Differential progression of unhealthy diet-induced hepatocellular carcinoma in obese and non-obese mice

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) ranks first among liver diseases in Western countries. NAFLD is typically associated with obesity and diabetes, however it also develops in lean individuals without metabolic syndrome. The prevalence of lean NAFLD is 7 percent in the U.S. and 25-30 percent in some Asian countries. NAFLD starts with excess liver fat accumulation (NAFL), progresses to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). The pathogenesis of lean NASH-HCC and how it differs from obese NASH-HCC is not well understood.

Methods: In this work, we generated a mouse model of lean and obese NASH-HCC using a choline deficient/high trans-fat/fructose/cholesterol diet and a choline supplemented/high trans-fat/fructose/cholesterol diet, respectively, to compare progression to NASH-HCC in lean versus obese mice. Comparisons were made at the organismal, histological, and molecular level by investigating fatty acid metabolism in the plasma of these mice.

Results: Obese mice showed more pronounced glucose intolerance and insulin resistance, higher levels of plasma cholesterol and triglycerides, and higher penetrance of NASH compared to lean mice. Despite the abnormal metabolic profile of obese mice, male obese and lean mice developed HCC with similar penetrance (53.3% and 53.8%, respectively), albeit lean mice showed faster tumor progression as evidenced by the larger tumor size and lower HCC-free survival. None of the female lean mice developed HCC, while 50% of female obese mice developed HCC. Both groups of mice showed a reduction in plasma polyunsaturated fatty acids (PUFAs), however, the levels were higher towards the endpoint in obese mice compared to lean mice.

Conclusions: Unhealthy diet composition appears to drive progression to NASH-HCC rather than the organismal effects of obesity. PUFA levels may increase due to systemic inflammation in obese mice and act as suppressors of tumor progression, thus delaying HCC progression in obese mice compared to lean mice. These models could be used to further dissect the molecular pathogenesis of lean and obese NASH-HCC and address the mechanisms whereby PUFAs may be implicated in hepatocarcinogenesis.

Fig 1. Weight gain and food consumption.

(A) Weight gain of mice fed with the control, CD-HFFC, and CS-HFFC diets. Male and female mice fed with the CS-HFFC diet gained significantly more weight than mice fed with the control or CD-HFFC diets. Male CS-HFFC fed with mice were considered obese at 14 weeks of age while females fed with the same diet became obese at 10 weeks of age. (B) and (C) Feed consumption assessment per cage. Overall, the three groups of mice seem to be consuming the same amount of feed.

Fig 2. Survival curves.

(A) HCC-free, and (B) Disease-free survival of male mice fed with the different diet types. Mice fed with the CD-HFFC diet had poorer disease-and HCC-related survival.

Fig 3. Metabolic function, plasma lipids, and plasma enzymes in mice fed with the control (C), CD-HFFC (CD), and CS-HFFC (CS) diets.

(A) Glucose tolerance. Both male and female mice fed with the CS-HFFC diet exhibited intolerance to glucose at 48 (p = 1.11 x 10–16, 1.02 x 10–13) and 60 (p = 1.11 x 10–16, 1.11 x 10–16) weeks of age. (B) Insulin resistance. No differences were observed until 48 weeks of age. Male and female mice fed with the CS-HFFC diet were insulin resistant at both 48 (p = 5.41 x 10–13, 5.31 x 10–6) and 60 (p = 7.59 x 10–13, 0.015) weeks of age. (C) Plasma cholesterol and (D) plasma triglyceride levels. (E) Plasma AST and (F) plasma ALT levels. Horizontal black lines indicate lower and upper limits of normal plasma lipid and enzyme levels. Statistical differences between diet groups are shown with either a,b,c or x,y,z, markings above the graphs (one set used for males and the other for females), where statistically significant differences would be denoted by different letters across diets.

Fig 4. Descriptive results from liver biopsy at study midpoint and necropsy at study endpoint.

(A) Distribution of steatosis, (B) inflammation, and (C) ballooning scores at study midpoint and endpoint. (D) Fibrosis scoring at midpoint and endpoint. Both male and female mice fed with the CD-HFFC diet developed a higher degree of fibrosis than mice fed with the CS-HFFC diet at the study midpoint, however CS-HFFC fed mice develop a higher degree of fibrosis by the study endpoint. All mice fed with the control diet had a fibrosis score of 0 and had normal liver phenotypes. (E) Liver disease diagnosis. All mice fed with the CS-HFFC diet developed NASH by study midpoint. All mice fed with both the CD-HFFC and CS-HFFC diet developed NASH by study endpoint. (F) Prevalence of HCC at study endpoint (64 weeks of age).

Fig 5. Histological pictures showing the various degrees of steatosis, inflammation, ballooning and fibrosis.

(A) Steatosis scores of 1–3. (B) Inflammation scores of 1–3. (C) Ballooning scores of 1–3. (D) Fibrosis scores of 1–3.

Fig 6. Histological pictures showing representative images of dysplastic nodules and HCC.

(A) Micrographs representing dysplastic nodules. (B) Micrographs of hepatocellular carcinoma.