Exercise and dietary intervention ameliorate high-fat diet-induced NAFLD and liver aging by inducing lipophagy

Abstract

Exercise and dietary intervention are currently available strategies to treat nonalcoholic fatty liver disease (NAFLD), while the underlying mechanism remains controversial. Emerging evidence shows that lipophagy is involved in the inhibition of the lipid droplets accumulation. However, it is still unclear if exercise and dietary intervention improve NAFLD through regulating lipophagy, and how exercise of skeletal muscle can modulate lipid metabolism in liver. Moreover, NAFLD is associated with aging, and little is known about the effect of lipid accumulation on aging process. Here in vivo and in vitro models, we found that exercise and dietary intervention reduced lipid droplets formation, decreased hepatic triglyceride in the liver induced by high-fat diet. Exercise and dietary intervention enhanced the lipophagy by activating AMPK/ULK1 and inhibiting Akt/mTOR/ULK1 pathways respectively. Furthermore, exercise stimulated FGF21 production in the muscle, followed by secretion to the circulation to promote the lipophagy in the liver via an AMPK-dependent pathway. Importantly, for the first time, we demonstrated that lipid accumulation exacerbated liver aging, which was ameliorated by exercise and dietary intervention through inducing lipophagy. Our findings suggested a new mechanism of exercise and dietary intervention to improve NAFLD through promoting lipophagy. The study also provided evidence to support that muscle exercise is beneficial to other metabolic organs such as liver. The FGF21-mediated AMPK dependent lipophagy might be a potential drug target for NAFLD and aging caused by lipid metabolic dysfunction.

Keywords: Aging; Exercise; FGF21; Lipophagy; Nonalcoholic fatty liver disease (NAFLD).

Graphical abstract

Fig. 1

Exercise and dietary intervention protected against the HFD-induced liver steatosis. (A and B): Representative H&E staining and Oil Red O staining in the liver from rats on HFD with exercise and dietary intervention. Scale: 200 μm for A, Scale: 50 μm for B·(C and D) TG level and PLIN2 protein level in livers from rats on HFD with exercise and dietary intervention. Data are shown as the means ± SEM, n = 8 per group. Significance was designated by asterisks with *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Exercise and dietary intervention improved the autophagy as well as lipophagy via different pathways. (A)Representative of autophagy-associated proteins immunoblotting and quantified data, (B) representative of lipophagy-associated protein PLIN2 immunoblotting in lysosome and quantified data, (C) representative of p-Akt (Ser473)/Akt total, p-mTORC1 (Ser2448)/mTORC1 total and p-ULK1 (Ser757)/ULK1 total immunoblotting and quantified data, (D) representative of p-AMPK (Ser487/Thr172)/AMPK total and p-ULK1 (Ser555)/ULK1 total immunoblotting and quantified data from rats on HFD with exercise and dietary intervention. Experiments were repeated three times. Data are shown as the means ± SEM. Significance was designated by asterisks with *p < 0.05.

Fig. 3

Exercise increased the FGF21 levels in serum and muscle. (A) FGF21 level in serum by ELISA. (B) FGF21 protein levels in liver and muscle by Western Blot. (C) FGF21 content in liver and muscle by ELISA. (D) FGF21 mRNA levels in liver and muscle by qPCR. Data are shown as the means ± SEM, n = 8 per group. Significance was designated by asterisks with *p < 0.05.

Fig. 4

FGF21 attenuated LDs accumulation in FFA-treated HepG2 cells through an autophagy dependent way. HepG2 cells were treated with FFA (400 μM). (A) Representative confocal images of LDs by BODIPY 493/503 staining. Scale: 50 μm. (B) TG level in HepG2 cells. (C) Representative immunoblotting of PLIN2, LC3II/LC3I and p62 in HepG2 cells. HepG2 cells were pre-transfected with Atg5 shRNA or empty vector by lenti-virus, then treated with FFA (400 μM) with or without FGF21 (1 μg/ml). (D) Representative confocal images of LDs by BODIPY 493/503 staining. Scale: 50 μm. (E) TG level in HepG2 cells. Experiments were repeated three times. Data are shown as the means ± SEM. Significance was designated by asterisks with *p < 0.05.

Fig. 5

FGF21 increased lipophagy through AMPK-dependent pathway in FFA-treated HepG2 cells. HepG2 cells were treated with FFA (400 μM), FGF21 (1 μg/ml) as well as AMPK inhibitor-Dorsomorphin (1 μmol/L). (A) Representative of p-AMPK (Thr172)/AMPK total, p-ULK1 (Ser555)/ULK1 total and LC3-II/LC3-I immunoblotting in HepG2 cells treated with FFA, FGF21 and Dorsomorphin, and the quantified data. (B) Representative confocal images of HepG2 cells expressing GFP-RFP-LC3 and quantitation of early autophagosome puncta and autolysosome puncta following FFA, FGF21 and Dorsomorphin treatment. Yellow showed co-localization of GFP and RFP, indicating early autophagosomes. Red only showed autolysosomes, Scale: 20 μm. Experiments were repeated three times. Data are shown as the means ± SEM. Significance was designated by asterisks with *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

FGF21 increased the lipophagy and reduced the lipid accumulation in FFA-treated HepG2 cells. HepG2 cells were treated with FFA (400 μM), FGF21 (1 μg/ml) as well as AMPK inhibitor-Dorsomorphin (1 μmol/L). (A) Representative confocal images of colocalization of LDs and autophagosomes by BODIPY 493/503 staining in GFP-LC3 protein expressed HepG2 cells. Scale: 50 μm. (B) TG level in HepG2 cells treated with FFA, FGF21 and Dorsomorphin. Experiments were repeated three times. Data are shown as the means ± SEM. Significance was designated by asterisks with *p < 0.05.

Fig. 7

Exercise and dietary intervention both ameliorated the liver aging caused by HFD. (A) Representative SA-β-gal staining, Scale: 200 μm, and (B) SA-β-gal activity in the liver from rats on HFD with exercise and dietary intervention. (C) Liver lipofuscin and (D) MDA contents in the liver from rats on HFD with exercise and dietary intervention. (E) p16 and (F) p27 mRNA levels in liver from rats on HFD with exercise and dietary intervention. Data are shown as the means ± SEM, n = 8 per group. Significance was designated by asterisks with *p < 0.05.

Fig. 8

FGF21 ameliorated the cell senescence through AMPK dependent autophagic pathway in FFA-treated WI-38 cells. WI-38 cells were treated with FFA (400 μM), FGF21 (1 μg/ml) as well as AMPK inhibitor-Dorsomorphin (1 μmol/L). (A) Representative of p-AMPK (Thr172)/AMPK total, p-ULK1 (Ser555)/ULK1 total and LC3-II/LC3-I immunoblotting in WI-38 cells and quantified data. (B) TG level in WI-38 cells. (C) Representative SA-β-gal staining (Scale: 50 μm), (D) SA-β-gal activity, (E) lipofuscin and (F) MDA contents in WI-38 cells. (G) p16 and (H) p27 mRNA levels in WI-38 cells. Experiments were repeated three times. Data are shown as the means ± SEM. Significance was designated by asterisks with *p < 0.05.

Fig. 9

Schematic representation of lipophagy induced by exercise and dietary intervention through different pathways in liver to ameliorate HFD-induced NAFLD and liver aging.