Hepatic regulation of VLDL receptor by PPARβ/δ and FGF21 modulates non-alcoholic fatty liver disease

Abstract

Objective: The very low-density lipoprotein receptor (VLDLR) plays an important role in the development of hepatic steatosis. In this study, we investigated the role of Peroxisome Proliferator-Activated Receptor (PPAR)β/δ and fibroblast growth factor 21 (FGF21) in hepatic VLDLR regulation.

Methods: Studies were conducted in wild-type and Pparβ/δ-null mice, primary mouse hepatocytes, human Huh-7 hepatocytes, and liver biopsies from control subjects and patients with moderate and severe hepatic steatosis.

Results: Increased VLDLR levels were observed in liver of Pparβ/δ-null mice and in Pparβ/δ-knocked down mouse primary hepatocytes through mechanisms involving the heme-regulated eukaryotic translation initiation factor 2α (eIF2α) kinase (HRI), activating transcription factor (ATF) 4 and the oxidative stress-induced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways. Moreover, by using a neutralizing antibody against FGF21, Fgf21-null mice and by treating mice with recombinant FGF21, we show that FGF21 may protect against hepatic steatosis by attenuating endoplasmic reticulum (ER) stress-induced VLDLR upregulation. Finally, in liver biopsies from patients with moderate and severe hepatic steatosis, we observed an increase in VLDLR levels that was accompanied by a reduction in PPARβ/δ mRNA abundance and DNA-binding activity compared with control subjects.

Conclusions: Overall, these findings provide new mechanisms by which PPARβ/δ and FGF21 regulate VLDLR levels and influence hepatic steatosis development.

Keywords: ATF4; ER stress; FGF21; PPAR; VLDLR.

Figure 1

VLDLR abundance is increased in liver of Pparβ/δ-null mice and in primary hepatocytes following knockdown of Pparβ/δ. Livers from male wild-type (WT) and Pparβ/δ-null mice were used (n = 6 per group). A, Assessment by quantitative real-time RT-PCR of hepatic Vldlr. B, Immunoblot analysis of liver VLDLR. C, Oil Red O and hematoxylin-eosin staining of livers. Scale bar: 100 μm. D, Liver triglyceride levels. Data are presented as the mean ± S.D. (n = 6 per group) relative to the wild-type mice. Vldlr mRNA abundance (E) and protein levels (F) in primary hepatocytes transfected with control siRNA or Pparβ/δ siRNA for 24 h. VLDLR mRNA levels (G) and Oil Red O staining (H) in Huh-7 hepatocytes transfected with control siRNA or Pparβ/δ siRNA for 24 h. Levels are presented as the mean ± S.D. (n = 3–5 per group). *p < 0.05 vs. wild-type mice or control siRNA. Scale bar: 100 μm.

Figure 2

HRI regulates VLDLR abundance in hepatocytes. A, primary hepatocytes were transfected with control or Hri siRNA for 24 h, and the mRNA abundance and protein levels of VLDLR were assessed. *p < 0.05 and **p < 0.01 vs. control siRNA. B, Huh-7 hepatocytes were incubated for 16 h in the absence (Control, CT) or presence of 10 μmol/L of either BTdCPU or BTCtFPU and the mRNA abundance and protein levels of VLDLR were analyzed. ***p < 0.001, **p < 0.01 and *p < 0.05 vs. control. C, mRNA abundance and protein levels of VLDLR in liver of mice treated with DMSO (vehicle) or BTdCPU (70 mg kg⁻¹ day⁻¹) for 7 days (n = 6 per group). ***p < 0.001, **p < 0.01 and *p < 0.05 vs. control cells or control mice. Immunoblot analyses of total and phospho-Nrf2 and NQO1 (D) and mRNA abundance of Nqo1 (E) in liver from male wild-type (WT) and Pparβ/δ-null mice (n = 6 per group). *p < 0.05 vs. control. F, VLDLR mRNA abundance in primary hepatocytes transfected with control, Pparδ, Atf4, Nrf2 and Hri siRNA for 24 h *p < 0.05 vs. control siRNA. ^#p < 0.05 vs. Pparδ siRNA.

Figure 3

Pparβ/δ deficiency exacerbates hepatic steatosis and VLDLR upregulation caused by ER stress. Oil Red O (A) and hematoxylin-eosin (B) staining of livers from male wild-type (WT) and Pparβ/δ-null mice treated for 24 h through i.p. injection with DMSO (vehicle) or tunicamycin (Tunic) (3 mg kg⁻¹ body weight). Scale bar: 100 μm. C, Liver triglyceride levels. D, Serum triglyceride levels. E, Immunoblot analyses of VLDLR. Trb3 (F) and Chop (G) mRNA abundance. H, immunoblot analyses of total and phospho-Nrf2 and NQO1. Data are presented as the mean ± S.D. (n = 6 per group). ***p < 0.001, **p < 0.01 and *p < 0.05 vs. wild-type animals treated with DMSO (vehicle). ^###p < 0.001, ^##p < 0.01 and ^#p < 0.05 vs. wild-type animals treated with tunicamycin. ^†††p < 0.001 and ^†p < 0.05 vs. Pparβ/δ-null mice treated with DMSO (vehicle).

Figure 4

Increased Fgf21 expression in liver of Pparβ/δ-null mice attenuates VLDLR abundance. A, Oil Red O and hematoxylin-eosin staining of livers from male wild-type (WT) and Pparβ/δ-null mice injected intraperitoneally with IgG (9 μg/mouse) or a neutralizing antibody (Ab) (9 μg/mouse) against FGF21 together with DMSO or tunicamycin (Tunic) (3 mg kg⁻¹ body weight). Scale bar: 100 μm. Mice were sacrificed at 14 h after treatment. B, Liver triglyceride levels. C, Vldlr mRNA abundance. ***p < 0.001, **p < 0.01 and *p < 0.05 vs. Pparβ/δ-null mice treated with IgG and DMSO. ^##p < 0.01 and ^#p < 0.05 vs. Pparβ/δ-null mice treated with neutralizing antibody against FGF21 and DMSO. ^†p < 0.05 vs. Pparβ/δ-null mice treated with IgG and tunicamycin. Liver triglyceride levels (D) and Vldlr mRNA abundance (E) in the liver from WT and Fgf21^−/− mice. Data are presented as the mean ± S.D. (n = 5 per group). ***p < 0.001, **p < 0.01 and *p < 0.05 vs. wild-type mice. Immunoblot analyses of VLDLR, total and phospho-eIF2α and ATF4 (F) and total and phospho-Nrf2 (G) were performed in liver lysates. Data are presented as the mean ± S.D. (n = 5 per group). ***p < 0.001, **p < 0.01 and *p < 0.05 vs. wild-type mice.

Figure 5

Recombinant FGF21 protein attenuates the increase in VLDLR levels caused by ER stress. Human Huh-7 hepatocytes were incubated with DMSO (vehicle, control, CT), tunicamycin (TUNIC) (1 μg/ml) or tunicamycin plus recombinant human FGF21 (1 μg/ml) and the mRNA abundance of VLDLR (A) and CHOP (B) and the protein levels of VLDLR and total and phospho-eIF2α (C) were assessed (n = 3 independent experiments). ***p < 0.001 vs. CT cells. ^#p < 0.05 vs. tunicamycin-treated cells. Analysis of the hepatic levels of VLDLR in mice injected intraperitoneally with vehicle or recombinant mouse FGF21 together with DMSO or tunicamycin. Data are presented as the mean ± S.D. (n = 5 per group). C, Vldlr and Chop mRNA abundance. D, Immunoblot analyses of hepatic VLDLR. ***p < 0.001, **p < 0.01 and *p < 0.05.

Figure 6

VLDLR upregulation is intensified by fructose feeding in the liver of Pparβ/δ-null mice. Oil Red O (A) and hematoxylin-eosin (B) staining of livers from male wild-type (WT) and Pparβ/δ-deficient mice (PPARβ/δ^−/−) fed with either water or water containing 30% fructose for eight weeks. Scale bar: 100 μm. C, Hepatic Vldlr mRNA abundance. Immunoblot analyses of hepatic VLDLR (D) and total and phospho-Nrf2 (E). F, Nqo1 mRNA abundance. MDA (G) and H₂O₂ (H) levels from liver of wild-type (WT) and Pparβ/δ-deficient mice fed with either water or water containing 30% fructose. Data are presented as the mean ± S.D. (n = 6 per group). **p < 0.01 and *p < 0.05 vs. water-fed WT mice. ^##p < 0.01 and ^#p < 0.05 vs. fructose-fed WT mice. ^††p < 0.01 and ^†p < 0.05 vs. water-fed Pparβ/δ^−/− mice. I, Vldlr mRNA abundance in primary hepatocytes transfected with control, Pparδ and Nrf2 siRNA for 24 h in the presence or absence of 25 mM fructose. *p < 0.05 vs. control siRNA. ^#p < 0.05 vs. Pparδ siRNA.

Figure 7

Liver of patients with steatosis show increased VLDLR content. A, Hematoxylin-eosin staining of liver biopsies from control subjects (Control, CT) and patients with moderate (30–66% of hepatocytes presenting steatosis) and severe (>66%) hepatic steatosis. Scale bar: 100 μm. Hepatic VLDLR mRNA (B) and protein (C) abundance. D, mRNA abundance of PPARβ/δ, PDK4 and CPT1α. E, autoradiograph of EMSA performed with a ³²P-labeled PPRE and crude nuclear protein extract (NE) from liver biopsies. One main specific complex (I) based on competition with a molar excess of unlabeled probe is shown. The supershift assay performed by incubating NE with an antibody (Ab) directed against PPARβ/δ shows a reduction in the band. F, mRNA abundance of HRI, TRB3 and NQO1. G, mRNA abundance of FGF21, β-KLOTHO and FGFR1c. Data are presented as the mean ± S.D. (n = 4 per group) relative to the control (CT) group. ***p < 0.001, **p < 0.01 and *p < 0.05 vs. CT group.

Figure 8

Proposed mechanisms by which PPARβ/δ regulates VLDLR levels and hepatic steatosis.Pparβ/δ deficiency may result in an increase in VLDLR levels and hepatic steatosis through several mechanisms. The activation of HRI caused by Pparβ/δ deficiency (reference [28]) and by activators of this kinase (BTdCPU) may increase the levels of VLDLR through the eIF2α-ATF4 pathway. ER stress can also activate the eIF2α-ATF4 pathway leading to an increase in the expression of VLDLR and FGF21. This hormone suppresses the eIF2α-ATF4 pathway through a negative feedback mechanism and thereby it also regulates the levels of VLDLR. ER stress also enhances the activity of Nrf2, a transcription factor reported to upregulate the expression of Fgf21 (reference [19]). Fructose feeding increases the levels of ROS and the activity of Nrf2 providing a mechanism for the increase of the levels of VLDLR. All these mechanisms may result in an increase in the levels of VLDLR causing hepatic steatosis. TG: triglyceride.

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