Impaired liver regeneration and lipid homeostasis in CCl4 treated WDR13 deficient mice

Abstract

WDR13 - a WD repeat protein, is abundant in pancreas, liver, ovary and testis. Absence of this protein in mice has been seen to be associated with pancreatic β-cell proliferation, hyperinsulinemia and age dependent mild obesity. Previously, we have reported that the absence of WDR13 in diabetic Lepr^db/db mice helps in amelioration of fatty liver phenotype along with diabetes and systemic inflammation. This intrigued us to study direct liver injury and hepatic regeneration in Wdr13^-/0 mice using hepatotoxin CCl₄. In the present study we report slower hepatic regeneration in Wdr13^-/0 mice as compared to their wild type littermates after CCl₄ administration. Interestingly, during the regeneration phase, hepatic hypertriglyceridemia was observed in Wdr13^-/0 mice. Further analyses revealed an upregulation of PPAR pathway in the liver of CCl₄- administered Wdr13^-/0 mice, causing de novo lipogenesis. The slower hepatic regeneration observed in CCl₄ administered Wdr13^-/0 mice, may be linked to liver hypertriglyceridemia because of activation of PPAR pathway.

Keywords: Fatty liver; Hepatosteatosis; Hepatotoxin; Hypertriglyceridemia; PPARg.

Fig. 1

Effect of Wdr13 deletion on liver regeneration after CCl₄ administration a Ki-67 immunostaining of liver sections from control and CCl₄ administered Wdr13^+/0 and Wdr13^−/0 mice. b Quantitative representation of Ki-67 positive cells from control and CCl₄ administered Wdr13^+/0 and Wdr13^−/0 liver_. c Liver/body weight ratios of control and CCl₄ administered Wdr13^+/0 and Wdr13^−/0 mice. d p53, Cyclin E and Cyclin D1 immunoblots performed on liver proteins. All the images presented here are cropped images of full length blots for better representation and all the gels were run under similar experimental conditions. e Relative protein levels of p53, Cyclin E and Cyclin D1 showed in (d) immunoblots. ImageJ analysis of western blots was performed for quantification normalised with respective β-actin as loading control. n = 5 mice for controls and n = 8 mice for treatment group, **p < 0.01, ***p < 0.001

Fig. 2

Effect of Wdr13 deletion on liver triglyceride levels after CCl₄ administration a Lipid droplets seen by Oil Red O staining of liver sections from control and CCl₄ treated Wdr13^+/0 and Wdr13^−/0 mice. Inset shows enlarged CCl₄ treated Wdr13^−/0 liver section indicating red lipid droplets. b Total triglyceride content of liver from control and CCl₄ treated Wdr13^+/0 and Wdr13^−/0 mice. c Relative mRNA expression levels of lipogenic genes in CCl₄ treated Wdr13^+/0 and Wdr13^−/0 mice. n = 5 mice for controls and n = 8 mice for treatment group, *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 3

Effect of Wdr13 deletion on liver PPAR pathway after CCl₄ administration a AC1, P-p38α, p38α and PPARγ immunoblots performed on liver proteins. All the images presented here are cropped images of full length blots for better representation and all the gels were run under similar experimental conditions. b Relative protein levels of AC1, p38α and PPARγ showed in (a) immunoblots. ImageJ analysis of western blots was performed for quantification normalised with respective β-actin as loading control. c Relative mRNA expression levels of PPAR pathway genes in CCl₄ treated Wdr13^+/0 and Wdr13^−/0 mice. d Relative mRNA expression levels of PPARγ target genes in CCl₄ treated Wdr13^+/0 and Wdr13^−/0 mice. n = 5 mice for controls and n = 8 mice for treatment group, *p < 0.05, **p < 0.01. (AC1- Adenylyl Cyclase 1)

Fig. 4

Effect of Wdr13 deletion on CCl₄ toxicity in primary hepatocytes a CCl₄ toxicity on primary hepatocytes analysed by MTT assay. b Relative mRNA expression levels of genes involved in lipid metabolism studied in hepatocytes treated with 0.1 M CCl₄ for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3

Fig. 5

Effect of Wdr13 deletion on serum insulin, glucose, lipids levels and mRNA expression levels of insulin responsive genes in liver after CCl₄ administration a Mean random serum insulin levels (ng/μl) in control and CCl₄ administered Wdr13^+/0 and Wdr13^−/0 mice. b Mean random serum glucose levels (mg/dl) in control and CCl₄ administered Wdr13^+/0 and Wdr13^−/0 mice. c Serum triglycerides and cholesterol levels (μg/ml) in control and CCl₄ administered Wdr13^+/0 and Wdr13^−/0 mice. d Relative mRNA expression levels of insulin responsive genes in CCl₄ administered Wdr13^+/0 and Wdr13^−/0 liver. n = 5 mice for controls and n = 8 mice for treatment group, *p < 0.01