Development of hepatocellular cancer induced by long term low fat-high carbohydrate diet in a NAFLD/NASH mouse model

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease. It can progress to nonalcoholic steatohepatitis (NASH) and, in a percentage of cases, to hepatocarcinogenesis. The strong incidence in western countries of obesity and metabolic syndrome, whose NAFLD is the hepatic expression, is thought to be correlated to consumption of diets characterized by processed food and sweet beverages. Previous studies described high-fat diet-induced liver tumors. Conversely, the involvement of low-fat/high-carbohydrate diet in the progression of liver disease or cancer initiation has not been described yet. Here we show for the first time hepatic cancer formation in low-fat/high-carbohydrate diet fed NAFLD/NASH mouse model. Animals were long term high-fat, low-fat/high-carbohydrate or standard diet fed. We observed progressive liver damage in low-fat/high-carbohydrate and high-fat animals after 12 and, more, 18 months. Tumors were detected in 20% and 50% of high-fat diet fed mice after 12 and 18 months and, interestingly, in 30% of low-fat/high-carbohydrate fed animals after 18 months. No tumors were detected in standard diet fed mice. Global increase of hepatic interleukin-1β, interleukin-6, tumor necrosis factor-α and hepatocyte growth factor was detected in low-fat/high-carbohydrate and high-fat with respect to standard diet fed mice as well as in tumor with respect to non-tumor bearing mice. A panel of 15 microRNAs was analyzed: some of them revealed differential expression in low-fat/high-carbohydrate with respect to high-fat diet fed groups and in tumors. Data here shown provide the first evidence of the involvement of low-fat/high-carbohydrate diet in hepatic damage leading to tumorigenesis.

Keywords: HF diet; LF-HC diet; NAFLD; NASH; hepatic cancer.

Figure 1

(A) Body weights. HF, LF-HC or SD (CTRL) control mice were weighed at the indicated time points (X axis). Values are mean ± SEM. Statistical significance as follows: HF vs. LF-HC P_{3M, 6M, 12M}<0.001, P_18M=0.004; HF vs. SD P_3M=0.0003, P_6M=0.0001, P_12M=0.0006; LF-HC vs. SD P_6M=0.002, P_18M=0.013. (B) Liver weights, expressed as mean ± SEM. Statistical significance as follows: *, P<0.01; **, P<0.006. (C) Livers from 12 and 18 months HF, LF-HC and SD mice. (D) Macroscopic nodules on livers from 18M HF (2 out of 5) and LF-HC mice (arrows).

Figure 2. Histological features of hepatic tissues from SD, LF-HC and HF mice

(A) Masson's trichrome staining (original magnification 10X, scale bar: 100 μm). Steatosis aspects were preeminent in 12 and 18 months HF mice compared to 12 and 18 months LF-HC. In SD mice ballooning degeneration was prevalent (black head arrow). Scattered inflammatory infiltrate (arrows) can be observed in LF-HC and, more severe, in HF mice. Fibrosis, was increasing in LF-HC and HF mice without resembling the typical aspect of cirrhosis. Bottom: graphical representation of the severity of pathological conditions in liver tissues from SD, LF-HC and HF mice. P as follows: * P<0.05, ** P<0.005. (B) Masson's trichrome (a,b), Sirius Red-Fast Green staining (c,d) and immunohistochemistry for CD31 (e,f) (original magnification 20X, scale bar 50 μm) of 18 months LF-HC and HF liver tissues. The microphotographs show irregular thin trabeculae (a,b,c,d) and a certain numbers of neovessels with CD31 immunopositivity (e,f). Masson's trichrome staining (g,h) of a macroscopic nodule's section (LF-HC T) from a 18 months LF-HC (original magnification 20X, scale bar 50 μm and 40X scale bar 25 μm) shows an increase of cellular density (asterisk: g,h) with aspects of atypia.

Figure 3. Cytokines and growth factor expression in liver tissues from mice without (NT) and with (T) tumors

(A) Comparison between pooled RNAs from non-tumor hepatic tissues, obtained from NT and T HF mice. Pooled RNAs from 12 months SD mice livers were used as reference. (B) Comparison between pooled RNAs from non-tumor hepatic tissues, obtained from NT and T HF or LF-HC mice. Pooled RNAs from 18 months SD mice livers were used as reference. HPRT was used as endogenous control. RQ: relative quantification. Data expressed as mean ± SE. P as follows: * P<0.05, ** P<0.005, *** P<0.0005. Black stars: significant differences resulting from T/NT LF-HC or T/NT HF vs. SD fed mice. Gray stars: significant differences resulting from the comparison between non tumor NT and tumor T bearing mice.

Figure 4. MiRNA expression analysis

(A) Expression of 15 miRNAs in non tumor hepatic tissues from 12 and 18 months HF vs. LF-HC mice. (B) MiRNAs expression in tumors from HF (12 and 18 months) and LF-HC (18 months) mice compared to respective normal HF and LF-HC hepatic tissues. Mamm-U6 was used as endogenous control. All the experiments are mean ± SE of three iterations. RQ: relative quantification.