β-Aminoisobutyric Acid Suppresses Atherosclerosis in Apolipoprotein E-Knockout Mice

Yuki Shimba; Keigo Katayama; Noriyuki Miyoshi; Masahiko Ikeda; Akihito Morita; Shinji Miura

doi:10.1248/bpb.b20-00078

Abstract

Endurance exercise training has been shown to induce peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in skeletal muscle. We recently reported that skeletal muscle-specific PGC-1α overexpression suppressed atherosclerosis in apolipoprotein E-knockout (ApoE^−/−) mice. β-Aminoisobutyric acid (BAIBA) is a PGC-1α-dependent myokine secreted from myocytes that affects multiple organs. We have also reported that BAIBA suppresses tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) gene expression in endothelial cells. In the present study, we hypothesized that BAIBA suppresses atherosclerosis progression, and tested that hypothesis with ApoE^−/− mice. The mice were administered water containing BAIBA for 14 weeks, and were then sacrificed at 20 weeks of age. Atherosclerotic plaque area, plasma BAIBA concentration, and plasma lipoprotein profiles were assessed. Immunohistochemical analyses of the plaque were performed to assess VCAM-1 and MCP-1 protein expression levels and macrophage infiltration. The results showed that BAIBA administration decreased atherosclerosis plaque area by 30%, concomitant with the elevation of plasma BAIBA levels. On the other hand, plasma lipoprotein profiles were not changed by the administration. Immunohistochemical analyses indicated reductions in VCAM-1, MCP-1, and Mac-2 protein expression levels in the plaque. These results suggest that BAIBA administration suppresses atherosclerosis progression without changing plasma lipoprotein profiles. We propose that the mechanisms of this suppression are reductions in both VCAM-1 and MCP-1 expression as well as macrophage infiltration into the plaque.

INTRODUCTION

Endurance exercise training induces transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in skeletal muscle.^1,2) We have previously reported that PGC-1α overexpressed specifically in skeletal muscle induces mitochondrial biosynthesis, muscle fiber-type switching from fast-twitch fiber to slow-twitch fiber, fatty acid transportation and oxidation, and increased endurance capacity.³⁾

Myokines, secreted from myocytes, regulate pleiotropic effects induced by exercise training.⁴⁾ PGC-1α expression in skeletal muscle increases the secretion of myokines such as irisin and β-aminoisobutyric acid (BAIBA).^{5, 6)} Irisin, which is secreted as a fragment of the FNDC5 protein, plays a role in the transformation of adipocytes into thermogenic cells,⁵⁾ as does BAIBA, which is a metabolite of valine⁶⁾ and thymine.⁷⁾

We have recently reported that skeletal muscle-specific PGC-1α overexpression suppresses atherosclerosis in apolipoprotein E-knockout (ApoE^−/−) mice.⁸⁾ In the same study,⁸⁾ we also demonstrated that irisin suppress tumor necrosis factor (TNF)-alpha-induced vascular cell adhesion molecule-1 (VCAM-1) and BAIBA suppress TNF-alpha-induced VCAM-1 and monocyte chemoattractant protein-1 (MCP-1) gene expression using human umbilical vein endothelial cells. Irisin injection has also been reported to suppress atherosclerosis progression in ApoE^−/− mice.⁹⁾ However, direct evidence that BAIBA administration suppresses atherosclerosis has not yet been reported. Therefore, in this study we investigated whether oral administration of BAIBA suppresses atherosclerosis in ApoE^−/− mice.

MATERIALS AND METHODS

Animals

ApoE^−/− mice were purchased from Charles River Laboratories Japan Inc. (Yokohama, Japan) and administered distilled water (ApoE^−/− group) or BAIBA (217794, Sigma-Aldrich, St. Louis, MO, U.S.A.)-supplemented water (ApoE^−/− + BAIBA group). The mice were housed in groups of 4 per cage in a room with a 12-h light/dark cycle at 22°C and provided with standard mouse chow (CE-2, CREA Japan Inc., Tokyo, Japan) and drinking water ad libitum. BAIBA was administered (170 mg/kg body weight/d) from 6 to 20 weeks of age. Bodyweight as well as liquid and food consumption were measured weekly. The mice were sacrificed at 20 weeks of age after 12 h of starvation, and their blood and hearts were collected. The animals were cared for according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and University of Shizuoka institutional guidelines. All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Shizuoka (registration numbers 165123 and 195230).

Blood Analysis

Blood samples were collected under isoflurane anesthesia after 12 h starvation. Plasma was separated using ethylenediaminetetraacetic acid and stored at −80°C until plasma BAIBA and lipid profile analyses were carried out. Plasma lipid profiles were analyzed by LipoSEARCH (Skylight Biotech Inc., Akita, Japan).¹⁰⁾

Plasma BAIBA Concentration

To extract BAIBA, 0.4 mol/L HClO₄ was mixed with the same volume of plasma. After filtration of the centrifuged supernatant, 20 µL of the sample was injected into an LC/MS system. Analysis was performed using a TSQ Quantum™ Access Max Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) equipped with an electrospray source ionization probe and an UltiMate 3000 RS Pump autosampler (Thermo Fisher Scientific Inc.). For LC analysis, an Intrada Amino Acids column (100 × 3 mm, Imtakt Corp., Kyoto, Japan) was used. Mobile phase A consisted of acetonitrile/formic acid = 100/0.1 (v/v) and mobile phase B consisted of 100 mM ammonium formate. The flow velocity was 0.4 mL/min. The gradient was as follows: 30–35% B (0–12 min), 100% B (12–15 min). The parameters of MS analysis were as follows: spray voltage, 4.0 kV; source temperature, 372°C; sheath gas flow rate, 50 arbitrary units; auxiliary gas flow rate, 15 arbitrary units; capillary temperature, 220°C; ion sweep gas pressure, 0 arbitrary units. MS data for BAIBA was obtained using selected reaction monitoring detecting in positive mode (m/z 104.2 > 30, m/z 104.2 > 57, m/z 104.2 > 86). BAIBA blood concentration was calculated from the peak area of the standard solution.

Atherosclerotic Plaque Area and Immunohistochemical Analyses

Atherosclerotic plaque area measurements and immunohistochemical analyses were performed as described previously.⁸⁾ Primary antibodies for immunofluorescence staining were used as follows: mouse anti-VCAM-1 (1 : 200, #550547, BD Bioscience, San Jose, CA, U.S.A.), anti-MCP-1 (1 : 50, #ab7202, Abcam, Cambridge, U.K.), anti-Mac-2 (1 : 16, #ab2785, Abcam), and anti-α-smooth muscle actin (α-SMA) (1 : 200, #ab7817, Abcam). Secondary antibodies for immunofluorescence staining were used as follows: goat anti-mouse immunoglobulin (Ig) M Alexa 488 (1 : 500, A21042, Invitrogen, Waltham, MA, U.S.A.), goat anti-rabbit IgG Alexa 647 (1 : 200, A21244, Invitrogen), goat anti-mouse IgG1 Alexa 555 (1 : 500, A21127, Invitrogen), and goat anti-mouse IgG2b Alexa 350 (1 : 200, A21140, Invitrogen).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, U.S.A.). Each group was compared with the other by Student’s t-test (for comparisons between the two groups). All data are expressed as the mean ± standard error of the mean (S.E.M.). The level of significance was set to p < 0.05.

RESULTS AND DISCUSSION

Atherosclerotic Plaque Area

There was no difference in bodyweight, liquid consumption, or food consumption between the two groups of mice (data not shown). Representative photomicrographs of the hematoxylin–eosin (H&E)-stained sections are presented in Fig. 1A. Quantitative atherosclerotic plaque area was 30% lower in the ApoE^−/− + BAIBA group compared to the ApoE^−/− group (Fig. 1B).

Fig. 1. Atherosclerotic Plaque Area

(A) Representative photomicrograph of an HE-stained aortic valve of the heart. The dashed line indicates atherosclerotic plaque area. The black line indicates 500 µm. (B) Quantitative value of the atherosclerotic plaque area. Mean ± S.E.M. * p < 0.05.

Plasma Lipid Profile

As shown in Table 1, no significant changes were observed in total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), non-HDL-C, or triglycerides (TG) between the two groups of mice.

Table 1. Plasma Lipid Profiles of the Two Groups of Mice

Parameters (mg/dL)	ApoE^−/−	ApoE^−/− + BAIBA	p
Parameters (mg/dL)	n = 8	n = 7	p
TC	449 ± 27	446 ± 18	0.94
HDL-C	21 ± 2	23 ± 2	0.64
Non-HDL-C	428 ± 26	424 ± 17	0.90
TG	56 ± 6	58 ± 4	0.78

TC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; TG, triglycerides. Data are expressed as the mean ± S.E.M.

Non-HDL-C and TG have been reported to accelerate atherosclerotic plaque formation.¹¹⁾ On the other hand, plasma HDL-C has been shown to be an anti-atherosclerotic lipid.¹²⁾ However, in the present study BAIBA administration suppressed atherosclerosis progression without changing the plasma lipid profile. Some reports have also indicated the suppression of atherosclerosis without changes in plasma lipid profile. For example, adiponectin, which is released from beige adipocytes,¹³⁾ shows an athero-protective effect in ApoE^−/− mice without changing the plasma levels of TC and HDL-C.¹⁴⁾

Plasma BAIBA Concentration

Plasma BAIBA was detected at a concentration of 3.4 ± 0.3 µM in the ApoE^−/− + BAIBA group, but was not detected in the plasma of the ApoE^−/− group, confirming that BAIBA was present in the plasma due to oral administration.

Immunohistochemical Analysis

Measurements were carried out for VCAM-1 and MCP-1 protein expression, markers of macrophage infiltration (Mac-2), and smooth muscle cell proliferation (α-SMA) in plaque. As shown in Fig. 2, VCAM-1, MCP-1, and Mac-2 protein expression levels in plaque were decreased by BAIBA administration. However, α-SMA levels were not changed by the administration.

Fig. 2. Immunohistochemical Analyses of Plaque

(A) Representative photomicrograph of immunofluorescence-stained atherosclerotic plaque. The white lines indicate plaque. (B) % positive area of the plaque. Mean ± S.E.M. * p < 0.05.

VCAM-1 and MCP-1 are known to be crucial for atherosclerosis initiation. VCAM-1-deficient mice have been shown to have lower levels of atherosclerotic plaque formation,¹⁵⁾ and MCP-1 is required for chronic macrophage infiltration during vascular lipid lesion formation in mice.¹⁶⁾ Thus, BAIBA-induced suppression of VCAM-1 and MCP-1 in the aorta may inhibit macrophage infiltration underneath the vascular endothelial cells.

CONCLUSION

In conclusion, we have found that the oral administration of BAIBA suppresses atherosclerosis progression in ApoE^−/− mice. We propose that the mechanism of this suppression is the suppression of VCAM-1 and MCP-1 expression and the inhibition of macrophage infiltration into atherosclerotic plaque. These results suggest that BAIBA is a food factor with the potential to prevent atherosclerosis.

Acknowledgments

We are indebted to Dr. Yusuke Sone (University of Shizuoka) and members of the Laboratory of Nutritional Biochemistry (Graduate School of Nutritional and Environmental Sciences, University of Shizuoka) for their technical assistance, and to Philip Hawke of the University of Shizuoka Scientific English Program for his comments on the English in the manuscript.

This study was financially supported by the Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP, No.14533567), and “Technologies for creating next-generation agriculture, forestry and fisheries” (funding agency: Bio-oriented Technology Research Advancement Institution, NARO). This study was also supported by JSPS KAKENHI (Grant Numbers 18H03181 and 26560400) and a University of Shizuoka Grant for Scientific and Educational Research.

Conflict of Interest

The authors declare no conflict of interest.

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