Protective effect of swimming exercise against diet-induced NAFLD in zebrafish. (A) Schematic of the swimming exercise strategy to study diet-induced NAFLD in zebrafish. (B) Body length, (C) body weight, and (D) body mass index of zebrafish during the experimental period. (E) HE staining, MASSON staining, and DHE visualization of zebrafish liver tissue in response to a high-fat diet and a high-fat diet combined with exercise (n=3). (F) NAS score, (G) fibrosis score, and (H) DHE integrated density in zebrafish livers. *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent the SEM. Scale bar, 20 μm. NAFLD, non-alcoholic fatty liver disease; N, normal diet; H, high fat diet; HE, high-fat diet plus exercise. ns, not significant.

Swimming exercise protects against inflammation and fibrosis in diet-induced NAFLD zebrafish livers. (A) Western-blot of CLO1A1, ACTA2, IL-1β, and IL-10. Protein expression levels of (B) COL1A1, (C) ACTA2, (D) IL-1β, and (E) IL-10. (n=6) *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. Scale bar, 20 μm. NAFLD, non-alcoholic fatty liver disease; N, normal diet; H, high fat diet; HE, high-fat diet plus exercise.

Swimming exercise maintains mitochondrial dynamic balance in diet-induced NAFLD zebrafish livers. (A) Representative TEM of zebrafish liver showing visible LDs in fish fed a high-fat diet. TEM micrographs were used to determine (B) mitochondrial numbers at 10000× magnification, (C) mitochondrial diameter at 40000× magnification, (D) mitochondrial length at 40000× magnification, and (E) mitochondrial area at 40000× magnification. (n=2) (F) Western blots of DRP1, OPA1, and MFN2. (n=6) The protein expression of (G) DRP1, (H) OPA1, and (I) MFN2 according to densitometry analysis. *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. Scale bar on TEM 10000× micrographs, 2 μm; scale bar on TEM 40000× micrographs, 0.5 μm. NAFLD, non-alcoholic fatty liver disease; N, normal diet; H, high fat diet; HE, high-fat diet plus exercise; M, mitochondria; LD, lipid droplet. ns, not significant.

Swimming exercise alleviates mitochondrial dysfunction in diet-induced NAFLD model zebrafish livers. (A) Western-blot of P-AMPK, AMPK, SIRT1, PGC1α, NRF1, NRF2, and TFAM. (n=6) The protein expression levels of (B) P-AMPK/AMPK, (C) SIRT1, (D) PGC1α, (E) NRF1, (F) NRF2, and (G) TFAM according to densitometry analysis. (H)Mtnd1 and mtnd6 mRNA expression. (n=6) (I) The mRNA expression levels of genes related to fatty acid oxidation. (n=6) (J) The mRNA expression levels of genes related to mitochondrial respiratory complex subunits. (n=6) *, p < 0.05, **, p < 0.01, ***, p < 0.001. Data represent the mean, and error bars represent SEM. NAFLD, non-alcoholic fatty liver disease; N, normal diet; H, high fat diet; HE, high-fat diet plus exercise; OXPHOS, oxidative phosphorylation; Mitochondrial NADH dehydrogenase 1,mtnd1; Mitochondrial NADH dehydrogenase 6, mtnd6. Acyl-CoA dehydrogenase medium chain, acadm; Carnitine palmitoyltransferase 1A, cpt1a; Peroxisome proliferator-activated receptor alpha b, pparab); NADH:ubiquinone oxidoreductase subunit A9a, ndufa9a; Succinate dehydrogenase complex, subunit A, sdha; Ubiquinol-cytochrome c reductase core protein 2b, uqcrc2b; cytochrome c oxidase subunit 4I1, cox4i1; ATP synthase F1 subunit beta, atp5f1b. ns, not significant.

Swimming exercise restores mitophagy in diet-induced NAFLD model zebrafish livers. (A) Representative TEM of zebrafish liver. (B) Western-blot of PINK1, PARKIN, and p62 (n=6). Protein expression levels of (C) PINK1, (D) PARKIN, and (E) p62 according to densitometry. * p < 0.05, ** p < 0.01, *** p < 0.001. Data represent the mean, and error bars represent SEM. Scale bar on TEM 20000× micrograph, 1 μm. The red arrows indicated the mitophagosomes. NAFLD, non-alcoholic fatty liver disease; N, normal diet; H, high fat diet; HE, high-fat diet plus exercise. ns, not significant.