Title: Melanocortin 4 Receptor–Deficient Mice as a Novel Mouse Model of Nonalcoholic Steatohepatitis
Abstract: Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the “two hit” hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptor–deficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies. Obesity may be viewed as a state of chronic low-grade inflammation that participates in the development of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is considered a hepatic phenotype of the metabolic syndrome and a high risk for progression to cirrhosis and hepatocellular carcinoma. Although the “two hit” hypothesis suggests involvement of excessive hepatic lipid accumulation and chronic inflammation, the molecular mechanisms underlying the development of NASH remain unclear, in part because of lack of appropriate animal models. Herein we report that melanocortin 4 receptor–deficient mice (MC4R-KO) develop steatohepatitis when fed a high-fat diet, which is associated with obesity, insulin resistance, and dyslipidemia. Histologic analysis reveals inflammatory cell infiltration, hepatocyte ballooning, and pericellular fibrosis in the liver in MC4R-KO mice. Of note, all of the MC4R-KO mice examined developed well-differentiated hepatocellular carcinoma after being fed a high-fat diet for 1 year. They also demonstrated enhanced adipose tissue inflammation, ie, increased macrophage infiltration and fibrotic changes, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Thus, MC4R-KO mice provide a novel mouse model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and hepatocellular carcinoma and to aid in understanding the pathogenesis of NASH, pursuing specific biomarkers, and evaluating potential therapeutic strategies. Nonalcoholic fatty liver disease (NAFLD) is characterized by increased accumulation of lipids in the liver without a history of excessive alcohol consumption or known liver disease.1Neuschwander-Tetri B.A. Caldwell S.H. Nonalcoholic steatohepatitis: summary of an AASLD single topic conference.Hepatology. 2003; 37: 1202-1219Crossref PubMed Scopus (1794) Google Scholar NAFLD often occurs with the metabolic syndrome, a constellation of visceral fat obesity, impaired glucose metabolism, atherogenic dyslipidemia, and elevated blood pressure, and is considered the hepatic manifestation of the metabolic syndrome.2Marchesini G. Bugianesi E. Forlani G. Cerrelli F. Lenzi M. Manini R. Natale S. Vanni E. Villanova N. Melchionda N. Rizzetto M. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome.Hepatology. 2003; 37: 917-923Crossref PubMed Scopus (2209) Google Scholar Patients with nonalcoholic steatohepatitis (NASH), a subset of NAFLD, are at high risk for progression to cirrhosis and hepatocellular carcinoma (HCC). However, the molecular mechanisms involved in disease progression from simple steatosis to NASH to HCC are currently unclear. This is in part because there are no appropriate animal models that reflect a liver condition of human NASH, although many attempts have been made to generate animal NASH models via genetic, dietary, and pharmacologic approaches.3Varela-Rey M. Embade N. Ariz U. Lu S.C. Mato J.M. Martinez-Chantar M.L. Non-alcoholic steatohepatitis and animal models: understanding the human disease.Int J Biochem Cell Biol. 2009; 41: 969-976Crossref PubMed Scopus (93) Google Scholar The pathogenesis of NASH is thought to involve a multistep process in which the first step is excessive accumulation of lipids in the liver. According to the “two hit” hypothesis, the development of NASH requires the presence of additional pathogenic factors such as oxidative stress, endotoxins, cytokines, chemokines, and lipotoxicity.4Day C.P. James O.F. Steatohepatitis: a tale of two “hits”?.Gastroenterology. 1998; 114: 842-845Abstract Full Text Full Text PDF PubMed Scopus (3473) Google Scholar, 5Browning J.D. Horton J.D. Molecular mediators of hepatic steatosis and liver injury.J Clin Invest. 2004; 114: 147-152Crossref PubMed Scopus (1757) Google Scholar, 6Neuschwander-Tetri B.A. Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites.Hepatology. 2010; 52: 774-788Crossref PubMed Scopus (745) Google Scholar Because NASH is often associated with visceral fat obesity, there should be a mechanistic link between the adipose tissue and the liver.7Day C.P. From fat to inflammation.Gastroenterology. 2006; 130: 207-210Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar Adipose tissue secretes a large number of bioactive substances or adipocytokines such as leptin and adiponectin. Unbalanced production of pro-inflammatory and anti-inflammatory adipocytokines in obesity has been implicated in the pathogenesis of obesity-related complications including NAFLD.7Day C.P. From fat to inflammation.Gastroenterology. 2006; 130: 207-210Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 8Marra F. Bertolani C. Adipokines in liver diseases.Hepatology. 2009; 50: 957-969Crossref PubMed Scopus (391) Google Scholar Indeed, deficiency of leptin signaling protects against hepatic fibrosis in several rodent models of chronic liver injury,9Leclercq I.A. Farrell G.C. Schriemer R. Robertson G.R. Leptin is essential for the hepatic fibrogenic response to chronic liver injury.J Hepatol. 2002; 37: 206-213Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 10Aleffi S. Petrai I. Bertolani C. Parola M. Colombatto S. Novo E. Vizzutti F. Anania F.A. Milani S. Rombouts K. Laffi G. Pinzani M. Marra F. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.Hepatology. 2005; 42: 1339-1348Crossref PubMed Scopus (296) Google Scholar, 11Saxena N.K. Ikeda K. Rockey D.C. Friedman S.L. Anania F.A. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice.Hepatology. 2002; 35: 762-771Crossref PubMed Scopus (355) Google Scholar which suggests that leptin may accelerate development of liver fibrosis. In contrast, studies in adiponectin-deficient mice have revealed that adiponectin is protective against development of hepatic fibrosis and inflammation.12Kamada Y. Matsumoto H. Tamura S. Fukushima J. Kiso S. Fukui K. Igura T. Maeda N. Kihara S. Funahashi T. Matsuzawa Y. Shimomura I. Hayashi N. Hypoadiponectinemia accelerates hepatic tumor formation in a nonalcoholic steatohepatitis mouse model.J Hepatol. 2007; 47: 556-564Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar The melanocortin 4 receptor (MC4R) is a seven-transmembrane G protein–coupled receptor that is expressed in the hypothalamic nuclei and is implicated in regulation of food intake and body weight.13Balthasar N. Dalgaard L.T. Lee C.E. Yu J. Funahashi H. Williams T. Ferreira M. Tang V. McGovern R.A. Kenny C.D. Christiansen L.M. Edelstein E. Choi B. Boss O. Aschkenasi C. Zhang C.Y. Mountjoy K. Kishi T. Elmquist J.K. Lowell B.B. Divergence of melanocortin pathways in the control of food intake and energy expenditure.Cell. 2005; 123: 493-505Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar Previous studies have identified many pathogenic mutations of the MC4R gene at a relatively high frequency in severe early-onset obesity, which suggests that MC4R mutations are the most common known monogenic cause of obesity in humans.14Vaisse C. Clement K. Durand E. Hercberg S. Guy-Grand B. Froguel P. Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity.J Clin Invest. 2000; 106: 253-262Crossref PubMed Scopus (729) Google Scholar Some leptin biological actions are mediated, at least in part, via the central melanocortin system.15Friedman J.M. Halaas J.L. Leptin and the regulation of body weight in mammals.Nature. 1998; 395: 763-770Crossref PubMed Scopus (4518) Google Scholar Indeed, mice with targeted disruption of MC4R have developed late-onset obesity associated with hyperphagia, hyperinsulinemia, and hyperglycemia.16Marsh D.J. Hollopeter G. Huszar D. Laufer R. Yagaloff K.A. Fisher S.L. Burn P. Palmiter R.D. Response of melanocortin-4 receptor–deficient mice to anorectic and orexigenic peptides.Nat Genet. 1999; 21: 119-122Crossref PubMed Scopus (466) Google Scholar, 17Huszar D. Lynch C.A. Fairchild-Huntress V. Dunmore J.H. Fang Q. Berkemeier L.R. Gu W. Kesterson R.A. Boston B.A. Cone R.D. Smith F.J. Campfield L.A. Burn P. Lee F. Targeted disruption of the melanocortin-4 receptor results in obesity in mice.Cell. 1997; 88: 131-141Abstract Full Text Full Text PDF PubMed Scopus (2553) Google Scholar MC4R-deficient mice (MC4R-KO mice) fed a high-fat diet (HFD) exhibit massive hepatic steatosis and altered gene expression related to lipid metabolism.18Albarado D.C. McClaine J. Stephens J.M. Mynatt R.L. Ye J. Bannon A.W. Richards W.G. Butler A.A. Impaired coordination of nutrient intake and substrate oxidation in melanocortin-4 receptor knockout mice.Endocrinology. 2004; 145: 243-252Crossref PubMed Scopus (87) Google Scholar, 19Sutton G.M. Trevaskis J.L. Hulver M.W. McMillan R.P. Markward N.J. Babin M.J. Meyer E.A. Butler A.A. Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or -4 receptors.Endocrinology. 2006; 147: 2183-2196Crossref PubMed Scopus (116) Google Scholar The role of MC4R in the pathogenesis of NASH, however, has not been elucidated. Herein we report for the first time that MC4R-KO mice develop a liver condition similar to human NASH when fed an HFD, which is associated with obesity, insulin resistance, and dyslipidemia. Of note, they also demonstrate enhanced adipose tissue inflammation, which may contribute to excessive lipid accumulation and enhanced fibrosis in the liver. Moreover, they develop well-differentiated HCC when fed the HFD for a prolonged time. Our data suggest that MC4R-KO mice would provide a novel rodent model of NASH with which to investigate the sequence of events that make up diet-induced hepatic steatosis, liver fibrosis, and HCC. MC4R-KO mice on the C57BL/6J background were a gift from Dr. Joel K. Elmquist (University of Texas Southwestern Medical Center, Dallas, TX).13Balthasar N. Dalgaard L.T. Lee C.E. Yu J. Funahashi H. Williams T. Ferreira M. Tang V. McGovern R.A. Kenny C.D. Christiansen L.M. Edelstein E. Choi B. Boss O. Aschkenasi C. Zhang C.Y. Mountjoy K. Kishi T. Elmquist J.K. Lowell B.B. Divergence of melanocortin pathways in the control of food intake and energy expenditure.Cell. 2005; 123: 493-505Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar Male C57BL/6J wild-type (WT) mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). The animals were housed in individual cages in a temperature-, humidity-, and light-controlled room (12-hour light and 12-hour dark cycle) and allowed free access to water and standard diet (CE-2; 343.1 kcal/100 g, 12.6% energy as fat; CLEA Japan, Inc.), unless otherwise noted. In the HFD feeding experiments, 8-week-old male mice were given free access to water and either standard diet or HFD (D12492; 524 kcal/100 g, 60% energy as fat; Research Diets, Inc., New Brunswick, NJ) for 8 or 20 weeks. Detailed dietary composition of the standard diet and HFD is given in Supplemental Table S1 (available on http://ajp.amjpathol.org). At the end of the experiments, the animals fed ad libitum were sacrificed after administration of 30 mg/kg i.p. pentobarbital anesthesia. All animal experiments were conducted in accordance with the guidelines of the Tokyo Medical and Dental University Committee on Animal Research (No. 100098). Blood glucose concentration was measured using the blood glucose test meter (Glutest PRO R; Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, Japan). Serum alanine aminotransferase, triglyceride (TG), free fatty acid (FFA), and total cholesterol concentrations were measured using the respective standard enzymatic assays. Serum concentrations of insulin and adipocytokines were determined using the respective enzyme-linked immunosorbent assay kits (insulin, Morinaga Co. Ltd., Tokyo, Japan; adiponectin, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan; and leptin and IL-6, R&D Systems, Inc., Minneapolis, MN). The homeostasis model assessment insulin resistance index was calculated as [fasting glucose (mg/dL) × fasting insulin (μU/mL)]/405. Total lipids in the liver were extracted using ice-cold chloroform and methanol, 2:1 (v/v). TG concentrations were measured using an enzymatic assay kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan).20Sakaida I. Terai S. Yamamoto N. Aoyama K. Ishikawa T. Nishina H. Okita K. Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice.Hepatology. 2004; 40: 1304-1311Crossref PubMed Scopus (475) Google Scholar The liver and epididymal white adipose tissue were fixed with neutral-buffered formalin and embedded in paraffin. Two-micrometer-thick sections of liver were stained using H&E, Masson's trichrome, and Sirius red.20Sakaida I. Terai S. Yamamoto N. Aoyama K. Ishikawa T. Nishina H. Okita K. Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice.Hepatology. 2004; 40: 1304-1311Crossref PubMed Scopus (475) Google Scholar, 21Tanaka M. Suganami T. Sugita S. Shimoda Y. Kasahara M. Aoe S. Takeya M. Takeda S. Kamei Y. Ogawa Y. Role of central leptin signaling in renal macrophage infiltration.Endocr J. 2010; 57: 61-72Crossref PubMed Scopus (23) Google Scholar The presence of α-smooth muscle actin (α-SMA) and α-fetoprotein was detected at immunohistochemistry using mouse monoclonal anti-human α-SMA antibody (Dako A/S, Glostrup, Denmark) and polyclonal goat anti-human α-fetoprotein antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), respectively.20Sakaida I. Terai S. Yamamoto N. Aoyama K. Ishikawa T. Nishina H. Okita K. Transplantation of bone marrow cells reduces CCl4-induced liver fibrosis in mice.Hepatology. 2004; 40: 1304-1311Crossref PubMed Scopus (475) Google Scholar Areas positive for Sirius red and α-SMA were measured using WinROOF software (Mitani Corp., Tokyo, Japan). Five-micrometer-thick sections of the epididymal white adipose tissue were stained using anti-mouse F4/80 antibody.22Kitagawa K. Wada T. Furuichi K. Hashimoto H. Ishiwata Y. Asano M. Takeya M. Kuziel W.A. Matsushima K. Mukaida N. Yokoyama H. Blockade of CCR2 ameliorates progressive fibrosis in kidney.Am J Pathol. 2004; 165: 237-246Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 23Itoh M. Suganami T. Satoh N. Tanimoto-Koyama K. Yuan X. Tanaka M. Kawano H. Yano T. Aoe S. Takeya M. Shimatsu A. Kuzuya H. Kamei Y. Ogawa Y. Increased adiponectin secretion by highly purified eicosapentaenoic acid in rodent models of obesity and human obese subjects.Arterioscler Thromb Vasc Biol. 2007; 27: 1918-1925Crossref PubMed Scopus (254) Google Scholar The number of nuclei surrounded by F4/80-positive cells was counted in a 10 mm2 area of each section and expressed as the mean per millimeter squared for quantification of F4/80-positive macrophages. Liver histologic features were assessed by two investigators (S.T. and I.S.) who had no knowledge of the origin of the slides according to the NASH clinical research network scoring system.24Juluri R. Vuppalanchi R. Olson J. Unalp A. Van Natta M.L. Cummings O.W. Tonascia J. Chalasani N. Generalizability of the Nonalcoholic Steatohepatitis Clinical Research Network Histologic Scoring System for Nonalcoholic Fatty Liver Disease.J Clin Gastroenterol. 2010; 45: 55-58Crossref Scopus (83) Google Scholar In brief, an NAFLD activity score higher than 5 was considered “definite NASH.”24Juluri R. Vuppalanchi R. Olson J. Unalp A. Van Natta M.L. Cummings O.W. Tonascia J. Chalasani N. Generalizability of the Nonalcoholic Steatohepatitis Clinical Research Network Histologic Scoring System for Nonalcoholic Fatty Liver Disease.J Clin Gastroenterol. 2010; 45: 55-58Crossref Scopus (83) Google Scholar The liver fibrosis score was determined semiquantitatively as follows: stage 0, no fibrosis; stage 1, periportal or perisinusoidal fibrosis; stage 2, periportal plus perisinusoidal fibrosis; and stage 3, bridging fibrosis.24Juluri R. Vuppalanchi R. Olson J. Unalp A. Van Natta M.L. Cummings O.W. Tonascia J. Chalasani N. Generalizability of the Nonalcoholic Steatohepatitis Clinical Research Network Histologic Scoring System for Nonalcoholic Fatty Liver Disease.J Clin Gastroenterol. 2010; 45: 55-58Crossref Scopus (83) Google Scholar Total RNA was extracted from the liver and epididymal white adipose tissue using Sepasol reagent (Nacalai Tesque, Inc., Kyoto, Japan). Quantitative RT-PCR was performed using the ABI Prism 7000 Sequence Detection System with PCR Master Mix Reagent (Applied Biosystems, Inc., Foster City, CA) as described previously.23Itoh M. Suganami T. Satoh N. Tanimoto-Koyama K. Yuan X. Tanaka M. Kawano H. Yano T. Aoe S. Takeya M. Shimatsu A. Kuzuya H. Kamei Y. Ogawa Y. Increased adiponectin secretion by highly purified eicosapentaenoic acid in rodent models of obesity and human obese subjects.Arterioscler Thromb Vasc Biol. 2007; 27: 1918-1925Crossref PubMed Scopus (254) Google Scholar Primers used are given in Table 1. mRNA levels were normalized to those of 36B4 mRNA.Table 1Primers Used in the Present StudyGenesPrimersACC1 Forward5′-TGAGATTGGCATGGTAGCCTG-3′ Reverse5′-CTCGGCCATCTGGATATTCAG-3′Catalase Forward5′-GGAGGCAGAAACTTTCCCATT-3′ Reverse5′-GGCCAAACCTTGGTCAGATC-3′COL1A1 Forward5′-CCTCAGGGTATTGCTGGACAAC-3′ Reverse5′-ACCACTTGATCCAGAAGGACCTT-3′CPT1A Forward5′-CCTGCATTCCTTCCCATTTG-3′ Reverse5′-TGCCCATGTCCTTGTAATGTG-3′F4/80 Forward5′-CTTTGGCTATGGGCTTCCAGT-3′ Reverse5′-GCAAGGAGGACAGAGTTTATCGTG-3′FAS Forward5′-CCTGGATAGCATTCCGAACCT-3′ Reverse5′-AGCACATCTCGAAGGCTACACA-3′gp91phox Forward5′-CCAGTGCGTGTTGCTCGA-3′ Reverse5′-AGTGAGGTTCCTGTCCAGTTGTCT-3′MMP-2 Forward5′-CCCCATGAAGCCTTGTTTACC-3′ Reverse5′-TTGTAGGAGGTGCCCTGGAA-3′MTP Forward5′-ACAGGTCCTCGAGCGTGTCT-3′ Reverse5′-CAGTGCTCCGCCAGAGAAG-3′p22phox Forward5′-CATGGAGCGATGTGGACAGA-3′ Reverse5′-CCCGAAAAGCTTCACCACAG-3′p40phox Forward5′-CAGCCAACATCGCTGACATC-3′ Reverse5′-CAAAGTGGCTGGTGAAGCCC-3′p47phox Forward5′-ACTCTCACTGAATACTTCAACG-3′ Reverse5′-TCATCAGGCCGCACTTT-3′p67phox Forward5′-AAGCAAAAAGAGCCCAAGGAA-3′ Reverse5′-CATGTAAGGCATAGGCACGCT-3′PPARα Forward5′-AGGAAGCCGTTCTGTGACAT-3′ Reverse5′-AATCCCCTCCTGCAACTTCT-3′SOD1 Forward5′-GCAGGACCTCATTTTAATCCTCACT-3′ Reverse5′-AGGTCTCCAACATGCCTCTCTTC-3′SREBP1c Forward5′-GGCACTAAGTGCCCTCAACCT-3′ Reverse5′-GCCACATAGATCTCTGCCAGTGT-3′TGFβ1 Forward5′-CCTGAGTGGCTGTCTTTTGACG-3′ Reverse5′-AGTGAGCGCTGAATCGAAAGC-3′TIMP1 Forward5′-CATCACGGGCCGCCTA-3′ Reverse5′-AAGCTGCAGGCACTGATGTG-3′TNFα Forward5′-ACCCTCACACTCAGATCATCTTC-3′ Reverse5′-TGGTGGTTTGCTACGACGT-3′36B4 Forward5′-GGCCCTGCACTCTCGCTTTC-3′ Reverse5′-TGCCAGGACGCGCTTGT-3′ Open table in a new tab The total amount of organic hydroperoxides in the serum was measured at spectrophotometry using the derivatives of reactive oxygen metabolites test (FREE Carpe Diem; Diacron International SAS, Grosseto, Italy). Hydroperoxides are intermediate oxidative products of lipids, peptides, and amino acids, and their concentrations represent an index of oxidative injury. This method is described in detail elsewhere.25Kondo K. Shibata R. Unno K. Shimano M. Ishii M. Kito T. Shintani S. Walsh K. Ouchi N. Murohara T. Impact of a single intracoronary administration of adiponectin on myocardial ischemia/reperfusion injury in a pig model.Circ Cardiovasc Interv. 2010; 3: 166-173Crossref PubMed Scopus (75) Google Scholar In brief, 20-μL serum samples were added to 1 mL assay mixture, gently mixed, and incubated for 3 minutes at 37°C. The absorbance increase at 505 nm was monitored for 2 minutes. The concentrations were expressed in conventional units [Carratelli units (U.CARR)], where 1 U.CARR corresponds to 0.8 mg/L H2O2. The TG secretion rate was measured as previously described.26Deushi M. Nomura M. Kawakami A. Haraguchi M. Ito M. Okazaki M. Ishii H. Yoshida M. Ezetimibe improves liver steatosis and insulin resistance in obese rat model of metabolic syndrome.FEBS Lett. 2007; 581: 5664-5670Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar In brief, 500 mg/kg body weight tyloxapol (Triton WR-1339; Sigma-Aldrich Corp., St. Louis, MO) was injected via the tail vein into mice that had been fasted for 5 hours. Serum TG concentrations were measured at 60 minutes after injection. The TG secretion rate was calculated from the increment in TG concentration per minute multiplied by the serum volume of mice (estimated as 3.5% of body weight in grams) and expressed in milligrams per minute per 100 g body weight. Data are given as mean ± SE. P < 0.05 was considered statistically significant. Statistical analysis was performed using analysis of variance followed by Scheffé's test. Throughout the experimental period, MC4R-KO mice demonstrated accelerated body weight gain relative to WT mice fed either the standard diet or the HFD (Figure 1A). Both the WT mice fed the HFD and the MC4R-KO mice fed the standard diet exhibited increased adiposity relative to the WT mice fed the standard diet (Figure 1B). Although increased adiposity was observed in MC4R-KO mice at 8 weeks of HFD feeding, there was no further increase or decrease in adipose tissue weight thereafter (Figure 1B). In contrast, MC4R-KO mice exhibited a time-dependent increase in liver weight and hepatic TG content relative to WT mice fed either diet (Figure 1, C and D; and data not shown). Both genotypes fed the HFD exhibited insulin resistance, and MC4R-KO mice fed the HFD exhibited a significant increase in serum FFA concentrations relative to WT mice (Table 2). Dysregulation of adipocytokines was also marked in MC4R-KO mice fed the HFD relative to WT mice (Table 2, and Supplemental Table S2 at http://ajp.amjpathol.org). Serum alanine aminotransferase concentrations were significantly increased in MC4R-KO mice fed the HFD relative to any other groups (Table 2). In essence, these observations are consistent with previous reports that described the metabolic phenotypes in the liver in MC4R-KO mice, which were generated using different strategies.18Albarado D.C. McClaine J. Stephens J.M. Mynatt R.L. Ye J. Bannon A.W. Richards W.G. Butler A.A. Impaired coordination of nutrient intake and substrate oxidation in melanocortin-4 receptor knockout mice.Endocrinology. 2004; 145: 243-252Crossref PubMed Scopus (87) Google Scholar, 19Sutton G.M. Trevaskis J.L. Hulver M.W. McMillan R.P. Markward N.J. Babin M.J. Meyer E.A. Butler A.A. Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or -4 receptors.Endocrinology. 2006; 147: 2183-2196Crossref PubMed Scopus (116) Google Scholar Collectively, our data indicate that MC4R-KO mice fed the HFD exhibit metabolic characteristics similar to those in obese humans.Table 2Serologic Parameters of MC4R-KO and WT Mice Fed the HFD for 20 WeeksWT miceMC4R-KO miceVariableSDHFDSDHFDBlood glucose (ad lib, mg/dL)117.3 ± 5.8133.0 ± 8.3167.4 ± 10.3⁎P < 0.05,170.1 ± 12.4HOMA-IR0.8 ± 0.315.5 ± 3.7⁎P < 0.05,17.0 ± 4.0†P < 0.01 versus WT-SD;21.8 ± 3.7TG (mg/dL)84.6 ± 9.846.3 ± 1.2⁎P < 0.05,135.8 ± 18.4⁎P < 0.05,92.7 ± 8.1FFA (mEq/L)0.27 ± 0.020.26 ± 0.010.34 ± 0.020.51 ± 0.06‡P < 0.01 versus WT-HFD;§P < 0.05;TC (mg/dL)51.6 ± 2.8189.7 ± 6.4†P < 0.01 versus WT-SD;143.0 ± 9.5⁎P < 0.05,294.5 ± 9.2‡P < 0.01 versus WT-HFD;¶P < 0.01 versus MC4R-SD.Adiponectin (μg/mL)15.4 ± 1.719.8 ± 1.713.5 ± 1.08.2 ± 0.9‡P < 0.01 versus WT-HFD;§P < 0.05;Leptin (ng/mL)1.7 ± 0.397.2 ± 8.3†P < 0.01 versus WT-SD;57.7 ± 6.0†P < 0.01 versus WT-SD;112.6 ± 9.2¶P < 0.01 versus MC4R-SD.IL-6 (pg/mL)0.84 ± 0.642.21 ± 1.683.25 ± 0.756.80 ± 0.71‡P < 0.01 versus WT-HFD;¶P < 0.01 versus MC4R-SD.ALT (IU/L)36.9 ± 1.1129.9 ± 19.6191.9 ± 29.2⁎P < 0.05,623.9 ± 50.8‡P < 0.01 versus WT-HFD;¶P < 0.01 versus MC4R-SD.n = 7–10. Data are expressed as mean ± SE.ALT, alanine aminotransferase; FFA, free fatty acid; HFD, high-fat diet; HOMA-IR, homeostasis model assessment–insulin resistance; IL-6, interleukin-6; SD, standard diet; TC, total cholesterol; TG, triglyceride; WT, wild-type. P < 0.05,† P < 0.01 versus WT-SD;‡ P < 0.01 versus WT-HFD;§ P < 0.05;¶ P < 0.01 versus MC4R-SD. Open table in a new tab n = 7–10. Data are expressed as mean ± SE. ALT, alanine aminotransferase; FFA, free fatty acid; HFD, high-fat diet; HOMA-IR, homeostasis model assessment–insulin resistance; IL-6, interleukin-6; SD, standard diet; TC, total cholesterol; TG, triglyceride; WT, wild-type. Next examined was lipid metabolism and oxidative stress in liver from MC4R-KO mice fed the HFD. Although histologic examinations revealed minimal lipid accumulation in liver from WT mice fed the HFD for 8 weeks, liver from MC4R-KO mice exhibited massive microvesicular steatosis in the centrilobular and portal areas (Figure 2A). Expression of mRNAs for de novo lipogenesis (fatty acid synthase and acetyl-CoA carboxylase 1) was markedly increased in liver from MC4R-KO mice relative to WT mice at 8 weeks (Figure 2B), as previously reported.18Albarado D.C. McClaine J. Stephens J.M. Mynatt R.L. Ye J. Bannon A.W. Richards W.G. Butler A.A. Impaired coordination of nutrient intake and substrate oxidation in melanocortin-4 receptor knockout mice.Endocrinology. 2004; 145: 243-252Crossref PubMed Scopus (87) Google Scholar, 19Sutton G.M. Trevaskis J.L. Hulver M.W. McMillan R.P. Markward N.J. Babin M.J. Meyer E.A. Butler A.A. Diet-genotype interactions in the development of the obese, insulin-resistant phenotype of C57BL/6J mice lacking melanocortin-3 or -4 receptors.Endocrinology. 2006; 147: 2183-2196Crossref PubMed Scopus (116) Google Scholar In addition, expression of mRNAs for fatty acid oxidation (peroxisome proliferator–activated receptor-α and carnitine palmitoyltransferase 1A), and TG secretion (microsomal triglyceride transport protein) and TG secretion rate were increased in liver from MC4R-KO mice (Figure 2, C–E). These observations are consistent with lipid metabolism in human NASH.27Marra F. Gastaldelli A. Svegliati Baroni G. Tell G. Tiribelli C. Molecular basis and mechanisms of progression of non-alcoholic steatohepatitis.Trends Mol Med. 2008; 14: 72-81Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar In contrast to the changes in hepatic expression of lipogenic genes in MC4R-KO mice (Figure 2B), WT mice exhibited up-regulation of lipogenic genes only after 20 weeks of HFD feeding (data not shown), which suggests that hepatic steatosis develops much faster in MC4R-KO mice than in WT mice. There was a marked increase in mRNA expression of the NADPH oxidase components (p40phox, p47phox, p67phox, gp91phox, and p22phox), and a slight increase in mRNA expression of antioxidant enzymes (superoxide dismutase 1 and catalase) in the HFD-fed MC4R-KO mice relative to any other groups (see Supplemental Figure S1, A and B, at http://ajp.amjpathol.org). Serum concentrations of derivatives of reactive oxidative metabolite were significantly increased in MC4R-KO mice fed the HFD (P < 0.01; see also Supplemental Figure S1C at http://ajp.amjpathol.org). After HFD feeding for 20 weeks, microvesicular steatosis was observed uniformly, and moderate inflammatory cell infiltration in liver from WT mice (Figure 3A), whereas liver fibrosis was rarely observed at this time point (Figure 3, B–D). In contrast, liver from MC4R-KO mice fed the HFD exhibited microvesicular and macrovesicular steatosis, ballooning degeneration, and massive infiltration of inflammatory cells (Figure 3A). Masson's trichrome and Sirius red staining revealed marked pericellular fibrosis in liver from MC4R-KO mice fed the HFD (Figure 3, B–D). In addition, the area positive for α-SMA was markedly increased in MC4R-KO mice relative to WT mice fed the HFD for 8 and 20 weeks (P < 0.05 and P < 0.01, respectively; Figure 3, E and F). Histologic analysis demonstrated a significant increase in the NAFLD activity and fibrosis scores in MC4R-KO mice at 8 and 20 weeks of HFD feeding (Figure 3, G and H). Expression of mRNAs for fibrogenic genes (transforming growth factor-β1; collagen, type 1, α1; metalloproteinase-2; and tissue inhibitor of metalloproteinase 1), and inflammatory genes (macrophage marker F4/80 and tumor necrosis factor-α) was increased in liver from MC4R-KO