Effects of HC, HF, and EF diets feeding on the growth of zebrafish larvae after 10 days feeding. (A) Description of the diets feeding protocol. (B) General observation of zebrafish in three groups. (C) Body length was measured at 15 dpf (n = 20). Ten fish for each sample were weighted from 100 fish per group at 15 dpf (n = 10). a, compared with control; b, compared with EF; c, compared with HF; *P < 0.05, ^#P < 0.01. Bar = 1 mm.

Assessment of lipid accumulation in zebrafish larvae. (A) The hepatic steatosis was observed by whole body ORO staining (A, below). The percent of larvae with hepatic steatosis was calculated from at least 100 fish each group. Data were representative of four independent experiments. (B) Showed fluorescent lipid accumulation deposits in the liver. Relative fluorescence intensities were quantified in the areas (n = 10). (C) Showed fluorescent lipid accumulation deposits in the blood vessel wall. Relative fluorescence intensities of the areas were quantified (n = 10). (D) Numerous lipid droplets in the hepatocytes were observed by ORO staining of frozen liver sections in HC, HF, and EF groups. The statistical results of positive area of ORO staining was showed in figure (F) (n = 10). (E) The lipid vacuoles were observed by H&E staining in HC, HF, and EF groups. Changes of cholesterol (G), triglyceride (H), and glucose (I) in livers of zebrafish larvae fed with control, HC, HF, and EF diets. Each sample were collected from 20 to 30 fish (n = 5). a, compared with control; b, compared with EF; c, compared with HF; *P < 0.05 vs. Control. Bar = 100 μm.

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Effects of HC, HF, and EF diets treatment on autophagy. (A) LC3 puncta formation in liver of GFP-LC3 transgenic zebrafish larvae fed with control, HC, HF, and EF diets. Bar = 100 μm. (B) Quantification of LC3 puncta abundance in (A) with software Image J. (C) The genes expression involved in autophagy (n = 4). (D) The protein expression of LC3BII, ATG3, and p62 were detected by western blot in liver of zebrafish larvae fed with control, HC, HF, and EF diets (n = 4) *P < 0.05, **P < 0.01 vs. control. (E) Autophagy pathway in HC group by KEGG. Genes with red-filled are up-regulated, while genes with green-filled are down-regulated.