Lipid overloading during liver regeneration causes delayed hepatocyte DNA replication by increasing ER stress in mice with simple hepatic steatosis

Abstract

Background and aim: Impaired fatty liver regeneration has already been reported in many genetic modification models. However, in diet-induced simple hepatic steatosis, which showed similar phenotype with clinical pathology, whether liver regeneration is impaired or not remains unclear. In this study, we evaluated liver regeneration in mice with diet-induced simple hepatic steatosis, and focused on excess lipid accumulation occurring during liver regeneration.

Methods: Mice were fed high fat diet (HFD) or control diet for 9-10 weeks. We analyzed intrahepatic lipid accumulation, DNA replication, and various signaling pathways including cell proliferation and ER stress during liver regeneration after partial hepatectomy. In addition, some of mice were pretreated with tauroursodeoxycholic acid (TUDCA), a chemical chaperone which alleviates ER stress, and then we estimated TUDCA effects on liver regeneration.

Results: The peak of hepatocyte BrdU incorporation, the expression of proliferation cell nuclear antigen (PCNA) protein, and the expressions of cell cycle-related genes were observed in delayed time in HFD mice. The expression of phosphorylated Erk1/2 was also delayed in HFD mice. The amounts of liver triglyceride were at least twofold higher in HFD mice at each time point. Intrahepatic palmitic acid was increased especially in HFD mice. ER stress induced during liver regeneration was significantly higher in HFD mice. In HFD mice, pretreatment with TUDCA reduced ER stress and resulted in improvement of delayed liver regeneration.

Conclusion: In simple hepatic steatosis, lipid overloading occurring during liver regeneration might be caused ER stress and results in delayed hepatocyte DNA replication.

Fig. 1

Delayed liver regeneration after PHx in simple fatty liver. a BrdU immunohistochemical staining (original magnification ×200), and BrdU labeling index. b The expression of PCNA protein detected by Western blotting. c The mRNA levels of cell-cycle related genes, cyclin D1, cyclin E2, Foxm1, cyclin A2, and cyclin B1 measured by real-time RT-PCR. d The activations of Akt and Erk1/2 evaluated by Western blotting (black squares Control, white squares HFD, n = 5–8; mean ± SE, *p < 0.05, **p < 0.01, and ***p < 0.005 HFD mice vs. CD mice in each time point by ANOVA and Wilcoxon test)

Fig. 2

Liver mass regeneration ratio and serum liver function examinations. a Liver mass regeneration ratio during liver regeneration. b The change of plasma ALT, total bilirubin (T-Bil), and albumin (Alb) levels during liver regeneration (black squares Control, white squares HFD, n = 5–8; mean ± SE, *p < 0.05 HFD mice vs. CD mice in each time point by ANOVA and Wilcoxon test)

Fig. 3

Lipid accumulation during liver regeneration. a Oil red O staining (original magnification ×400). b Hepatic tryglyceride contents (black squares Control, white squares HFD, n = 5–8; mean ± SE, **p < 0.01 HFD mice vs. CD mice in each time point by ANOVA and Wilcoxon test)

Fig. 4

Enhanced ER stress was observed in simple fatty liver during liver regeneration. a The mRNA levels of ER stress-related genes, GRP78, IRE1α, ATF6, PERK, sXBP-1, and CHOP measured by real-time RT-PCR. b The expression of GRP78 protein detected by Western blotting and immunohistochemical staining (original magnification ×100). c The expression of sXBP-1 protein detected by Western blotting and immunohistochemical staining (original magnification ×200) (black squares Control, white squares HFD, n = 5–8; mean ± SE, *p < 0.05 and **p < 0.01 HFD mice vs. CD mice in each time point by ANOVA and Wilcoxon test)

Fig. 5

TUDCA pretreatment prevented ER stress induces during liver regeneratin in simple fatty liver. a The mRNA levels of GRP78, sXBP-1, and CHOP at 24 h after PHx with or without TUDCA pretreatment measured by real-time RT-PCR. b The expression of GRP78 protein at 24 h after PHx with or without TUDCA pretreatment detected by Western blotting and immunohistochemical staining (original magnification ×100). c The expression of sXBP-1 protein at 24 h after PHx with or without TUDCA pretreatment detected by Western blotting and immunohistochemical staining (original magnification ×200) (black squares Control, dark gray squares Control with TUDCA, white squares HFD, light gray squares HFD with TUDCA, n = 4–6; mean ± SE, *p < 0.05 mice with TUDCA pretreatment vs. mice without TUDCA pretreatment fed the same diet at each time point by ANOVA and Wilcoxon test)

Fig. 6

TUDCA pretreatment improved delayed liver regeneration in simple fatty liver. a The activation of Erk1/2 at 24 h after PHx with or without TUDCA pretreatment evaluated by Western blotting. b BrdU immunohistochemical staining (original magnification ×200) with TUDCA pretreatment, and BrdU labeling index at 36 and 48 h after PHx with or without TUDCA pretreatment. c The expression of PCNA protein at 24 and 36 h after PHx with or without TUDCA pretreatment detected by Western blotting, densitometric analysis was performed at 36 h after PHx. d The mRNA levels of Foxm1 and cyclin A2 at 36 h after PHx with or without TUDCA pretreatment measured by real-time RT-PCR (black squares Control, dark gray squares Control with TUDCA, white squares HFD, light gray squares HFD with TUDCA, n = 4–6; mean ± SE, *p < 0.05 mice with TUDCA pretreatment vs. mice without TUDCA pretreatment fed same diet in each time point by ANOVA and Wilcoxon test)