CD36 plays a negative role in the regulation of lipophagy in hepatocytes through an AMPK-dependent pathway - PubMed (original) (raw)

CD36 plays a negative role in the regulation of lipophagy in hepatocytes through an AMPK-dependent pathway

Yun Li et al. J Lipid Res. 2019 Apr.

Abstract

Fatty acid translocase cluster of differentiation (CD36) is a multifunctional membrane protein that facilitates the uptake of long-chain fatty acids. Lipophagy is autophagic degradation of lipid droplets. Accumulating evidence suggests that CD36 is involved in the regulation of intracellular signal transduction that modulates fatty acid storage or usage. However, little is known about the relationship between CD36 and lipophagy. In this study, we found that increased CD36 expression was coupled with decreased autophagy in the livers of mice treated with a high-fat diet. Overexpressing CD36 in HepG2 and Huh7 cells inhibited autophagy, while knocking down CD36 expression induced autophagy due to the increased autophagosome formation in autophagic flux. Meanwhile, knockout of CD36 in mice increased autophagy, while the reconstruction of CD36 expression in CD36-knockout mice reduced autophagy. CD36 knockdown in HepG2 cells increased lipophagy and β-oxidation, which contributed to improving lipid accumulation. In addition, CD36 expression regulated autophagy through the AMPK pathway, with phosphorylation of ULK1/Beclin1 also involved in the process. These findings suggest that CD36 is a negative regulator of autophagy, and the induction of lipophagy by ameliorating CD36 expression can be a potential therapeutic strategy for the treatment of fatty liver diseases through attenuating lipid overaccumulation.

Keywords: adenosine monophosphate-activated protein kinase; autophagy; cluster of differentiation 36; hepatic steatosis; lipid droplets; β-oxidation.

Copyright © 2019 Li et al.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1.

Fig. 1.

Hepatic CD36 expression is increased and autophagy is decreased in response to HFD treatment in mice. A: HE staining (original magnification: ×400) and ORO staining (original magnification: ×400). B: TG and FFA levels in mouse liver tissues. C: Representative Western blotting analysis of CD36, LC3II, and p62 expression in mouse livers. Data are presented as means ± SDs, n = 6. *P < 0.05 and **P < 0.01 versus NCD groups. FFA, free fatty acid; HE, hematoxylin and eosin staining; ORO, Oil Red O.

Fig. 2.

Fig. 2.

Downregulation of CD36 increases autophagy and upregulation of CD36 inhibits autophagy in HepG2 and Huh7 cells with PA treatment. A, B: LC3II and p62 protein levels after CD36 knockdown. C, D: LC3II and p62 levels after CD36 overexpression. E: Immunofluorescence staining with LC3 antibody. Fifty cells were used for statistical analysis of the number of LC3-positive puncta per cell. F: LC3II and p62 protein levels after treatment with CQ in siNeg and siCD36 HepG2 or Huh7 cells. G: Analysis of double-fluorescent mRFP-enhanced GFP-LC3 fusion protein expression to detect autophagic flux (yellow puncta represent autophagosomes and red puncta represent autolysosomes) in siNeg and siCD36 HepG2 cells. Fifty cells were used for the statistical analysis of the number of autophagosomes and autolysosomes per transfected cell. Data are presented as means ± SDs, n = 3; for A, B, E, and G, *P < 0.05 versus siNeg groups; for C and D, *P < 0.05 versus NCD groups; and for F, *P < 0.05 versus siNeg cells without CQ. #P < 0.05 versus siNeg cells with CQ.

Fig. 3.

Fig. 3.

CD36 knockout in vivo increases autophagy of mouse livers and its reconstruction in CD36 knockout mice decreases autophagy. A: Western blotting analysis of LC3II and p62 levels in CD36−/− and WT mice fed the NCD. B: Western blotting analysis of LC3II and p62 levels in CD36−/− and WT mice fed the HFD. C: Ultrastructural analysis of the livers of the CD36−/− mice treated with lentivirus empty vector or lentivirus vector containing CD36. The white arrowhead represents autophagic vacuoles (defined to include autophagosomes and autolysosomes), and the red arrowhead represents intralysosomal lipids. Twenty fields were used for the statistical analysis of the number of autophagic vacuoles per field. Data are presented as means ± SDs, n = 6. *P < 0.05 and **P < 0.01 versus respective control groups.

Fig. 4.

Fig. 4.

Knockdown of CD36 increases lipophagy and β-oxidation and inhibition of autophagy reduces lipophagy and β-oxidation. A–C: Autophagosomal (LC3/BODIPY) and lysosomal (LysoTracker/BODIPY) colocalization with lipids under PA treatment. Thirty cells were used for the statistical analysis of the number of confocal puncta per cell. D, E: Oxygen consumption rate in HepG2 cells. F: Intracellular TG content in HepG2 cells with PA treatment. G: Western blotting analysis of CPT1a in WT and CD36−/− mice fed the HFD. H: CPT1a protein level in HepG2 cells with PA treatment. I: CPT1a mRNA level in WT and CD36−/− mice fed the HFD. Data are presented as means ± SDs; n = 3 for A–F and H, *P < 0.05 versus siNeg groups and #P < 0.05 versus siCD36 groups; n = 6 for G and I, *P < 0.05 and **P < 0.01 versus WT HFD groups.

Fig. 5.

Fig. 5.

AMPK signaling is mechanistically involved in CD36-regulated autophagy. A: Representative Western blotting analysis of p-AMPK and total AMPK levels in NCD and HFD mouse livers. B: p-AMPK and total AMPK levels in CD36−/− and WT mice fed the HFD. C: p-AMPK, total AMPK, and LKB1 levels in HepG2 cells with PA treatment. D: p-AMPK, total AMPK, LC3II, and p62 levels in siCD36 HepG2 cells with or without CC under PA treatment. E: Lipid droplets in siCD36 HepG2 cells with or without CC under PA treatment. Data are presented as means ± SDs; n = 6 for A,*P < 0.05 versus the NCD group; n = 6 for B, *P < 0.05 versus the WT HFD group; n = 3 for C–E, *P < 0.05 versus siNeg groups; and #P < 0.05 versus siCD36 groups.

Fig. 6.

Fig. 6.

Reducing CD36 expression increased autophagy through the phosphorylation of ULK1/Beclin1 in the presence of PA. A: Representative Western blotting analysis of p-mTOR, mTOR, FOXO1, p-FOXO1, and ATG7 levels in siCD36 HepG2 cells. B: mRNA level of FOXO1 in siCD36 HepG2 cells. C, D: mRNA levels of several important ATGs, including ULK1, Beclin1, ATG5, ATG7, ATG12, ATG16L1, and ATG16L2, in siCD36 HepG2, and Huh7 cells. E: mRNA levels of ULK1 and Beclin1 in CD36−/− and WT mice fed the HFD. F: p-ULK1 and total ULK1 protein levels in siCD36 HepG2 cells. G: p-ULK1 and total ULK1 protein levels in siCD36 HepG2 cells with or without CC. H: p-Beclin1 and total Beclin1 protein levels in siCD36 Huh7 cells. Data are presented as means ± SDs; n = 3 for A–D and F–H, *P < 0.05 versus siNeg groups; n = 6 for E, *P < 0.05 versus WT HFD group; #P < 0.05 versus siCD36 group; I: The proposed model of CD36-mediated lipophagy.

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