Hepatic Deletion of Carbohydrate Response Element Binding Protein Impairs Hepatocarcinogenesis in a High-Fat Diet-Induced Mouse Model

Abstract

The transcription factor carbohydrate response element binding protein (ChREBP) has emerged as a crucial regulator of hepatic glucose and lipid metabolism. The increased ChREBP activity involves the pro-oncogenic PI3K/AKT/mTOR signaling pathway that induces aberrant lipogenesis, thereby promoting hepatocellular carcinomas (HCC). However, the molecular pathogenesis of ChREBP-related hepatocarcinogenesis remains unexplored in the high-fat diet (HFD)-induced mouse model. Male C57BL/6J (WT) and liver-specific (L)-ChREBP-KO mice were maintained on either a HFD or a control diet for 12, 24, and 48 weeks, starting at the age of 4 weeks. At the end of the feeding period, mice were perfused, and liver tissues were formalin-fixed, paraffin-embedded, sectioned, and stained for histological and immunohistochemical analysis. Biochemical and gene expression analysis were conducted using serum and frozen liver tissue. Mice fed with HFD showed a significant increase (p < 0.05) in body weight from 8 weeks onwards compared to the control. WT and L-ChREBP-KO mice also demonstrated a significant increase (p < 0.05) in liver-to-body weight ratio in the 48-week HFD group. HFD mice exhibited a gradual rise in hepatic lipid accumulation over time, with 24-week mice showing a 20-30% increase in fat content, which further advanced to 80-100% fat accumulation at 48 weeks. Both dietary source and the increased expression of lipogenic pathways at transcriptional and protein levels induced steatosis and steatohepatitis in the HFD group. Moreover, WT mice on a HFD exhibited markedly higher inflammation compared to the L-ChREBP-KO mice. The enhanced lipogenesis, glycolysis, persistent inflammation, and activation of the AKT/mTOR pathway collectively resulted in significant metabolic disturbances, thereby promoting HCC development and progression in WT mice. In contrast, hepatic loss of ChREBP resulted in reduced hepatocyte proliferation in the HFD group, which significantly contributed to the impaired hepatocarcinogenesis and a reduced HCC occurrence in the L-ChREBP-KO mice. Our present study implicates that prolonged HFD feeding contributes to NAFLD/NASH, which in turn progresses to HCC development in WT mice. Collectively, hepatic ChREBP deletion ameliorates hepatic inflammation and metabolic alterations, thereby impairing NASH-driven hepatocarcinogenesis.

Keywords: ChREBP; MLXIPL; NAFLD; NASH; PI3K/AKT/mTOR; fatty liver diseases; hepatocarcinogenesis; hepatocellular carcinoma; high-fat diet; lipogenesis; liver cancer.

Figure 1

HFD feeding resulted in diet-induced obesity and hepatomegaly in WT and L-ChREBP-KO mice over a 48-week period. (A) Body weight changes of mice over 48 weeks, starting at 4 weeks of age. (B) Percent change in body weight from baseline. (C) Gross morphology of liver in each group. (D) Changes in liver weight in each group over 48 weeks. (E) Liver-to-body weight ratio in each group ((A,B,D,E): n = 264 per group in HFD; n = 25 per group in control). Values of the data are expressed as mean ± SEM. Significant differences are indicated as follows: *** p < 0.001 vs. the control group.

Figure 2

Effect of diet and genotype on biochemical parameters. (A) Changes in blood glucose levels over a 48-week period among the groups (n = 264 per group in HFD; n = 25 per group in control). (B,C) Serum ALT and AST levels in WT and L-ChREBP-KO mice at the age of 48 weeks (n = 5–6 per group). AST: Aspartate Aminotransferase; ALT: Alanine Aminotransferase. (D–F) Hepatic glycogen, triglyceride, and total cholesterol levels in 48-week WT and L-ChREBP-KO mice (n = 4–6 per group). Values of the data are expressed as mean ± SEM. Significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the control group.

Figure 3

Effects of HFD on the expression of hepatic lipid metabolism-related genes in 48-week mice. (A–C) Expression of key genes (FASN, ACACA, and SCD1) involved in de novo. (D) Lipogenic transcription factor SREBP-1c expression in 48-week mice. (E) Intracellular fat transport-associated gene CD36 expression in response to HFD feeding (n = 3–4 per group). Values of the data are expressed as mean ± SEM. Significant differences are indicated as follows: ** p < 0.01, and *** p < 0.001 vs. the control group.

Figure 4

Changes in expression levels of AKT1/mTOR, insulin signaling, glycolysis, and inflammation-associated genes in 48-week mice. (A,B) The mRNA expression of AKT1 and mTOR in 48-week mice. (C) Insulin signaling gene expression levels. (D) Altered expression of PKM2; (E,F) Upregulation of inflammation-related genes, TNFalpha and IL6 (n = 3–4 per group). Values of the data are expressed as mean ± SEM. Significant differences are indicated as follows: ** p < 0.01, and *** p < 0.001 vs. the control group.

Figure 5

Histological assessment of NAFLD/NASH at 48 weeks in WT and L-ChREBP-KO mice. (A) Histological scoring of HFD-induced steatosis at 48 weeks within each group (n = 264 per group in HFD; n = 25 per group in control). (B) HFD feeding led to inflammation in mice at 48 weeks within each group (n = 264 per group in HFD; n = 25 per group in control). (C) NAFLD scores at 48 weeks within each group of mice (n = 264 per group in HFD; n = 25 per group in control). (D) Representative images of H&E-stained histological slides illustrating the enhanced fat accumulation in mice in response to HFD exposure. The black arrow in WT and L-ChREBP-KO indicates steatohepatitis in the HFD group. Scale bar: 100 µm. Values of the data are expressed as mean ± SEM. Significant differences are indicated as follows: *** p < 0.001 vs. the control group.

Figure 6

Representative images of immunohistochemical staining in liver tissue of WT and L-ChREBP-KO mice at 48 weeks. Activation of AKT/mTOR pathway in WT. Alteration in the expression of HK2, PKM2, and IRS1 in WT mice. Upregulation of de novo lipogenesis candidates FASN expression in WT mice. Scale bar: 100 µm.

Figure 7

Tumor development, hepatocyte proliferation activity, and histological staining of WT and L-ChREBP-KO mice. (A) Representative macroscopic appearance of the liver with HCA and HCC in WT and L-ChREBP-KO mice. The red circle marks the HCA and HCC development within liver. (B) Ki-67 proliferation index in tumor tissue of WT mice (n = 55 in WT normal liver; n = 4 in WT tumor). The proliferation index for the L-ChREBP-KO HFD group was excluded from statistical analysis because only one tumor was observed in this group. (C) Representative immunohistochemical image with Ki-67 staining in WT HFD and WT HFD tumor tissue. The proliferation index of Ki-67-stained hepatocytes was determined at 40× magnification and the nuclei were counted in three randomly chosen fields, which was approximately 2000 cells per section. Positive hepatocytes for Ki-67 are stained in brown. (D) H&E staining of WT and L-ChREBP-KO mice. The black dashed line demarcates HCC development from normal liver (left picture). Magnifications for H&E staining: 10×, 40×, and 100×. Scale bar: 1000 µm for 10× and 100 µm for 40× and 100×. Values of the data are expressed as mean ± SEM. Significant differences are indicated as follows: *** p < 0.001 vs. the control group.

Figure 8

Representative immunohistochemical staining in HCC tissue of WT and L-ChREBP-KO mice. Alteration in the expression of HK2, PKM2 and IRS1 in WT. Upregulated expression of pAKT, pmTOR, and p4EBP1 in WT. Increased expression of de novo lipogenesis proteins, ACAC, and FASN, in WT mice. Scale bar: 100 µm.