Hepatic cellular senescence pathway genes are induced through histone modifications in a diet-induced obese rat model

Abstract

Overnutrition, such as a high-fat (HF) diet, is a feature followed by some in developed nations that leads to obesity and fatty liver disease. In rats, when fed a fat-high diet, some develop obesity (obesity prone, OP) while others display an obesity-resistant (OR) phenotype. The present study investigated the differences between OP and OR rats on their activation of hepatic cellular senescence pathways on a HF diet. Male OP and OR rats were fed a HF diet containing 45% kcal from fat for 13 wk, and livers were collected for analysis by quantitative real-time PCR, Western blot, and chromatin immunoprecipitation. OP rats were 41% heavier than OR rats, despite consuming the same amount of food. Triacylglycerol levels were increased significantly in both plasma and liver of OP rats. Gene analysis demonstrated a significant increase of both the amount and the transcription rates of p16(INK4a) and p21(Cip1) mRNA in OP rats. The increased p16(INK4a) and p21(Cip1) also caused a significant decrease in the level of phosphorylation of retinoblastoma protein. In OP rats, the increase of p16(INK4a) was associated with the higher acetylation levels of histone H4 at the p16(INK4a) promoter and coding region and lower methylation level of histone H3 lysine-27 in the p16(INK4a) coding region. The increase of p21(Cip1) was associated with increased acetylation of both histone H3 and H4 and decreased trimethylation of histone H3 lysine-27 at the p21(Cip1) promoter. In the p21(Cip1) coding region, dimethylation of histone H3 lysine-4 was significantly higher (P <0.05) in livers of OP rats compared with OR rats.

Fig. 1.

Physiological outcomes of obesity-prone (OP) and obesity-resistant (OR) rats following 13 wk of high-fat (HF) feeding. A: food intake/g body wt of OR (n = 8) and OP (n = 7) rats. B: differences in body wt of OR and OP rats. C: triglyceride concentration in plasma. D: triglyceride (TAG) concentration in liver. Values are means ± SE. *P <0.05.

Fig. 2.

Hepatic steatosis by hematoxylin and eosin staining. A: hepatic pathology of OR rat liver showing mild liver steatosis. B: hepatic pathology of OP rat liver showing steatohepatitis and ballooning degeneration. The magnification of photomicrographs is ×40.

Fig. 3.

Liver cell cycle control genes in OP and OR rats. Shown are p16^INK4a and p21^Cip1 (A) and p27 and p53 (B) mRNA levels in OR (n = 8) and OP (n = 7) rats as determined by quantitative real-time PCR. Values are means ± SE. *P <0.05. C: p16^INK4a and p21^Cip1 transcription rate under HF diet in male OR and OP rats as determined by quantitative real-time PCR. Values are means ± SE. *P <0.05.

Fig. 4.

Phospho-retinoblastoma (Rb) pathway in livers of OR and OP rats. A: expression of p21^Cip1 protein in liver of OR and OP rats analyzed by Western blotting. NC, normal control. B: expression of phospho-Rb protein in liver of OR and OP rats analyzed by Western blotting. C: graph presents protein expression level as mean ± SE (n = 4). *P <0.05.

Fig. 5.

Chromatin modification at the p16^INK4a promoter and coding region in OR and OP rats. In vivo binding of specific factors to different regions of the p16^INK4a gene in OR (n = 8) and OP (n = 7) rats as determined by chromatin immunoprecipitation (ChIP) assay. F, forward; R, reverse. Data are presented as the ratio to the value obtained with input DNA. Values are means ± SE. *P <0.05.

Fig. 6.

Chromatin modification at the p21^Cip1 promoter and coding region in OR and OP rats. In vivo binding of specific factors to different regions of the p21^Cip1 gene in OR (n = 8) and OP (n = 7) rats as determined by ChIP assay. Data are presented as the ratio to the value obtained with input DNA. Values are means ± SE. *P <0.05.