HCC Development Is Associated to Peripheral Insulin Resistance in a Mouse Model of NASH

Abstract

NAFLD is the most common liver disease worldwide but it is the potential evolution to NASH and eventually to hepatocellular carcinoma (HCC), even in the absence of cirrhosis, that makes NAFLD of such clinical importance. Aim: we aimed to create a mouse model reproducing the pathological spectrum of NAFLD and to investigate the role of possible co-factors in promoting HCC. Methods: mice were treated with a choline-deficient L-amino-acid-defined-diet (CDAA) or its control (CSAA diet) and subjected to a low-dose i.p. injection of CCl4 or vehicle. Insulin resistance was measured by the euglycemic-hyperinsulinemic clamp method. Steatosis, fibrosis and HCC were evaluated by histological and molecular analysis. Results: CDAA-treated mice showed peripheral insulin resistance at 1 month. At 1–3 months, extensive steatosis and fibrosis were observed in CDAA and CDAA+CCl4 groups. At 6 months, equal increase in steatosis and fibrosis was observed between the two groups, together with the appearance of tumor. At 9 months of treatment, the 100% of CDAA+CCl4 treated mice revealed tumor versus 40% of CDAA mice. Insulin-like Growth Factor-2 (IGF-2) and Osteopontin (SPP-1) were increased in CDAA mice versus CSAA. Furthermore, Immunostaining for p-AKT, p-c-Myc and Glypican-3 revealed increased positivity in the tumors. Conclusions: the CDAA model promotes the development of HCC from NAFLD-NASH in the presence of insulin resistance but in the absence of cirrhosis. Since this condition is increasingly recognized in humans, our study provides a model that may help understanding mechanisms of carcinogenesis in NAFLD.

Hypoxia‐inducible factor 2α drives nonalcoholic fatty liver progression by triggering hepatocyte release of histidine‐rich glycoprotein

“…Mice carrying a hepatocyte‐specific deletion of HIF‐2α (hHIF‐2α –/– ) were obtained by breeding HIF‐2α fl/fl C57BL/6 mice with mice on the same genetic background expressing Cre‐recombinase under the control of the albumin promoter (Alb/Cre +/+ mice; Jackson Laboratories, Bar Harbor, ME). Eight‐week‐old male hHIF‐2α –/– mice and related control sibling littermates not carrying the HIF‐2α deletion were fed either a methionine/choline deficient (MCD) diet for 4 or 8 weeks or a choline‐deficient L‐amino acid–defined (CDAA) diet (Laboratorio Dottori Piccioni, Gessate, Italy) for 12 and 24 weeks . Control littermates received the corresponding methionine/choline‐sufficient or the choline‐sufficient L‐amino acid–defined (CSAA) diet.…”

“…Eightweek-old male hHIF-2a -/mice and related control sibling littermates not carrying the HIF-2a deletion were fed either a methionine/choline deficient (MCD) diet for 4 or 8 weeks or a choline-deficient L-amino acid-defined (CDAA) diet (Laboratorio Dottori Piccioni, Gessate, Italy) for 12 and 24 weeks. (26,27) Control littermates received the corresponding methionine/choline-sufficient or the choline-sufficient L-amino acid-defined (CSAA) diet. In preliminary experiments 8-week-old normal C57BL/6 mice were fed the MCD diet or the methionine/choline-sufficient control diet for 4 and 8 weeks.…”

Hepatocellular carcinoma in a mouse model fed a choline‐deficient, L‐amino acid‐defined, high‐fat diet

“…; De Minicis et al . ). Unfortunately, because these chemical substances themselves are genotoxic, there is a possibility that the cancer is caused by these substances.…”

“…; De Minicis et al . ) or a high‐fat, high‐fructose diet (Murine ALIOS Model) (Dowman et al . ), can induce NASH‐associated tumour development in wild‐type mice.…”

An oxidative stress paradox: time for a conceptual change?

“…Patients with diabetes are much more likely to have steatosis and NASH than the general population, yet a clear reason for this link remains undetermined [11]. Here, the authors challenged mice with a CDAA diet, which induces NASH pathology in the context of normal weight gain (not obesity) [12]. Despite relatively small numbers of mice, they surprisingly found that HGKO mice had less lobular inflammation and circulating levels of the inflammatory cytokines, TNFα, IL-6 and IFN-γ, with similar levels of steatosis.…”