20°C until used. A veterinarian at Saint Louis University filled a prescription for fenofibrate. Fenofibrate pills were pulverized, homogenized in saline, and used within 24 h. Animals were maintained in a 12-h/12-h light/dark cycle with ad libitum access to food and water. Six-week-old, male C57BL/6 mice (Jackson Laboratories, stock 000664) were fed NASH diet [Research Diets D09100301, containing 20% fat (65% trans-fat from Primex® shortening; 20% saturated fat from lard), 1.8% cholesterol, and 20% fructose] for 8 weeks, then kept on NASH diet or crossed over to standard chow diet (PicoLab 5353) for 6 additional weeks. Where indicated, mice were dosed with 25 mg/kg ASO-ctrl or ASO-Fsp27 [Monday and Thursday, intraperitoneally (IP)], and 100 μl vehicle or 40 mg/kg fenofibrate (daily, oral gavage). ASO dose was chosen on the basis of our previous work (20Langhi C. Arias N. Rajamoorthi A. Basta J. Lee R.G. Baldan A. Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance.J. Lipid Res. 2017; 58: 81-91Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Fenofibrate dose is 0.7 times the maximum recommended human dose equivalent for mice, based on milligram per square meter of surface area and did not elicit liver carcinomas in long-term preclinical studies in rodents (US Food and Drug Administration [FDA] Pharmacology Review for fenofibrate, available at accessdata.fda.gov). For each experimental condition, n = 7. See Fig. 1A for details. Mice were sacrificed between 8:00 AM and 9:00 AM following an overnight fasting. Body composition was measured by NMR using an LF50 Analyzer (Bruker BioSpin, Billerica, MA). Animal studies were approved by the IACUC at Saint Louis University. Livers and epididymal WAT (eWAT) were fixed in 10% formalin, postfixed in 50% ethanol, and embedded in paraffin blocks. Sections (4 μm) were processed for hematoxylin and eosin, picrosirius (Polysciences; Warminister, PA), or F4/80 (Bio-Rad MCA497GA; 1:150 dilution; Raleigh, NC) staining. Under polarized light microscopy, type I and type II collagen fibers appear bright yellow/orange and green, respectively, in picrosirius-stained micrographs, and were quantified using ImageJ software, as has been described (21Griffett K. Welch R.D. Flaveny C.A. Kolar G.R. Neuschwander-Tetri B.A. Burris T.P. The LXR inverse agonist SR9238 suppresses fibrosis in a model of non-alcoholic steatohepatitis.Mol. Metab. 2015; 4: 353-357Crossref PubMed Scopus (49) Google Scholar). F4/80-positive macrophages were stained as has been described (20Langhi C. Arias N. Rajamoorthi A. Basta J. Lee R.G. Baldan A. Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance.J. Lipid Res. 2017; 58: 81-91Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Adipocyte areas in hematoxylin/eosin-stained micrographs were calculated by using ImageJ software. Tissue lipids were extracted into chloroform by a modified Folch method and resolubilized in water, as has been described (22Allen R.M. Marquart T.J. Albert C.J. Suchy F.J. Wang D.Q. Ananthanarayanan M. Ford D.A. Baldan A. miR-33 controls the expression of biliary transporters, and mediates statin- and diet-induced hepatotoxicity.EMBO Mol. Med. 2012; 4: 882-895Crossref PubMed Scopus (137) Google Scholar). Specific lipid classes were quantified using enzymatic kits for triglycerides, total cholesterol, FFA, and phosphatidyl choline (Wako Chemicals, Richmond, VA). Results were normalized to total protein. Blood was collected from the inferior vena cava after an overnight fasting. Circulating lipids were quantified enzymatically in 5–20 μl of plasma with Wako kits. Ketone bodies were determined enzymatically in 10 μl of plasma with a kit from Cayman. Fast performance LC (FPLC) lipoprotein profiles from plasma samples were determined by a modified Column Lipoprotein Profile method. Briefly, plasma samples were pooled, diluted in saline (1:5 for cholesterol, 1:2 for triglycerides), and 40 μl injected into a Superose-6 column (GE Healthcare, Chicago, IL) by using elution buffer (saline, 2 mmol/l EDTA, 0.01% sodium azide, pH = 7.4) at 0.6 ml/min flow rate at 40°C. The eluate was mixed with cholesterol or TAG reagent (Pointe Scientific, Canton, MI) and incubated at 40°C in a 5-m KOT coiled reactor. The mixture entered a capillary spectrophotometer at 0.3 ml/min, and the signal was collected in real time using LC Solution software (Shimadzu, Kyoto, Japan). RNA was isolated from liver tissue with a Direct-zol RNA Miniprep kit (ZYMO Research, Irvine, CA) and from eWAT with RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA). The relative abundance of selected transcripts was determined by real-time quantitative PCR (qPCR), using PowerSybrGreen (Life Technologies, Carlsbad, CA) in a LightCycler LC480 instrument (Roche, Indianapolis, IN). Values were normalized to 36b4, and relative expression was calculated with the ΔΔCT method. Primer sets are available upon request. Liver proteins were extracted in 150 mmol/l NaCl, 1% NP-40, 0.1% SDS, 100 mmol/l Tris-HCl, pH 7.4, supplemented with protease inhibitors (Roche), and cleared by centrifugation at 4°C for 10 min at 10,000 g. Fifty micrograms of protein were resolved in either 4%–12% Bis-Tris or 3%–8% Tris-Acetate gels (Invitrogen), transferred to PVDF membranes, and probed with different primary and secondary antibodies in TBS-Tween20 containing 4% nonfat dry milk. Catalog numbers for each antibody and dilutions are provided in supplemental Table S1. Immune complexes were detected with SuperSignal West Pico chemiluminescent substrate (Pierce) and normalized to vinculin. Protein carbonylation was assessed in 20 μg of liver extract using the OxyBlot Protein Oxidation Detection kit (Millipore), following the manufacturer's instructions. Data are shown as mean ± SEM. Differences were analyzed by two-way ANOVA followed by posthoc Bonferroni's test. A P value of ≤ 0.05 was considered statistically significant. To determine the effects of a combined therapy (Fsp27 silencing and PPARα activation) on diet-induced steatohepatitis, we fed C57BL/6 mice a high-trans-fat, high-cholesterol, high-fructose diet that has been shown to result in severe hepatosteatosis and mild hepatic inflammation and fibrosis (18Neuschwander-Tetri B.A. Ford D.A. Acharya S. Gilkey G. Basaranoglu M. Tetri L.H. Brunt E.M. Dietary trans-fatty acid induced NASH is normalized following loss of trans-fatty acids from hepatic lipid pools.Lipids. 2012; 47: 941-950Crossref PubMed Scopus (40) Google Scholar, 19Tetri L.H. Basaranoglu M. Brunt E.M. Yerian L.M. Neuschwander-Tetri B.A. Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a high-fructose corn syrup equivalent.Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: G987-G995Crossref PubMed Scopus (334) Google Scholar, 21Griffett K. Welch R.D. Flaveny C.A. Kolar G.R. Neuschwander-Tetri B.A. Burris T.P. The LXR inverse agonist SR9238 suppresses fibrosis in a model of non-alcoholic steatohepatitis.Mol. Metab. 2015; 4: 353-357Crossref PubMed Scopus (49) Google Scholar, 23Charlton M. Krishnan A. Viker K. Sanderson S. Cazanave S. McConico A. Masuoko H. Gores G. Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 301: G825-G834Crossref PubMed Scopus (272) Google Scholar). After 8 weeks on the NASH diet, mice were randomized into four groups and kept on the same diet for 6 additional weeks while dosed with ASO-ctrl or ASO-Fsp27 and saline or fenofibrate (Fig. 1A). An additional age-matched group was fed the NASH diet for 8 weeks, then switched to chow (Fig. 1A). Data in Fig. 1B and supplemental Fig. S1A show that control mice on the NASH diet gained weight during the last 6 weeks of the experiment, as opposed to those switched to chow. Treatment with fenofibrate or ASO-Fsp27 alone resulted in significantly less weight gain, in comparison with NASH-fed control mice. Combining both treatments, however, resulted in weight loss similar to that in mice switched to chow. Consistent with these data, analysis of body composition by NMR revealed a significant decrease in fat mass upon treatment with either fenofibrate or ASO-Fsp27, in comparison with control NASH-fed mice, but combined treatment led to a synergistic reduction in fat mass, again similar to that in mice switched to chow diet (Fig. 1C). As was expected, lean mass contents reverse-mirrored the data on fat contents (Fig. 1C). Importantly, these changes in body weight and body composition in NASH-fed mice occurred despite the fact that caloric intake was not significantly different among groups (supplemental Fig. S1B). As was expected, fenofibrate robustly reduced plasma TAG (Fig. 1D and supplemental Fig. S1C). Silencing Fsp27 also decreased circulating TAG, similar to fenofibrate, but no synergistic effect was noted with the combined therapy (Fig. 1D and supplemental Fig. S1C). In contrast, neither fenofibrate nor ASO-Fsp27 changed total cholesterol nor FFA in plasma (Fig. 1D). Notably, β-hydroxy-butyrate was elevated in plasma samples of mice dosed with both ASO-Fsp27 and fenofibrate (Fig. 1D), suggesting accelerated hepatic fatty acid oxidation. Plasma lipids were also analyzed by FPLC to determine lipoprotein profiles. The data confirmed the changes in VLDL-TAG (Fig. 1E) and revealed a large change in the HDL/LDL distribution of cholesterol in response to fenofibrate (Fig. 1F). Consistent with the NMR data above, supplemental Fig. S2A, B shows a robust reduction in eWAT in NASH-fed mice dosed with either fenofibrate or ASO-Fsp27 and a trend for further reduction in mice receiving the double treatment. These data are consistent with our previous report of a robust decrease in visceral fat pads after sustained silencing of Fsp27 in HFD-fed mice (20Langhi C. Arias N. Rajamoorthi A. Basta J. Lee R.G. Baldan A. Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insulin resistance.J. Lipid Res. 2017; 58: 81-91Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Lipid analysis of eWAT samples from NASH-fed mice revealed that TAG contents mirrored the changes in tissue mass, whereas cholesterol levels were highly variable within each experimental group, and phosphatidylcholine contents remained unchanged (supplemental Fig. S2C). Histologically, white adipocytes were larger in NASH-fed mice than were those in mice switched to chow, and reduced average cell size was markedly noticeable in samples from mice dosed with both ASO-Fsp27 and fenofibrate (supplemental Fig. S2D). A selected panel of lipid-related transcripts was analyzed in the same eWAT samples (supplemental Fig. S3). Data show that ASO-Fsp27 was effective in reducing Fsp27 expression (80%–95%). The expression of canonical PPARα targets (Fsp27, Cpt1a, Mcad, Acox), however, remained largely unaltered by the fenofibrate treatment alone. This apparent lack of effect of fenofibrate could be the consequence of the relatively low levels of PPARα in the adipocyte; alternatively, perhaps diet-derived PPARα ligands already provide maximal induction of these transcripts in adipose tissue. In contrast, ASO-Fsp27, or a combination of ASO-Fsp27 and fenofibrate, resulted in the decrease of multiple lipid droplet-associated (Cidea, Plin1) and lipogenic (Srebp1c, Fasn, Scd1) transcripts. The large macroscopic reduction in eWAT in mice dosed with ASO-Fsp27, fenofibrate, or both are likely explained by the previously reported reduction in lipid storage, synthesis, or accelerated mobilization of FFA, or all of these, from the lipid droplet upon loss of adipocyte FSP27 activity (24Tanaka N. Takahashi S. Matsubara T. Jiang C. Sakamoto W. Chanturiya T. Teng R. Gavrilova O. Gonzalez F.J. Adipocyte-specific disruption of fat-specific protein 27 causes hepatosteatosis and insulin resistance in high-fat diet-fed mice.J. Biol. Chem. 2015; 290: 3092-3105Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) or following PPARα activation (25Ferreira A.V. Parreira G.G. Green A. Botion L.M. Effects of fenofibrate on lipid metabolism in adipose tissue of rats.Metabolism. 2006; 55: 731-735Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). As was expected in mice (26Yang Q. Nagano T. Shah Y. Cheung C. Ito S. Gonzalez F.J. The PPAR alpha-humanized mouse: a model to investigate species differences in liver toxicity mediated by PPAR alpha.Toxicol. Sci. 2008; 101: 132-139Crossref PubMed Scopus (127) Google Scholar), fenofibrate treatment resulted in hepatomegaly (Fig. 2A, B). The livers of mice that received the combined therapy were consistently darker than were those in the other experimental groups (Fig. 2A), suggesting that the lipid contents might be reduced. Data in Fig. 2C show that hepatic glycogen contents in mice dosed with ASO-Fsp27 and fenofibrate were elevated, in comparison with the rest of NASH-fed animals, and similar to those from mice switched to chow. Enzymatic assays in lipid extracts confirmed the robust decrease in hepatic TAG contents in mice treated with both ASO-Fsp27 and fenofibrate (Fig. 2D). Neither treatment alone reduced steatosis (Fig. 2D). Unexpectedly, the switch from NASH diet to chow resulted only in relatively modest changes in liver weight and steatosis (Fig. 2A, B). The amounts of FFA were increased in livers from mice treated with ASO-Fsp27 (Fig. 2D), which is consistent with previous reports of defective expression of hepatic PPARα fatty acid oxidation targets upon sustained loss of FSP27 (20Langhi C. Arias N. Rajamoorthi A. Basta J. Lee R.G. Baldan A. Therapeutic silencing of fat-specific protein 27 improves glycemic control in mouse models of obesity and insuli
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