Peroxisome proliferator-activated receptor α agonist induces mouse hepatomegaly through the spatial hepatocyte enlargement and proliferation

Abstract

Peroxisome proliferator-activated receptor alpha (PPARα) activation-induced hepatomegaly is accompanied by hepatocyte hypertrophy around the central vein (CV) area and hepatocyte proliferation around the portal vein (PV) area. However, the molecular mechanisms underlying this spatial change of hepatocytes remains unclear. In this study, we examined the characteristics and possible reasons for the zonation distinction of hypertrophy and proliferation during PPARα activation-induced mouse liver enlargement. Mice were injected with corn oil or a typical mouse PPARα agonist WY-14643 (100 mg·kg^-1·d^-1, i.p.) for 1, 2, 3, 5 or 10 days. At each time point, the mice were sacrificed after the final dose, and liver tissues and serum were harvested for analysis. We showed that PPARα activation induced zonal changes in hepatocyte hypertrophy and proliferation in the mice. In order to determine the zonal expression of proteins related to hepatocyte hypertrophy and proliferation in PPARα-induced liver enlargement, we performed digitonin liver perfusion to separately destroy the hepatocytes around the CV or PV areas, and found that PPARα activation-induced increase magnitude of its downstream targets such as cytochrome P450 (CYP) 4 A and acyl-coenzyme A oxidase 1 (ACOX1) levels around the CV area were higher compared with those around the PV area. Upregulation of proliferation-related proteins such as cell nuclear antigen (PCNA) and cyclin A1 (CCNA1) after WY-14643-induced PPARα activation mainly occurred around the PV area. This study reveals that the zonal expression of PPARα targets and proliferation-related proteins is responsible for the spatial change of hepatocyte hypertrophy and proliferation after PPARα activation. These findings provide a new insight into the understanding of PPARα activation-induced liver enlargement and regeneration.

Keywords: hepatocyte hypertrophy; hepatocyte proliferation; hepatomegaly; peroxisome proliferator-activated receptor α; spatial distribution.

Fig. 1. PPARα activation induces hepatomegaly without liver injury in mice.

a Animal experiment scheme. C57BL/6 male mice were intraperitoneally injected with corn oil or WY-14643 (100 mg·kg⁻¹·d⁻¹) for 1, 2, 3, 5, 7, 10 d, respectively. b Representative morphological images of livers of different groups. c Liver-to-body weight ratios of different groups. d The levels of serum ALT, AST and ALP in the vehicle and WY-14643 groups. e The mRNA levels of hepatic Il-6, Tnf-α and Ifn-γ were determined by qRT-PCR analysis. Data are presented as means ± SD (n = 5). ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001 compared to the vehicle group.

Fig. 2. PPARα activation induces hepatocyte enlargement mainly around the CV area.

a CTNNB1 staining around the CV area. b Quantification of CTNNB1 staining in a. c CTNNB1 staining around the PV area. d Quantification of CTNNB1 staining in c. Data are presented as means ± SD (n = 3), ^*P < 0.05 compared to the vehicle group. Scale bar = 50 μm. CV Central vein, PV Portal vein.

Fig. 3. PPARα activation induces hepatocyte proliferation mainly around the PV area.

a Ki67 staining around the CV area. b Ki67 staining around the PV area. c Quantification of Ki67 staining in a, b (n = 3). Red arrows: brown-stained Ki67 positive cells. Data are presented as means ± SD. *P < 0.05; ***P < 0.001 compared to the vehicle group. Scale bar = 50 μm. CV Central vein, PV Portal vein.

Fig. 4. Dynamic changes of other PPARα downstream targets and proliferation-related proteins.

a Western blot analysis of CYP4A, ACOX1, catalase and EHHADH. b Western blot analysis of PCNA, CCNA1, MCM2 and YAP. c, d Quantification of protein of a/b expression levels in different groups (n = 5). Data are presented as means ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared to the vehicle group. ACTB Actin beta, CV Central vein, PV Portal vein.

Fig. 5. Hepatic digitonin perfusion destroys hepatocytes around the CV or PV areas.

a Representative morphological photograph of livers during digitonin perfusion through the portal vein. b Representative morphological photograph of livers during digitonin perfusion through the central vein. c Western blot analysis of zonal markers GS and ASS1 levels in liver tissues after digitonin perfusion through the portal vein (n = 3). d Western blot analysis of GS and ASS1 levels in liver tissues after digitonin perfusion through the central vein (n = 3). Data are presented as means ± SD. ^**P < 0.01, ^***P < 0.001 compared to the vehicle group. ACTB Actin beta, CV Central vein, PV Portal vein.

Fig. 6. The expression of PPARα downstream targets and proliferation-related proteins around hepatocytes surrounding the CV and PV areas.

a, b Western blot analysis and quantification of PPARα downstream targets in whole liver tissues, CV area-retained liver tissues and PV area-retained liver tissues. c, d Western blot analysis and quantification of proliferation-related proteins PCNA, CCNA1, MCM2 and total YAP in whole liver tissues, PV area-retained liver tissues and CV area-retained livers. e, f Representative photographs and quantification of PCNA staining. Red arrows: brown-stained PCNA-positive cells. Scale bar = 100 μm. Data are presented as means ± SD (n = 3), ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. ACTB Actin beta, CV Central vein, ns no significance, PV Portal vein.