Protective role of endogenous plasmalogens against hepatic steatosis and steatohepatitis in mice

Abstract

Free cholesterol (FC) accumulation in the liver is an important pathogenic mechanism of nonalcoholic steatohepatitis (NASH). Plasmalogens, key structural components of the cell membrane, act as endogenous antioxidants and are primarily synthesized in the liver. However, the role of hepatic plasmalogens in metabolic liver disease is unclear. In this study, we found that hepatic levels of docosahexaenoic acid (DHA)-containing plasmalogens, expression of glyceronephosphate O-acyltransferase (Gnpat; the rate-limiting enzyme in plasmalogen biosynthesis), and expression of Pparα were lower in mice with NASH caused by accumulation of FC in the liver. Cyclodextrin-induced depletion of FC transactivated Δ-6 desaturase by increasing sterol regulatory element-binding protein 2 expression in cultured hepatocytes. DHA, the major product of Δ-6 desaturase activation, activated GNPAT, thereby explaining the association between high hepatic FC and decreased Gnpat expression. Gnpat small interfering RNA treatment significantly decreased peroxisome proliferator-activated receptor α (Pparα) expression in cultured hepatocytes. In addition to GNPAT, DHA activated PPARα and increased expression of Pparα and its target genes, suggesting that DHA in the DHA-containing plasmalogens contributed to activation of PPARα. Accordingly, administration of the plasmalogen precursor, alkyl glycerol (AG), prevented hepatic steatosis and NASH through a PPARα-dependent increase in fatty acid oxidation. Gnpat^+/- mice were more susceptible to hepatic lipid accumulation and less responsive to the preventive effect of fluvastatin on NASH development, suggesting that endogenous plasmalogens prevent hepatic steatosis and NASH.

Conclusion: Increased hepatic FC in animals with NASH decreased plasmalogens, thereby sensitizing animals to hepatocyte injury and NASH. Our findings uncover a novel link between hepatic FC and plasmalogen homeostasis through GNPAT regulation. Further study of AG or other agents that increase hepatic plasmalogen levels may identify novel therapeutic strategies against NASH. (Hepatology 2017;66:416-431).

FIG. 1

Pparα expression and the plasmalogen levels in the liver of animal models of nonalcoholic fatty liver disease (NAFLD). (A) Representative hematoxylin & eosin (H&E) (top) and Masson’s Trichrome (MT) staining (bottom) of livers of mice fed the normal diet (ND), high-fat diet (HFD), and methionine- and choline-deficient diet (MCDD) for 8 weeks. Scale bars, 50 μm. (B) Plasma alanine aminotransferase (ALT) level. (C) Relative mRNA expression levels of genes responsible for inflammation and fibrosis, specifically, Tnfα, Ccl2, Il6, Tgfβ, and α-Sma (encoded by Acta2). (C) Relative mRNA expression levels of Pparα and genes involved in mitochondrial and peroxisomal FAO, and Srebp1c and Srebp2. (D) Relative mRNA expression levels of Gnpat and Agps. (E) Levels of hepatic phosphatidylcholine (PC)- and ethanolamine (PE)-plasmalogens in ND-, HFD-, and MCDD-fed mice. (F) Free cholesterol (FC) levels in the liver of mice fed the ND, HFD and MCDD. n = 8 each. Data are shown as the mean ± s.e.m. *P < 0.05 versus ND-fed mice; ^#P < 0.05 versus HFD-fed mice.

FIG. 2

HFHCD feeding decreases hepatic plasmalogen levels and Δ-6 desaturase (Fads2) gene expression. (A) Relative Pparα mRNA expression and FAO in mitochondria and peroxisomes. (B) Plasma total cholesterol and hepatic FC levels, and relative mRNA expression of Srebp2. (C) Relative gene expression levels of Gnpat and Agps, and hepatic plasmalogen contents. (D) Relative mRNA expression levels of Fads1 and Fads2 in HFHCD-fed mice (n = 8 each). Data are shown as the mean ± s.e.m. *P < 0.05 versus ND-fed mice (E) Relative gene expression levels of Fads1, Fads2, Gnpat, and Pparα in siFads1- or siFads2-transfected AML12 hepatocytes (n = 6). (F) DHA restores Gnpat expression in siFads2-transfected AML12 hepatocytes, and increases promoter activity of Gnpat (n = 5). Data are shown as the mean ± s.e.m. *P < 0.05 versus control, ^#P < 0.05 versus control siRNA (siCON), ^§P < 0.05 versus Fads2 siRNA.

FIG. 3

Molecular link between higher hepatic FC level and lower Gnpat expression in NASH. (A) Effect of cholesterol depletion by cyclodextrin (CDX) on Srebp2 and Fads2 mRNA expression in AML12 cells. (B) Cyclodextrin increased FADS2 promoter activity, and knockdown of Srebp2 by siRNA inhibited cyclodextrin-induced Fads2 transcription activity. Data are shown as the mean ± s.e.m. n = 5 each. *P < 0.05 versus vehicle, ^#P < 0.05 versus siCON. (C) Schematic flow that explains how cholesterol depletion increases GNPAT transcription activity.

FIG. 4

GNPAT regulates Pparα expression. (A) Effects of siRNA-mediated knockdown of Pparα (siPparα) and Gnpat (siGnpat) versus control (siCON) in AML12 cells. Relative Pparα and Gnpat mRNA expression and FAO-related gene expression. (B) Mitochondrial and peroxisomal FAO measured by ¹⁴C-palmitate oxidation and total cell death in siGnpat-treated cells (n = 6 each). Data are shown as the mean ± s.e.m. *P < 0.05 versus siCON. (C) DHA increases Pparα and its target genes expression. Data are shown as the mean ± s.e.m. *P < 0.05 versus vehicle. (D) DHA increases PPARα transcription activity and promoter activity. Data are shown as the mean ± s.e.m. n = 5 each. *P < 0.05 versus vehicle, ^#P < 0.05 versus vehicle-treated PPARα expression plasmid.

FIG. 5

Changes in hepatic metabolism in Gnpat-knockout (KO) mice. (A) Relative Gnpat mRNA expression of whole liver tissue in wild-type (WT; Gnpat^+/+ littermates) and Gnpat^+/− mice (n = 8). (B) Representative H&E staining of the livers of WT and Gnpat^+/− mice fed the high-fat diet (HFD, top) or methionine- and choline-deficient diet (MCDD, bottom) for 8 weeks. Scale bars, 50 μm. (C,D) Relative Gnpat and Pparα mRNA expression (left) and hepatic plasmalogen levels (middle and right) in HFD-fed (C) and MCDD-fed (D) WT and Gnpat^+/− mice (n = 8 each). Data are shown as the mean ± s.e.m. *P < 0.05 versus ND-fed WT mice; ^#P < 0.05 HFD-fed WT versus Gnpat^+/− mice. (E) Fenofibrate treatment prevented hepatic steatosis in HFD-fed Gnpat^+/− mice. WT and Gnpat^+/− mice were fed a HFD supplemented without or with 0.1% or 0.5% (wt/wt) fenofibrate for 8 weeks (n = 5). (F) Fenofibrate treatment increases hepatic mRNA expression of Pparα target genes. *P < 0.05 versus HFD-fed WT mice, ^#P < 0.05 HFD-fed Gnpat^+/− mice without Fenofibrate treatment.

FIG. 6

Administration of AG prevents steatosis and NASH in MCDD-fed mice. (A) H&E and MT staining of the livers of MCDD-fed mice with or without AG supplementation (100 mg/kg/day) for 8 weeks. Scale bars, 50 μm. (B) Plasmalogen contents in the liver of ND- and MCDD-fed mice with or without AG treatment. (C) Mitochondrial and peroxisomal FAO, and relative expression of Pparα and its target genes involved in FAO (n = 8). Data are shown as the mean ± s.e.m. *P < 0.05 versus ND-fed mice; ^#P < 0.05 versus MCDD-fed mice. (D) Abolishment of the preventive effect of AG on hepatic steatosis and inflammation in Pparα-KO mice (n = 5). (E) Hepatic FC is not decreased by AG treatment in MCDD-fed mice. Data are shown as the mean ± s.e.m. *P < 0.05 versus ND-fed mice.

FIG. 7

The preventive effect of fluvastatin on NASH is partially abrogated in Gnpat^+/− mice. (A) Representative H&E and MT staining of the livers of WT mice fed the MCDD, and WT and Gnpat^+/− mice fed MCDD with fluvastatin (15 mg/kg/day), for 8 weeks. Scale bars, 50 μm. (B) Plasma ALT levels of WT and Gnpat^+/− mice fed the ND and MCDD with or without fluvastatin. (C) Relative Tnfα and Ccl₂ mRNA expression levels. (D) Liver free cholesterol. (E) Gnpat and Pparα and mRNA expression levels. (F) Hepatic plasmalogen levels in each experimental group (n = 8 each). Data are shown as the mean ± s.e.m. *P < 0.05 versus ND-fed mice, ^#P < 0.05 versus MCDD-fed mice, ^§P < 0.05 WT versus Gnpat^+/− mice.

FIG. 8

Conceptual model for the role of hepatic FC and DHA-plasmalogens in the pathogenesis of NASH. (A) HFD increases TG synthesis to induce hepatic steatosis. However, NASH does not develop in this model if hepatic FC, plasmalogen levels, and Pparα expression are unaltered. (B) MCDD and HFHCD increase hepatic FC, which decreases Srebp2 expression to decrease Fads2 expression and DHA synthesis. DHA transcriptionally activates GNPAT and PPARα to increase plasmalogen synthesis and FAO, respectively. DHA is selectively targeted to plasmalogens during de novo synthesis of plasmalogens, and DHA liberated from DHA-containing plasmalogens may be responsible for increased PPARα-dependent FAO. Collectively, MCDD and HFHCD increase hepatic FC, which is responsible for decreased hepatic DHA-containing plasmalogens and development of NASH. (C) AG treatment increases DHA-plasmalogen levels to activate PPARα-dependent FAO. In addition, plasmalogens can act as endogenous antioxidants. Taken together, these mechanisms result in the prevention of NASH development.