Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice

Abstract

Aim: Recent evidence suggests that the essential amino acid leucine may be involved in systemic cholesterol metabolism. In this study, we investigated the effects of leucine supplementation on the development of atherosclerosis in apoE null mice.

Methods: ApoE null mice were fed with chow supplemented with leucine (1.5% w/v) in drinking water for 8 week. Aortic atherosclerotic lesions were examined using Oil Red O staining. Plasma lipoprotein-cholesterol levels were measured with fast protein liquid chromatography. Hepatic gene expression was detected using real-time PCR and Western blot analyses.

Results: Leucine supplementation resulted in 57.6% reduction of aortic atherosclerotic lesion area in apoE null mice, accompanied by 41.2% decrease of serum LDL-C levels and 40.2% increase of serum HDL-C levels. The body weight, food intake and blood glucose level were not affected by leucine supplementation. Furthermore, leucine supplementation increased the expression of Abcg5 and Abcg8 (that were involved in hepatic cholesterol efflux) by 1.28- and 0.86-fold, respectively, and significantly increased their protein levels. Leucine supplementation also increased the expression of Srebf1, Scd1 and Pgc1b (that were involved in hepatic triglyceride metabolism) by 3.73-, 1.35- and 1.71-fold, respectively. Consequently, leucine supplementation resulted in 51.77% reduction of liver cholesterol content and 2.2-fold increase of liver triglyceride content. Additionally, leucine supplementation did not affect the serum levels of IL-6, IFN-γ, TNF-α, IL-10 and IL-12, but markedly decreased the serum level of MCP-1.

Conclusion: Leucine supplementation effectively attenuates atherosclerosis in apoE null mice by improving the plasma lipid profile and reducing systemic inflammation.

Figures

Figure 1

Leucine reduced aortic atherosclerotic lesions in apoE null mice. ApoE null mice were fed with chow and supplemented with or without leucine (1.5%) in drinking water for 8 weeks. (A) Representative images of en face Oil Red O staining in the full aorta. (B) Relative quantification of lesion area in the control and leucine treatment mice; data are presented as the percentages of total en face aortic area. (C) Representative images of Oil Red O staining in the frozen section of the left ventricular outflow tract. (D) Quantification of lesion area in the control and leucine treatment mice, data are presented as the lesion areas (×105 μm2). (E) Food intake was measured at the first week of leucine supplementation. (F) Body weight was measured every two weeks. Data are presented as the mean±SEM. n=8 in control group. n=9 in leucine group. cP<0.01 vs control.

Figure 2

Effect of leucine on plasma lipid profile and glucose level of apoE null mice. After leucine supplementation (1.5%) in drinking water for 8 weeks, (A) serum total triglyceride (TG), (B) total cholesterol (TC), (C) low-density lipoprotein cholesterol (LDL-C) and (D) high density lipoprotein cholesterol (HDL-C) concentrations were measured. (E) Distribution of cholesterol in plasma lipoproteins from control and leucine treated apoE null mice was measured by use of fast protein liquid chromatography (n=3). (F) Blood glucose levels at fasted and fed states were measured. Data are presented as the mean±SEM. n=8–9. bP<0.05 vs control.

Figure 3

Lipid content and ABCG5/ABCG8 protein levels in the livers of apoE null mice. (A, B) After leucine supplementation (1.5%) in drinking water for 8 weeks, liver lipids were extracted. Triglyceride (TG) and total cholesterol (TC) contents were measured. (C, D) Western blot analyses detected liver protein levels of ABCG5 and ABCG8; protein levels relative to β-actin are shown. Data are presented as the mean±SEM. n=8–9. bP<0.05 vs control.

Figure 4

Effect of leucine on plasma inflammatory cytokine levels in apoE null mice. The serum inflammatory cytokines MCP-1, IL-6, IFN-γ, TNF-α, IL-10, IL-12 levels were measured. Data are presented as the mean±SEM. n=8–9. cP<0.01 vs control.