Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis using a high-fat, choline-deficient diet and intraperitoneal injection of diethylnitrosamine


Background:
The incidence of hepatocellular carcinoma with nonalcoholic steatohepatitis is increasing, and its clinicopathological features are well established. Several animal models of nonalcoholic steatohepatitis have been developed to facilitate its study; however, few fully recapitulate all its clinical features, which include insulin resistance, inflammation, fibrosis, and carcinogenesis. Moreover, these models require a relatively long time to produce hepatocellular carcinoma reliably. The aim of this study was to develop a mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis that develops quickly and reflects all clinically relevant features.

Methods:
Three-week-old C57BL/6J male mice were fed either a standard diet (MF) or a choline-deficient, high-fat diet (HFCD). The mice in the MF + diethylnitrosamine (DEN) and HFCD + DEN groups received a one-time intraperitoneal injection of DEN at the start of the respective feeding protocols.

Results:
The mice in the HFCD and HFCD + DEN groups developed obesity early in the experiment and insulin resistance after 12 weeks. Triglyceride levels peaked at 8 weeks for all four groups and decreased thereafter. Alanine aminotransferase levels increased every 4 weeks, with the HFCD and HFCD + DEN groups showing remarkably high levels; the HFCD + DEN group presented the highest incidence of nonalcoholic steatohepatitis. The levels of fibrosis and steatosis varied, but they tended to increase every 4 weeks in the HFCD and HFCD + DEN groups. Computed tomography scans indicated that all the HFCD + DEN mice developed hepatic tumors from 20 weeks, some of which were glutamine synthetase-positive.

Conclusions:
The nonalcoholic steatohepatitis-hepatocellular carcinoma model we describe here is simple to establish, results in rapid tumor formation, and recapitulates most of the key features of nonalcoholic steatohepatitis. It could therefore facilitate further studies of the development, oncogenic potential, diagnosis, and treatment of this condition.

Keywords:
Nonalcoholic steatohepatitis, Hepatocellular carcinoma, Diethylnitrosamine, High-fat choline-deficient diet, Mouse model


Autoři: Norihiro Kishida 1;  Sachiko Matsuda 1,2;  Osamu Itano 1*;  Masahiro Shinoda 1;  Minoru Kitago 1;  Hiroshi Yagi 1;  Yuta Abe 1;  Taizo Hibi 1;  Yohei Masugi 3;  Koichi Aiura 4;  Michiie Sakamoto 3;  Yuko Kitagawa 1
Působiště autorů: Department of Surgery, School of Medicine, Keio University, 5 Shinanomachi, Shinjuku-ku, Tokyo 160-858 , Japan. 1;  Chugai Pharmaceutical Endowed Research Chair in Molecular Targeted Therapy of Gastrointestinal Cancer, School of Medicine, Keio University, Tokyo, Japan. 2;  Department of Pathology, School of Medicine, Keio University, Tokyo, Japan. 3;  Department of Surgery, Kawasaki Municipal Hospital, Kawasaki-ku, Japan. 4
Vyšlo v časopise: BMC Gastroenterology 2016, 16:61
Kategorie: Research article
prolekare.web.journal.doi_sk: 10.1186/s12876-016-0477-5

© 2016 The Author(s).
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
The electronic version of this article is the complete one and can be found online at: http://bmcgastroenterol.biomedcentral.com/articles/10.1186/s12876-016-0477-5

Souhrn

Background:
The incidence of hepatocellular carcinoma with nonalcoholic steatohepatitis is increasing, and its clinicopathological features are well established. Several animal models of nonalcoholic steatohepatitis have been developed to facilitate its study; however, few fully recapitulate all its clinical features, which include insulin resistance, inflammation, fibrosis, and carcinogenesis. Moreover, these models require a relatively long time to produce hepatocellular carcinoma reliably. The aim of this study was to develop a mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis that develops quickly and reflects all clinically relevant features.

Methods:
Three-week-old C57BL/6J male mice were fed either a standard diet (MF) or a choline-deficient, high-fat diet (HFCD). The mice in the MF + diethylnitrosamine (DEN) and HFCD + DEN groups received a one-time intraperitoneal injection of DEN at the start of the respective feeding protocols.

Results:
The mice in the HFCD and HFCD + DEN groups developed obesity early in the experiment and insulin resistance after 12 weeks. Triglyceride levels peaked at 8 weeks for all four groups and decreased thereafter. Alanine aminotransferase levels increased every 4 weeks, with the HFCD and HFCD + DEN groups showing remarkably high levels; the HFCD + DEN group presented the highest incidence of nonalcoholic steatohepatitis. The levels of fibrosis and steatosis varied, but they tended to increase every 4 weeks in the HFCD and HFCD + DEN groups. Computed tomography scans indicated that all the HFCD + DEN mice developed hepatic tumors from 20 weeks, some of which were glutamine synthetase-positive.

Conclusions:
The nonalcoholic steatohepatitis-hepatocellular carcinoma model we describe here is simple to establish, results in rapid tumor formation, and recapitulates most of the key features of nonalcoholic steatohepatitis. It could therefore facilitate further studies of the development, oncogenic potential, diagnosis, and treatment of this condition.

Keywords:
Nonalcoholic steatohepatitis, Hepatocellular carcinoma, Diethylnitrosamine, High-fat choline-deficient diet, Mouse model


Zdroje

1. El-Serag HB. Hepatocellular carcinoma. N Engl J Med. 2011;365:1118–27.

2. Amarapurkar DN, Hashimoto E, Lesmana LA, Sollano JD, Chen PJ, Goh KL. How common is non-alcoholic fatty liver disease in the Asia-Pacific region and are there local differences? J Gastroenterol Hepatol. 2007;22:788–93.

3. Chitturi S, Farrell GC, Hashimoto E, Saibara T, Lau GK, Sollano JD. Nonalcoholic fatty liver disease in the Asia-Pacific region: definitions and overview of proposed guidelines. J Gastroenterol Hepatol. 2007;22:778–87.

4. Yasui K, Hashimoto E, Tokushige K, Koike K, Shima T, Kanbara Y, et al. Clinical and pathological progression of non-alcoholic steatohepatitis to hepatocellular carcinoma. Hepatol Res. 2012;42:767–73.

5. Polyzos SA, Kountouras J, Zavos C, Deretzi G. Nonalcoholic fatty liver disease: multimodal treatment options for a pathogenetically multiple-hit disease. J Clin Gastroenterol. 2012;46:272–84.

6. Okanoue T, Umemura A, Yasui K, Itoh Y. Nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in Japan. J Gastroenterol Hepatol. 2011;26 Suppl 1:153–62.

7. Yasui K, Hashimoto E, Komorizono Y, Koike K, Arii S, Imai Y, et al. Characteristics of patients with nonalcoholic steatohepatitis who develop hepatocellular carcinoma. Clin Gastroenterol Hepatol. 2011;9:428–33. quiz e450.

8. Hashimoto E, Yatsuji S, Tobari M, Taniai M, Torii N, Tokushige K, et al. Hepatocellular carcinoma in patients with nonalcoholic steatohepatitis. J Gastroenterol. 2009;44 Suppl 19:89–95.

9. Yatsuji S, Hashimoto E, Tobari M, Taniai M, Tokushige K, Shiratori K. Clinical features and outcomes of cirrhosis due to non-alcoholic steatohepatitis compared with cirrhosis caused by chronic hepatitis C. J Gastroenterol Hepatol. 2009;24:248–54.

10. Shimada M, Hashimoto E, Taniai M, Hasegawa K, Okuda H, Hayashi N, et al. Hepatocellular carcinoma in patients with non-alcoholic steatohepatitis. J Hepatol. 2002;37:154–60.

11. Marrero JA, Fontana RJ, Su GL, Conjeevaram HS, Emick DM, Lok AS. NAFLD may be a common underlying liver disease in patients with hepatocellular carcinoma in the United States. Hepatology. 2002;36:1349–54.

12. Bugianesi E, Leone N, Vanni E, Marchesini G, Brunello F, Carucci P, et al. Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology. 2002; 123:134–40.

13. Horie Y, Suzuki A, Kataoka E, Sasaki T, Hamada K, Sasaki J, et al. Hepatocytespecific Pten deficiency results in steatohepatitis and hepatocellular carcinomas. J Clin Invest. 2004;113:1774–83.

14. Anstee QM, Goldin RD. Mouse models in non-alcoholic fatty liver disease and steatohepatitis research. Int J Exp Pathol. 2006;87:1–16.

15. Sahai A, Malladi P, Pan X, Paul R, Melin-Aldana H, Green RM, et al. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin. Am J Physiol Gastrointest Liver Physiol. 2004;287:G1035–43.

16. Deng QG, She H, Cheng JH, French SW, Koop DR, Xiong S, et al. Steatohepatitis induced by intragastric overfeeding in mice. Hepatology. 2005;42:905–14.

17. Hill-Baskin AE, Markiewski MM, Buchner DA, Shao H, DeSantis D, Hsiao G, et al. Diet-induced hepatocellular carcinoma in genetically predisposed mice. Hum Mol Genet. 2009;18:2975–88.

18. Raubenheimer PJ, Nyirenda MJ, Walker BR. A choline-deficient diet exacerbates fatty liver but attenuates insulin resistance and glucose intolerance in mice fed a high-fat diet. Diabetes. 2006;55:2015–20.

19. Weltman MD, Farrell GC, Liddle C. Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation. Gastroenterology. 1996;111:d1645–53.

20. Okumura K, Ikejima K, Kon K, Abe W, Yamashina S, Enomoto N, et al. Exacerbation of dietary steatohepatitis and fibrosis in obese, diabetic KK-A(y) mice. Hepatol Res. 2006;36:217–28.

21. Fujii M, Shibazaki Y, Wakamatsu K, Honda Y, Kawauchi Y, Suzuki K, et al. A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma. Med Mol Morphol. 2013;46:141–52.

22. Gans JH. Diethylnitrosamine-induced changes in mouse liver morphology and function. Proc Soc Exp Biol Med. 1976;153:116–20.

23. Travis CC, McClain TW, Birkner PD. Diethylnitrosamine-induced hepatocarcinogenesis in rats: a theoretical study. Toxicol Appl Pharmacol. 1991;109:289–304.

24. He XY, Smith GJ, Enno A, Nicholson RC. Short-term diethylnitrosamine-induced oval cell responses in three strains of mice. Pathology. 1994;26:154–60.

25. Onishi M, Sokuza Y, Nishikawa T, Mori C, Uwataki K, Honoki K, et al. Different mutation patterns of mitochondrial DNA displacement-loop in hepatocellular carcinomas induced by N-nitrosodiethylamine and a cholinedeficient l-amino acid-defined diet in rats. Biochem Biophys Res Commun. 2007;362:183–7.

26. Shimizu K, Onishi M, Sugata E, Sokuza Y, Mori C, Nishikawa T, et al. Disturbance of DNA methylation patterns in the early phase of hepatocarcinogenesis induced by a choline-deficient L-amino acid-defined diet in rats. Cancer Sci. 2007;98:1318–22.

27. de Lima VM, Oliveira CP, Alves VA, Chammas MC, Oliveira EP, Stefano JT, et al. A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma. J Hepatol. 2008;49:1055–61.

28. Dowman JK, Hopkins LJ, Reynolds GM, Nikolaou N, Armstrong MJ, Shaw JC, et al. Development of hepatocellular carcinoma in a murine model of nonalcoholic steatohepatitis induced by use of a high-fat/fructose diet and sedentary lifestyle. Am J Pathol. 2014;184:1550–61.

29. Nakagawa H, Umemura A, Taniguchi K, Font-Burgada J, Dhar D, Ogata H, et al. ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development. Cancer Cell. 2014;26:331–43.

30. Itoh M, Suganami T, Nakagawa N, Tanaka M, Yamamoto Y, Kamei Y, et al. Melanocortin 4 receptor-deficient mice as a novel mouse model of nonalcoholic steatohepatitis. Am J Pathol. 2011;179:2454–63.

31. Paula Santos N, Colaco A, da Costa RM G, Manuel Oliveira M, Peixoto F, Alexandra Oliveira P. N-diethylnitrosamine mouse hepatotoxicity: Timerelated effects on histology and oxidative stress. Exp Toxicol Pathol. 2014; 66(9-10):429–36.

32. Teufelhofer O, Parzefall W, Kainzbauer E, Ferk F, Freiler C, Knasmuller S, et al. Superoxide generation from Kupffer cells contributes to hepatocarcinogenesis: studies on NADPH oxidase knockout mice. Carcinogenesis. 2005;26:319–29.

33. Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005;115:1343–51.

34. Chitturi S, Abeygunasekera S, Farrell GC, Holmes-Walker J, Hui JM, Fung C, et al. NASH and insulin resistance: Insulin hypersecretion and specific association with the insulin resistance syndrome. Hepatology. 2002;35:373–9.

35. Brennan AM, Mantzoros CS. Drug Insight: the role of leptin in human physiology and pathophysiology–emerging clinical applications. Nat Clin Pract Endocrinol Metab. 2006;2:318–27.

36. Imajo K, Fujita K, Yoneda M, Nozaki Y, Ogawa Y, Shinohara Y, et al. Hyperresponsivity to low-dose endotoxin during progression to nonalcoholic steatohepatitis is regulated by leptin-mediated signaling. Cell Metab. 2012;16:44–54.

37. Park EJ, Lee JH, Yu GY, He G, Ali SR, Holzer RG, et al. Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell. 2010;140:197–208.

38. Wolf MJ, Adili A, Piotrowitz K, Abdullah Z, Boege Y, Stemmer K, et al. Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes. Cancer Cell. 2014;26:549–64.

Štítky
Gastroenterológia a hepatológia
Prihlásenie
Zabudnuté heslo

Nemáte účet?  Registrujte sa

Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa