Exercise intervention improves mitochondrial quality in non-alcoholic fatty liver disease zebrafish

Abstract

Introduction: Recent reports indicate that mitochondrial quality decreases during non-alcoholic fatty liver disease (NAFLD) progression, and targeting the mitochondria may be a possible treatment for NAFLD. Exercise can effectively slow NAFLD progression or treat NAFLD. However, the effect of exercise on mitochondrial quality in NAFLD has not yet been established.

Methods: In the present study, we fed zebrafish a high-fat diet to model NAFLD, and subjected the zebrafish to swimming exercise.

Results: After 12 weeks, swimming exercise significantly reduced high-fat diet-induced liver injury, and reduced inflammation and fibrosis markers. Swimming exercise improved mitochondrial morphology and dynamics, inducing upregulation of optic atrophy 1(OPA1), dynamin related protein 1 (DRP1), and mitofusin 2 (MFN2) protein expression. Swimming exercise also activated mitochondrial biogenesis via the sirtuin 1 (SIRT1)/ AMP-activated protein kinase (AMPK)/ PPARgamma coactivator 1 alpha (PGC1α) pathway, and improved the mRNA expression of genes related to mitochondrial fatty acid oxidation and oxidative phosphorylation. Furthermore, we find that mitophagy was suppressed in NAFLD zebrafish liver with the decreased numbers of mitophagosomes, the inhibition of PTEN-induced kinase 1 (PINK1) - parkin RBR E3 ubiquitin protein ligase (PARKIN) pathway and upregulation of sequestosome 1 (P62) expression. Notably, swimming exercise partially recovered number of mitophagosomes, which was associated with upregulated PARKIN expression and decreased p62 expression.

Discussion: These results demonstrate that swimming exercise could alleviate the effects of NAFLD on the mitochondria, suggesting that exercise may be beneficial for treating NAFLD.

Keywords: exercise; mitochondria quality control; mitochondrial dysfunction; non-alcoholic fatty liver disease; zebrafish.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.