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

Norihiro Kishida, Sachiko Matsuda, Osamu Itano, Masahiro Shinoda, Minoru Kitago, Hiroshi Yagi, Yuta Abe, Taizo Hibi, Yohei Masugi, Koichi Aiura, Michiie Sakamoto, Yuko Kitagawa

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. 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. 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. 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.