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, 23 (5), 318-325
eCollection

Endogenous Leptin Promotes Autophagy in EBSS-induced PFCs

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Endogenous Leptin Promotes Autophagy in EBSS-induced PFCs

Deling Jiao et al. Anim Cells Syst (Seoul).

Abstract

Leptin is an important adipokine and plays a vital role in animals. However, the role of leptin in the autophagic response of pig fibroblast cells (PFCs) has not been fully elucidated. In this study, we investigated the relationship between leptin and autophagy as well as underlying molecular basis. We found that PFCs treated with EBSS could secrete leptin, and the leptin concentration in the supernatant of leptin transgenic PFCs was higher than that of WT PFCs. We found an increase in LC3-II protein level and a decrease in p62 protein level in treated leptin transgenic PFCs compared with treated WT PFCs. Meanwhile, we observed an increase of autophagosomes by transmission electron microscopy and an enhancement of the accumulation of LC3 puncta in the cytoplasm of treated leptin transgenic PFCs, and these effects were further augmented by Baf A1 treatment. Furthermore, we detected the expression levels of 7 autophagy signaling pathway genes and 17 autophagy-related (ATG) genes by q-PCR. We found that between the two types of EBSS-treated cells 3 genes expression pattern were significantly different among the 7 autophagy signaling pathway genes and 8 genes expression pattern were significantly differernt among the ATG genes. These results indicated that leptin may promote autophagy and involving the downregulation of FOXO1 and LMNA genes via an unknown pathway which causes the upregulation of the 4 genes and the downregulation of 4 genes.

Keywords: Leptin; PFCs; autophagy; autophagy signaling pathway-related genes; autophagy-related genes.

Figures

Figure 1.
Figure 1.
Starvation induces autophagy in WT and leptin transgenic PFCs. (A) Quantification of the leptin concentration in culture supernatants of WT and leptin transgenic PFCs. ** p < 0.01 compared to WT PFCs. (B) Both PFCs were treated with EBSS for the indicated times in the presence or absence of Baf A1 (100 nM); then, the protein expression levels of LC3B and p62 were analyzed by immunoblotting. β-actin was used as an internal control. (C-D) Quantification of the LC3-II and P62 protein expression level in both treated cells. *p < 0.05 and **p < 0.01 compared to EBSS-treated PFCs or EBSS with Baf A1-treated PFCs at 0 h, #p < 0.05 and ## p < 0.01 compared to EBSS-treated PFCs at the indicated time. (E) Representative electron micrographs for both cells. WT and leptin transgenic PFCs were treated with EBSS for 4 h. Red arrows refer to autophagic vacuoles. Scale bars = 0.5 μm.
Figure 2.
Figure 2.
Confocal microscopy images of LC3 in WT and leptin transgenic PFCs. Both leptin transgenic and WT PFCs were treated with EBSS or a combination of EBSS with Baf A1 for the indicated times, and the accumulation of LC3 puncta was analyzed with confocal microscopy. (A) Representative images of LC3 puncta in WT PFCs. (B) Representative images of LC3 puncta in leptin transgenic PFCs. Cells were stained with antibodies against LC3 (green), and nuclei were stained blue with DAPI. Scale bars = 20 μm.
Figure 3.
Figure 3.
Effect of leptin on the mRNA expression levels of autophagy signaling pathway-related genes in PFCs. Both types of cells were treated with EBSS for 0 and 4 h. (A, B) The mRNA expression levels of the autophagy signaling pathway-related genes mTOR, FOXO1, EPG5, LMNA, PRKAA1, DRAM1 and AMBRA in both treated cells. *p < 0.05 compared to the EBSS treatment at 0 h in both PFCs.
Figure 4.
Figure 4.
Effects of leptin on the mRNA level of ATG genes in PFCs. The cells were treated as described above. (A) The mRNA expression levels of the autophagosome initiation-related genes ULK1, ULK2 and ATG13 in both treated cell. (B) The mRNA expression levels of the nucleation-related genes ATG14, BECN1, VPS15 and PIK3C3 in both treated cells. (C) The mRNA expression levels of the elongation-related genes ATG4A, ATG4B and ATG4D in both treated cells. (D) The mRNA expression levels of the elongation-related genes ATG5, ATG16L1 and ATG16L2 in both treated cells. (E) The mRNA expression levels of the degradation cycling-related genes ATG2A, ATG2B, ATG9A and P62 in both treated cells. (F) The protein level of ATG4B in both treated cells was analyzed by immunoblotting. β-actin was used as an internal control. *p < 0.05 and **p < 0.01 compared to the EBSS treatment at 0 h in both PFCs.

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References

    1. Cassano S, Pucino V, La Rocca C, Procaccini C, De Rosa V, Marone G, Matarese G. 2014. Leptin modulates autophagy in human CD4+CD25− conventional T cells. Metabolism: Clinical and Experimental. 63:1272–1279. doi: 10.1016/j.metabol.2014.06.010 - DOI - PMC - PubMed
    1. Chen J, Zeng W, Pan W, Peng C, Zhang J, Su J, Long W, Zhao H, Zuo X, Xie X, et al. 2018. Symptoms of systemic lupus erythematosus are diagnosed in leptin transgenic pigs. PLoS Biol. 16:e2005354. doi: 10.1371/journal.pbio.2005354 - DOI - PMC - PubMed
    1. Choi JC, Worman HJ. 2013. Reactivation of autophagy ameliorates LMNA cardiomyopathy. Autophagy. 9:110–111. doi: 10.4161/auto.22403 - DOI - PMC - PubMed
    1. Deretic V, Saitoh T, Akira S. 2013. Autophagy in infection, inflammation and immunity. Nat Rev Immunol. 13:722–737. doi: 10.1038/nri3532 - DOI - PMC - PubMed
    1. Dudley LJ, Cabodevilla AG, Makar AN, Sztacho M, Michelberger T, Marsh JA, Houston DR, Martens S, Jiang X, Gammoh N. 2019. Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy. EMBO J. 38:e100554. doi: 10.15252/embj.2018100554 - DOI - PMC - PubMed

Grant support

This work was supported by the Natural Science Foundation of Yunnan Province [Grant Number 2016FA010], and National Natural Science Foundation of China [Grant Number 31560636 and 81872452].

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