Inhibited Lipophagy Suppresses Lipid Metabolism in Zebrafish Liver Cells

Abstract

Lipophagy degrades lipid droplets (LDs) through the lysosomal degradative pathway, thus plays important roles in regulating lipid metabolism in mammals. However, information on the existence and functions of lipophagy in fish lipid metabolism is still limited. In the present study, we confirmed the existence of lipophagy by observing the structures of LDs sequestered in autophagic vacuoles in the zebrafish liver cell line (ZFL) via electronic microscopy. Moreover, starved cells increased the mRNA expression of the microtubule-associated protein 1A/1B light chain 3 beta (LC3), which is a marker protein for autophagy and protein conversion from LC3-I to LC3-II. Inhibiting autophagy with chloroquine increased significantly the LDs content and decreased fatty acid β-oxidation and esterification activities in the ZFL cells cultured in the fed state. Furthermore, inhibiting autophagy function downregulated the mRNA expression of the genes and their proteins related to lipid metabolism. Altogether, the present study verified the existence of lipophagy and its essential regulatory roles in lipid metabolism in fish cells.

Keywords: esterification; fatty acid β-oxidation; lipid metabolism; lipophagy; zebrafish liver cells.

FIGURE 1

Starvation induces lipophagy in ZFL cell line. (A) Lipid droplets (LDs) stained with BODIPY 493/503 (green) in ZFL cell line in starvation condition. (B) The mRNA expression, immunoblot and densitometric analysis of LC3 in the samples in ZFL cell line. (^∗P < 0.05, ^∗∗P < 0.01, n = 6). Error bars, SEM. (C) Co-localization of BODIPY 493/503 (green) with LC3 (red) in ZFL cell line in starvation condition. Nuclei are highlighted with 4, 6-diamidino-2-phenylindole (DAPI).

FIGURE 2

Electron micrographs of ZFL cells. (A,B) The structure of an autophagic vesicole (AV). (C–H) The structure of an autophagosome containing a lipid droplet (AP). N, nucleus; ER, endoplasmic reticulum; Mit, mitochondria.

FIGURE 3

Inhibited lipophagy causes accumulation of lipid droplets in ZFL cell line. (A,B) Lipid droplets (LDs) stained with BODIPY 493/503 and TG contents in the ZFL cells. (C) Immunoblot and densitometric analysis of LC3 and p62 in the ZFL cells. (^∗P < 0.05, n = 6). Error bars, SEM. (D) Co-localization of BODIPY 493/503 (green) with LC3 (red) in treated samples with CQ.

FIGURE 4

Inhibited lipophagy reduces activities of lipid catabolism and esterification in ZFL cell line. (A) The β-oxidation activities of [1-¹⁴C] oleic acid in the ZFL cells. (B) The esterification activities of [1-¹⁴C] oleic acid in the ZFL cells. (^∗P < 0.05, n = 4). Error bars, SEM.

FIGURE 5

Inhibited lipophagy suppresses expression of lipid metabolism-related genes and protein levels in ZFL cell line. (B–E,G) The mRNA expression of the genes related to lipid metabolism. (A,F) The protein levels of PPARa, ATGL and p-HSL. ^∗P < 0.05, ^∗∗P < 0.01, n = 6. Error bars, SEM.