Figure 1.
Effect of diet treatment on metabolic parameters and body weight.
A–B) Mice treated with CDAA diet for 1 month develop peripheral insulin resistance as shown by the lower glucose uptake vs CSAA-treated mice (M value) with similar levels of FPG. CCl4 treatment induces an increase in fasting plasma glucose and enhances the condition of insulin resistance, reducing M value. C) Insulin levels measured at 1 month of treatment. D) The graphic shows a gradual increase in body weight in both CDAA and CSAA diet. CCl4 injection similarly reduces body weight in both diets. E) Epididymal fat/body weight ratio appears to be similar between the two different diets both at 3 and 9 months of treatment, with similar reduction in the presence of CCl4. Data represent mean ± SD; §p<0.05 vs CSAA; ù p<0.05 vs CDAA; *p<0.05 vs CSAA 3 months; #p<0.05 vs CSAA+CCl4 3 months; ò p<0.05 vs CDAA 3 months; ç p<0.05 vs CDAA+CCl4 3 months; b p<0.05 vs CSAA+CCl4 6 months; c p<0.05 vs CSAA+CCl4 9 months; d p<0.05 vs CSAA.
Figure 2.
Treatment with CDAA determines a progressive increase of hepatic steatosis.
A) Increased levels of liver/body weight fraction are observed in the group of mice treated with CDAA diet, either with or without CCl4 administration, in comparison to mice treated with control diet, starting at 1 month. B) Histochemical staining and relative morphometric evaluation for Oil Red O and C) hepatic triglyceride measurement show increased deposition of lipids in the liver parenchyma of CDAA and CDAA+CCl4 treated animals in comparison to CSAA treated mice. Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.
Figure 3.
Gene expression of elements involved in carbohydrate and lipid metabolism.
qRT-PCR expression shows a progressive decrease of the amount of PEPCK (A), G6Pase (B), ChREBP (D), ACOX-1 (E) and CPT1A (F) mRNA expression in the groups of CDAA or CDAA+ CCl4, while only a slight decrease is observed for mRNA expression of SREBP-1c (C). Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.
Figure 4.
Treatment with CDAA or CDAA+CCl4 induces hepatic inflammation in mice.
A) Immunohistochemistry for pan-macrophage marker F4/80. B) TNFα and (C) MCP-1 mRNA levels are increased in CDAA or CDAA+ CCl4 already after 1 month of treament. D–F) Markers of Inflammasome activation ASC, Caspase-1 and IL-1β show increased expression in CDAA or CDAA+ CCl4 groups. Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.
Figure 5.
Treatment with CDAA or CDAA+CCl4 induces liver injury and expression of markers of hepatic fibrogenesis in mice.
A) The total degree of liver injury (hepatic steatosis and necroinflammation) was determined according to the NAS score. B) The number of apoptotic cells was measured with TUNEL and expressed as apoptotic cell/field. C–E) qRT-PCR shows a progressive increase, in a time dependent manner, of the amount of TIMP-1 (C), CTGF (D) and TGFβ (E) mRNA expression in the group of CDAA, with the evidence of a promoting effect exerted by CCl4 starting from month 3. Data represent mean ± SD; ò p<0.05 vs CSAA 1 month; *p<0.05 vs CSAA 3 months; §p<0.05 vs CSAA 6 months; #p<0.05 vs CSAA 9 months; ç p<0.05 vs CDAA 6 months; ù p<0.05 vs CDAA 9 months.
Figure 6.
Treatment with CDAA or CDAA+CCl4 induces hepatic fibrosis in mice.
A–B) qRT-PCR expression shows a progressive increase, in a time dependent manner, of the amount of Collagen α1(I) (A) and αSMA (B) mRNA in the group of CDAA, with the evidence of a promoting effect exerted by CCl4 starting from month 6. Conversely, mice treated with control diet (CSAA) do not show any time-dependent increase neither in the level of Collagen α1(I) nor of αSMA, not even after chronic CCl4 injection. C–D) Morphometric analysis shows increased collagen deposition by Sirius Red staining (C) and increased HSC activation by αSMA Immunohistochemistry (D) in the liver of CDAA treated mice in comparison to CSAA treated mice. This condition is even enhanced in the liver of mice treated with CDAA+CCl4. Data represent mean ± SD; *p<0.05 vs CSAA; #p<0.05 vs CSAA+ CCl4; ò p<0.05 vs CDAA.
Figure 7.
Treatment with CDAA+CCl4 induces development of HCC after 9 months.
A) Mice sacrificed after 1 and 3 months from the beginning of the treatment do not show any nodular lesions in the parenchyma. Conversely, mice sacrificed after 6 months of treatment show few nodular lesions compatible with HCC. After 9 months of treatment mice show a liver parenchyma completely damaged. B) Histochemical staining for H&E and Sirius Red show respectively no fat deposition and absence of collagen content into the nodules. C) After 6 months of treatment, about 30% of mice show HCC development both with CDAA and CDAA+CCl4. After 9 months of treatment, 100% of mice from the CDAA+CCl4 group show at least one nodule into the liver parenchyma. D) After 6 months, the average of nodules dimension in the group of CDAA mice is significantly lower in comparison to that of the group of CDAA+CCl4 mice. After 9 months, the average of nodules dimension in the group of CDAA+CCl4 mice results of 9 mm, in comparison to 3 mm of the group of mice treated with CDAA alone. Data represent mean ± SD; *p<0.05 vs CDAA.
Figure 8.
The expression of pro-carcinogenic genes is modified already after 3 months of treatment.
A) Immunohistochemistry for p-AKT and p-c-Myc shows positive staining in the liver parenchyma of CDAA+CCl4 mice, in correspondence of HCC foci (20X magnification) B) Glypican-3 Immunohistochemistry shows positive staining in the tumor area (5X and 20X magnification). C) Mice treated with CDAA or CDAA+CCl4 show, starting at 3 months, increased levels of mRNA for IGF-2 and (D) SPP-1 and (E) decreased mRNA levels of PTEN. Data represent mean ± SD; *p<0.05 vs CSAA; #p<0.05 vs CSAA+CCl4.