Exercise Ameliorates Atherosclerosis via Up-Regulating Serum β-Hydroxybutyrate Levels

Abstract

Atherosclerosis, accompanied by inflammation and metabolic disorders, is the primary cause of clinical cardiovascular death. In recent years, unhealthy lifestyles (e.g., sedentary lifestyles) have contributed to a worldwide epidemic of atherosclerosis. Exercise is a known treatment of atherosclerosis, but the precise mechanisms are still unknown. Here, we show that 12 weeks of regular exercise training on a treadmill significantly decreased lipid accumulation and foam cell formation in ApoE^-/- mice fed with a Western diet, which plays a critical role in the process of atherosclerosis. This was associated with an increase in β-hydroxybutyric acid (BHB) levels in the serum. We provide evidence that BHB treatment in vivo or in vitro increases the protein levels of cholesterol transporters, including ABCA1, ABCG1, and SR-BI, and is capable of reducing lipid accumulation. It also ameliorated autophagy in macrophages and atherosclerosis plaques, which play an important role in the step of cholesterol efflux. Altogether, an increase in serum BHB levels after regular exercise is an important mechanism of exercise inhibiting the development of atherosclerosis. This provides a novel treatment for atherosclerotic patients who are unable to undertake regular exercise for whatever reason. They will gain a benefit from receiving additional BHB.

Keywords: ABCA1; ABCG1; BHB; SR-BI; atherosclerosis; autophagy; exercise; foam cell formation.

Figure 1

Exercise inhibits the development of atherosclerosis and increases serum BHB levels. (A) ApoE^−/− mice were fed normal food (ND) or a Western diet (WD), and either had access to exercise (E) for 12 weeks or did not (sedentary) (B) Body weight of mice (n = 11). (C) Representative images of the cross-sections of the aortic root stained with Oil Red O. Scale bars, 100 μm. (D) Representative images of the whole aorta stained by Oil Red O. Scale bars, 1 mm. (E) Quantification of cross-sections of the lesion area in the aortic root and whole aorta (n = 6). (F) Total cholesterol (TC) and triglycerides (TG) in the serum of ApoE^−/− mice (n = 11). (G) BHB levels in the serum from the mice (n = 11). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 vs. ND mice, and # p < 0.05, ## p < 0.01 vs. WD mice, ns is no differences.

Figure 2

Exercise increases the protein levels of cellular cholesterol transporters and autophagy flux. (A–C,E) Representative immunofluorescence images of aortic root frozen sections from ApoE^−/− mice: (A) ABCA1 stains (red) and DAPI nuclear stains (blue); scale bars, 50 μm. (B) ABCG1 stains (red) and DAPI nuclear stains (blue); scale bars, 50 μm. (C) SR-BI stains (red) and DAPI nuclear stains (blue); scale bars, 50 μm. (E) LC3 stains (green), p62 stains (red), and DAPI nuclear stains (blue); scale bars, 50 μm. (D,F) Quantification of immunofluorescence images (n = 6). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 vs. ND mice; # p < 0.05, ## p < 0.01 vs. WD mice.

Figure 3

BHB increases the expression of cholesterol transporters and attenuates foam cell for-mation. (A) Western blot analysis of the protein expression of cellular cholesterol transporters including ABCA1, ABCG1, and SR-B1 in PMs treated with OxLDL (50 μg/mL) and BHB (0.5 mM, 1 mM) for 24 h. (B) Quantification of relative protein expression (n ≥ 3). (C) Oil Red O staining of PMs in the presence or absence of OxLDL (50 μg/mL) or BHB for 24 h. Scale bars, 5 μm. (D) Quantification of Oil Red O staining (n = 6). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. OxLDL treated group.

Figure 4

BHB ameliorates autophagy in vitro. (A,C,E) Western blot analysis of p62 and LC3 protein levels in PMs. (A) Western blot analysis of the expression of p62 and LC3 in PMs pretreated with or without OxLDL (50 μg/mL) for 6 h, 12 h, and 24 h. (C) Western blot analysis of the expression of p62 and LC3 in PMs pretreated with or without OxLDL (50 μg/mL) for 24 h and treated with BHB (0.5 mM, 1 mM) for 24 h. (E) Expression of p62 and LC3 in PMs pre-incubated with Baf a1 (200 nM) for 6 h before treatment with BHB (1 mM). (B,D,F) Quantification of relative protein levels (n ≥ 3). (G) Oil Red O staining of PMs in the presence or absence of OxLDL (50 μg/mL), BHB (1 mM), or 3-MA (1 mM) for 6 h. Scale bars, 5 μm. (H) Quantification of Oil Red O staining (n = 6). * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. OxLDL-treated group or BHB-treated group.

Figure 5

BHB is atheroprotective in ApoE^−/− mice fed a Western diet. (A) ApoE^−/− mice were fed a Western diet for 16 weeks and administered the vehicle or BHB (1 mm/kg daily, i.p.) in the final 4 weeks of the diet. (B) Body weight of the mice. (C) Representative images of the cross-sections of the aortic root stained with Oil Red O. Scale bar, 100 μm. (D) Representative images of the whole aorta stained by Oil Red O. Scale bar, 1 mm. (E) Quantification of the cross-sections of the lesion area in the aortic root and whole aorta (n = 6). (F) Total cholesterol (TC) and triglycerides (TG) in the serum from the mice (n = 9). (G) BHB level in the serum from the mice (n = 9). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 vs. ND mice; # p < 0.05, ## p < 0.01 vs. WD mice, ns is no differences.

Figure 6

BHB increases the expression of cholestene transporters and induces autophagy. (A–C,E) Representative immunofluorescence images of frozen sections of aortic roots from ApoE^−/− mice. (A) ABCA1 stains (red) and DAPI nuclear stains (blue). Scale bars, 50 μm. (B) ABCG1 stains (red) and DAPI nuclear stains (blue). Scale bars, 50 μm. (C) SR-BI stains (red) and DAPI nuclear stains (blue). Scale bars, 50 μm. (E) LC3 stains (green), p62 stains (red), and DAPI nuclear stains (blue). Scale bars, 50 μm. (D,F) Quantification of immunofluorescence images (n = 6). Data are represented as means ± SEM. * p < 0.05, ** p < 0.01 vs. ND mice; # p < 0.05, ## p < 0.01 vs. WD mice.