Reduction of Polyunsaturated Fatty Acids with Tumor Progression in a Lean Non-Alcoholic Steatohepatitis-Associated Hepatocellular Carcinoma Mouse Model

Abstract

Background and Aim: Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. While obesity and diabetes are the hallmarks of NAFLD, it also develops in lean individuals in the absence of metabolic syndrome, with a prevalence of 7 percent in the U.S. and 25-30 percent in some Asian countries. NAFLD represents the spectrum of liver disease, starting with excess liver fat accumulation (NAFL) that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis and ultimately hepatocellular carcinoma (HCC). To date, the pathogenesis of lean NASH-HCC is poorly understood and a mouse model is lacking. We aimed to develop a mouse model of lean NASH-HCC using a choline deficient and high trans-fat/sucrose/cholesterol diet to enable better understanding of its molecular pathogenesis. Methods: C57BL/6N mice were fed this diet starting at 4 weeks of age for 52 weeks and were compared to mice fed an isocaloric low fat control diet for the same duration. C57BL/6N mice were chosen instead of the C57BL/6J mice due to the high susceptibility of C57BL/6J mice to diet-induced obesity. The plasma and tumor fatty acid profile of these mice was also investigated. Results: Nearly 61% of the mice developed lean NASH-HCC. These mice showed reduction of plasma polyunsaturated fatty acids (PUFAs) (linolenic acids (α and γ, ω-3 and ω-6, respectively), eicosapentanoic acid (ω-3), docosahexanoic acid (ω-3), and linoleic acid (ω-6)) and increasing levels over time in mice with pre-malignant lesions. Conclusions: We developed a novel high penetrance diet-induced lean NASH-HCC mouse model. Plasma PUFA levels were reduced with tumor progression in parallel with reduced expression of genes controlling desaturase expression suggesting their potential use as biomarkers for lean NASH-HCC progression as well as chemopreventive molecules.

Keywords: fat; liver cancer; non-alcoholic fatty liver disease; sugar; unhealthy diet.

Figure 1

Weight gain and food consumption. A. Weight of mice fed the control diet (black) and the CD-HFSC diet (grey). Mice fed the CD-HFSC diet gained weight but had lower weight compared to mice fed the control diet (P < 0.001 after week 4). B. Feed consumption was assessed per cage. Overall the two groups of mice seem to be consuming the same amount of food. * = p < 0.05.

Figure 2

Glucose tolerance tests in mice fed the control diet (black) and the CD-HFSC diet (grey). Mice fed the CD-HFSC diet show no difference in glucose tolerance compared to mice fed the control diet.

Figure 3

A. Plasma lipid and B. plasma enzyme profile in mice fed the control diet (black) and the CD-HFSC diet (grey). Mice fed the CD-HFSC diet show lower plasma triglyceride levels and higher levels of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) compared to mice fed the control diet, likely reflecting lipid retention in the liver and liver damage, respectively. Horizontal black lines indicate lower and upper limits of normal plasma lipid and enzyme levels.

Figure 4

Mice fed the CD-HFSC diet developed NASH-HCC with high penetrance by 55-56 weeks of age. A. H&E stain of normal hepatic tissue; B. NAFL; H&E stain displaying some fat with little inflammation or hepatocyte damage; C. NASH; H&E stain with inflammation and hepatocyte damage with fat in liver; D. regenerative nodule compressing surrounding parenchyma with an intact reticulin network; E. H&E stain of dysplastic nodule compressing hepatocytes; F. H&E stain of HCC compressing surrounding hepatocytes with focal destruction of reticulin framework.

Figure 5

Fatty acid levels in mice fed the CD-HFSC diet with HCC and pre-malignant (PRE) nodules, and mice with NAFLD fed the control diet (NAFLD-CON). A and B. Mice with nodules (PRE and HCC) had significantly lower plasma levels of specific fatty acids than control mice with NAFLD at 32 and 55 weeks (* = p < 0.05). C. The levels of polyunsaturated fatty acids increased from 32 weeks to 55 weeks in mice with premalignant nodules. Polyunsaturated fatty acids = PUFA; monounsaturated fatty acids = MUFA; saturated fatty acids = SFA; +µg/mg.