Higher cardiorespiratory fitness attenuates arterial stiffening associated with the Ala54Thr polymorphism in FABP2

Shumpei Fujie, Motoyuki Iemitsu, Haruka Murakami, Kiyoshi Sanada, Hiroshi Kawano, Yuko Gando, Ryoko Kawakami, Motohiko Miyachi

Abstract

Fatty acid binding protein 2 (FABP2) Ala54Thr polymorphism is a candidate gene associated with the risk of cardiovascular disease. Habitual exercise brings higher cardiorespiratory fitness and results in the improvement of cardiovascular disease risk. However, the effect of cardiorespiratory fitness level and FABP2 Ala54Thr polymorphism on the risk of cardiovascular diseases remains unclear. In the present study, a cross-sectional investigation of 837 Japanese men and women was performed to clarify the effects of cardiorespiratory fitness on the relationship between risk of cardiovascular disease and FABP2 Ala54Thr gene polymorphism. The study subjects were divided into high-cardiorespiratory fitness (High-Fit) and low-cardiorespiratory fitness (Low-Fit) groups based on the median value of peak oxygen uptake in each sex and decade. The FABP2 Ala54Thr polymorphism did not significantly affect carotid β-stiffness or blood pressure. In the Low-Fit group, carotid β-stiffness, systolic blood pressure, and diastolic blood pressure were higher for individuals with the Ala/Ala genotype compared with those with the Ala/Thr or Thr/Thr genotype, whereas no differences were observed in the High-Fit group. Additionally, serum triglyceride and plasma glucose levels were lower and serum high-density lipoprotein cholesterol levels were higher in the High-Fit group compared with the Low-Fit group; the FABP2 Ala54Thr polymorphism did not significantly affect these parameters. These results suggest that the higher cardiorespiratory fitness may attenuate the changes in central arterial stiffness and blood pressure that are associated with the FABP2 genotype.

  • peak oxygen uptake
  • systolic blood pressure
  • diastolic blood pressure
  • arteriosclerosis

the aorta and large arteries (i.e., central arteries) are conduits delivering blood to the tissues and organs, and the central arteries act as mechanical buffers to normalize fluctuations in blood pressure created by cardiac pulsation and intermittent blood flow. Increased arterial stiffness impairs this buffering function, leading to increased systolic blood pressure (SBP) and left ventricular afterload. Blacher et al. (6) and Laurent et al. (22) proposed arterial stiffness as an independent predictor of cardiovascular risk as well as all-cause and cardiovascular mortality. Increased arterial stiffness is associated with the development of several pathologic conditions, including hypertension, atherosclerosis, congestive heart failure, stroke, and aortic root regurgitation (2, 6, 22, 27). Several studies have shown that arterial stiffness is lower in physically active individuals compared with sedentary individuals (26, 32). Furthermore, aerobic exercise training reduces arterial stiffness (32, 33). Thus, regular exercise prevents or improves arterial stiffness.

Fatty acid binding protein 2 (FABP2) is an intestinal protein that is expressed primarily in mature enterocytes of the small intestine (5). FABP2 shows high affinity for saturated and unsaturated long-chain fatty acids and plays a key role in the absorption and intracellular transport of dietary fatty acids in the small intestine (1). A transition from G to A at codon 54 in exon 2 resulting alanine to threonine (Ala54Thr) of the gene encoding FABP2 is associated with increased affinity for long-chain fatty acid compared with G genotype (Ala containing protein) in the nondiabetic subjects of the Pima Indian population (4). Furthermore, several studies have suggested that individuals who are homozygous for the 54Thr codon in FABP2 had an increased dyslipidemia risk owing to elevated total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, and triglyceride (TG) levels, or reduced high-density lipoprotein (HDL) cholesterol levels (13, 15, 16, 38). Other studies, however, showed the FABP2 Ala54Thr polymorphism did not affect serum lipid profiles (14, 19, 29, 30). Thus, the effects of the FABP2 Ala54Thr polymorphism on the risk of cardiovascular disease have been conflicting. Additionally, individuals who were homozygous for the 54Thr codon in FABP2 did not show significantly different SBP or diastolic blood pressure (DBP) compared with 54Ala codon homozygotes (10, 19, 21, 35). Habitual exercise brings higher cardiorespiratory fitness and results in the prevention and/or improvement of cardiovascular disease risk, such as enhanced arterial stiffness, hypertension, dyslipidemia, and atherosclerosis (9, 11, 18, 33). However, it remains unclear whether cardiorespiratory fitness levels affect the relationship between the risk of cardiovascular disease and genetic variations in FABP2.

We hypothesized that the genotype at the Ala54Thr single nucleotide polymorphism (SNP; G→A) in exon 2 of FABP2 on chromosome 4 and cardiorespiratory fitness level may affect risks of arterial stiffness in healthy Japanese subjects. The present study was a cross-sectional investigation of 837 Japanese men and women (18–76 yr) subjects to clarify the effects of cardiorespiratory fitness on the relationship between arterial stiffness and the FABP2 Ala54Thr genotype.

METHODS

Subjects.

A total of 837 Japanese subjects (244 men and 593 women) between 18 and 76 yr of age participated in this cross-sectional study (mean age: 44 ± 1 yr). These subjects were sedentary or moderately active who participated in swimming, stretching, and moderate/light physical activities (at least 60 min session per week), and they did not participate any other vigorous sports activity. Subjects were divided into low cardiorespiratory fitness (Low-Fit) and high cardiorespiratory fitness (High-Fit) groups, based on the median value of peak oxygen uptake (V̇o2 peak), an index of cardiorespiratory fitness, in each gender and decade. Subjects were recruited for the present study by advertisement. All subjects were free of any overt signs or symptoms of chronic disease and were nonsmoking. Carotid β-stiffness (β-stiffness) and common carotid intima-media thickness (ccIMT) were examined as indexes of arterial stiffness. SBP, DBP, percent body fat, and genotype at the FABP2 Ala54Thr polymorphism were also determined. Total body fat mass was determined using dual-energy X-ray absorptiometry (Hologic QDR-4500A scanner; Hologic, Waltham, MA). Body composition was determined by Hologic software version 11.2:3 for windows (Hologic). Brachial SBP and DBP were measured in the supine position at rest with a vascular testing device (Colin Medical Technology, Tokyo, Japan). Serum cholesterol and TG levels and plasma glucose levels were also measured.

The study was approved by the Ethical Review Board of the National Institute of Health and Nutrition. Written informed consent was obtained from all subjects before inclusion in the study.

Measurement of V̇o2 peak.

o2 peak was measured using an incremental cycle exercise test on a cycle ergometer (828E; Monark, Varberg, Sweden). Incremental cycle exercise began at a work rate of 90 W (60–120 W) for men and 60 W (30–90 W) for women, and power output was increased by 15 W/min until the subjects could not maintain a fixed pedaling frequency of 60 rpm. The subjects were encouraged during the ergometer test to exercise at maximum intensity. Heart rate and rating of perceived exertion (RPE) were monitored minute by minute during exercise. RPE was obtained using the modified Borg scale (7). VO2 was monitored during the last 30 s of each period of increased work rate. Subjects breathed through a low-resistance two-way valve, and the expired air was collected in Douglas bags. Expired O2 and CO2 concentrations were measured by mass spectrometry (ARCO-1000A; Arco System, Chiba, Japan), and gas volume was determined with a dry gas meter (DC-5C; Shinagawa Seiki, Tokyo, Japan). The highest value of VO2 during the exercise test was designated as V̇o2 peak if three out of four of the following criteria were met: 1) a plateau in VO2 with increases in external work, 2) a maximal respiratory exchange ratio ≥1.1, 3) a maximal heart rate ≥90% of the age-predicted maximum (208 − 0.7 × age) (34), and 4) RPE ≥18.

Measurement of ccIMT.

ccIMT was measured from the images obtained by use of a Vivid i ultrasound system (GE Medical Systems, Milwaukee, WI) equipped with a high-resolution linear array broadband transducer as described previously (20, 25). Ultrasound images were analyzed using image analysis software (Image J; National Institutes of Health, Bethesda, MD). At least 10 measurements of ccIMT were obtained for each segment, and mean values were used for further analyses. This technique has excellent day-to-day reproducibility (coefficient of variation, 3 ± 1%).

Measurement of the β-stiffness index.

A combination of ultrasound imaging of the pulsatile common carotid artery and simultaneous applanation of tonometrically obtained arterial pressure from the contralateral carotid artery allowed noninvasive determination of arterial compliance (33). The carotid artery diameter was measured from images obtained from an ultrasound system equipped with a high-resolution linear array transducer. A longitudinal image of the cephalic portion of the common carotid artery was acquired 1–2 cm proximal to the carotid bulb. All image analyses were performed by the same investigator.

Pressure waveforms and amplitudes were obtained from the common carotid artery by use of a pencil-shaped probe with a high-fidelity strain gauge transducer (SPT-301; Millar Instruments, Houston, TX) (33). Because baseline blood pressure levels are subjected to hold-down force, the pressure signal obtained via tonometry was calibrated by equating the carotid mean arterial blood pressure and DBP to brachial artery values (20, 25, 33). The β-stiffness index was calculated using the equation [ln(P1/P0)]/[(D1 − D0)/D0], where D1 and D0 are the maximum (systolic) and minimum (diastolic) diameters, and P1 and P0 are the highest (systolic) and lowest (diastolic) blood pressures, respectively. The day-to-day coefficients of variation for the carotid artery diameter, pulse pressure, and β-stiffness were 2 ± 1, 7 ± 3, and 5 ± 2%, respectively.

SNP genotyping.

Genomic DNA was extracted from plasma buffy coats and buccal cells using a QIAamp DNA Blood Maxi Kit (Qiagen, Tokyo, Japan). Genotypes at the FABP2 Ala54Thr SNP were determined in real-time PCRs using TaqMan probes and an Applied Biosystems 7500 Fast real-time PCR system (Life Technologies Japan, Tokyo, Japan) as described previously with minor modifications (17, 18). Gene-specific primers and TaqMan probes for each SNP were synthesized using Primer Express v.1.5 software (Perkin-Elmer Applied Biosystems) according to published DNA sequences for each SNP as follows: Ala54Thr (G→A) in exon 2 of FABP2 (NCBI accession #rs1799883). The sequences of the oligonucleotides were as follows: FABP2 forward: 5′-TGAAGCTGACAATTACACAAGAAGGA-3′, FABP2 reverse: 5′-AGGTGACACCAAGTTCAAAAACAAC-3′, FABP2/G probe: 5′-AATCAAGCGCTTTTC-3′, FABP2/A probe: 5′-AATCAAGCACTTTTC-3′.

To examine 96-well PCR plates we used an Applied Biosystems 7500 Fast real-time PCR system in end-point analysis mode of the SDS v.1.7a software package (Life Technologies Japan). Genotypes were determined automatically by the single processing algorithms in the software.

Measurements of serum cholesterol and TG levels and plasma glucose level.

Fasting serum concentrations of cholesterol (TC and HDL) and TG, and plasma concentrations of glucose were determined by standard enzymatic techniques.

Statistical analysis.

The frequencies of FABP2 alleles were calculated using a gene-counting method, and the Hardy-Weinberg equilibrium was confirmed using the χ2 test. Differences between the High-Fit and Low-Fit groups were examined with Student's t-tests for unpaired values. One-way ANOVA was used to evaluate differences among FABP2 genotypes. Differences between fitness groups categorized based on genotype were assessed by two-way analysis of covariance model that included age and sex as covariates, and then Fisher's post hoc test applied when the difference was significant. Values are expressed as means ± SE. P < 0.05 was defined as statistically significant. All statistical analyses were performed by use of StatView (5.0; SAS Institute, Tokyo, Japan).

RESULTS

Comparison of characteristics in low and high cardiorespiratory fitness groups.

In the High-Fit group, body weight, body fat, blood glucose, and TG levels were significantly lower than in the Low-Fit group. HDL levels and V̇o2 peak values were significantly higher in the High-Fit group than in the Low-Fit group (Table 1). No significant differences between the High-Fit and Low-Fit groups were noted for age, height, SBP, DBP, β-stiffness, ccIMT, or TC levels (Table 1).

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Table 1.

Characteristics of subjects in the high cardiorespiratory fitness and low cardiorespiratory fitness groups

Comparison of characteristics between each genotype.

We analyzed FABP2 genotypes of the study subjects (Table 2). No significant differences between males and females were found for the frequencies of the various alleles at the FABP2 Ala54Thr polymorphism. In addition, allele frequencies did not deviate from the expected Hardy-Weinberg equilibrium.

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Table 2.

Distribution of gene polymorphisms of FABP2 Ala54Thr and allele frequency in the study subjects

We next compared the characteristics of subjects with the different FABP2 alleles (Table 3). No significant differences were found among the genotypes for all parameters.

View this table:
Table 3.

Genotypes of FABP2 Ala54Thr and subject characteristics

Comparison of characteristics between genotypes and cardiorespiratory fitness groups.

We compared the characteristics of subjects with different genotypes and levels of fitness (Table 4). The interaction between FABP2 genotypes and fitness groups was significant for body weight (F = 4.70, DF = 2, P = 0.009) and body mass index (BMI) (F = 5.52, DF = 2, P = 0.004). Compared with the Low-Fit group, individuals in High-Fit group showed significantly lower body weight (Ala/Thr genotypes) and BMI (Ala/Ala and Ala/Thr genotypes). Additionally, all High-Fit subjects had lower body fat and higher V̇o2 peak values than Low-Fit group; there was a significant main effect of fitness level that was independent of FABP2 genotype (F = 147.1, DF = 1, P < 0.05). No significant differences were noted between individuals in the two cardiorespiratory fitness groups and with various genotypes for age, height, TC, HDL, TG, or glucose levels.

View this table:
Table 4.

Characteristics of subjects in each cardiorespiratory fitness and genotypes of FABP2 Ala54Thr group

Comparison of β-stiffness, blood pressure, and ccIMT between genotypes and cardiorespiratory fitness groups.

The interaction between FABP2 genotypes and fitness groups was significant for β-stiffness (F = 3.14, DF = 2, P = 0.044). β-Stiffness values for Low-Fit subjects with the Ala/Ala genotype at the FABP2 Ala54Thr polymorphism were significantly higher than those of individuals with the Thr/Thr or Ala/Thr genotype, whereas no significant differences were observed for β-stiffness among the three genotypes in the High-Fit group (Fig. 1).

Fig. 1.

Carotid β-stiffness values are shown for each fitness group and for individuals with various genotypes at a polymorphism in the FABP2 gene (encoding Ala/Ala, Ala/Thr, or Thr/Thr at amino-acid position 54). Data are expressed as means ± SE. Low-Fit, low cardiorespiratory fitness group; High-Fit, high cardiorespiratory fitness group.

Additionally, there were significant interactions in both FABP2 genotypes and fitness groups in SBP (F = 3.61, DF = 2, P = 0.027). SBP in Low-Fit subjects with the Ala/Ala or Ala/Thr genotypes was significantly higher than in individuals with the Thr/Thr genotype, but no significant differences were detected among the three genotypes in the High-Fit group (Fig. 2A). Moreover, there were significant interactions in both FABP2 genotypes and fitness groups in DBP (F = 3.39, DF = 2, P = 0.034). DBP in Low-Fit subjects with the Ala/Ala genotype at the FABP2 Ala54Thr polymorphism was significantly higher than in individuals with the Thr/Thr genotype; again, the High-Fit group did not show significant differences in DBP among individuals with any of the three genotypes (Fig. 2B). Finally, ccIMT was not different among the three Ala54Thr genotypes in the Low-Fit or High-Fit group (Table 4).

Fig. 2.

Brachial systolic blood pressure (SBP, A) and diastolic blood pressure (DBP, B) are shown for each fitness group and individuals with various genotypes at a polymorphism in the FABP2 gene (encoding Ala/Ala, Ala/Thr, or Thr/Thr at amino-acid position 54). Data are expressed as means ± SE.

DISCUSSION

The present cross-sectional study of Japanese subjects demonstrated associations among arterial stiffness (β-stiffness and blood pressure), cardiorespiratory fitness, and genotype at the FABP2 Ala54Thr polymorphism. Interestingly, in the Low-Fit subjects, carotid β-stiffness and brachial blood pressure were higher in individuals with the Ala/Ala genotype than in those with the Thr/Thr genotype. These differences were not observed in High-Fit subjects, however. Conversely, lipid parameters, such as TC, HDL, and TG levels, were not affected by the combined effects of the FABP2 Ala54Thr genotype and cardiorespiratory fitness. Moreover, no significant differences were noted for ccIMT, an index of atherosclerosis, based on FABP2 Ala54Thr genotype and cardiorespiratory fitness. Thus, the beneficial effects of cardiorespiratory fitness on central arterial stiffness and blood pressure may be related to the FABP2 Ala54Thr genotype, independent of such atherosclerotic risk factors as ccIMT and lipid profiles.

In this study, genotypes at the Ala54Thr polymorphism in the FABP2 gene were not associated with specific serum lipid profiles. Thr54-containing FABP2 shows a twofold increase in its affinity for long-chain fatty acids compared with the Ala54-containing protein, which increases the absorption of fatty acids (4). In a meta-analysis, it has been shown that the Thr54 genotype of FABP2 Ala54Thr polymorphism is associated with increased TC and LDL levels and decreased HDL levels (38). However, some studies have reported that levels of these lipids profiles did not significantly differ based on the genotype at the FABP2 Ala54Thr polymorphism (14, 19, 29, 30). Inconsistent results were also reported for Japanese subjects (19, 31). Thus, the relationship between FABP2 Ala54Thr polymorphism and lipids profile was not consistent. Differences in dietary lipid intake among various cultures may explain, at least in part, the conflicting results observed in previous studies. Although dietary lipid intake is a major determinant of blood cholesterol levels, we could not assess this parameter in all subjects.

Differences in β-stiffness and blood pressure were not seen among FABP2 Ala54Thr polymorphism unless subjects were divided into High-Fit and Low-Fit groups in the present study. Several studies have reported that the FABP2 Ala54Thr polymorphism did not affect individual SBP or DBP (10, 19, 21, 35). In this study, β-stiffness and blood pressure were elevated in Low-Fit individuals with the Ala/Ala genotype compared with Thr-allele. Consequently, lifestyle differences that reduce fitness levels may be causal factor of these differences in β-stiffness and blood pressure.

Although carotid β-stiffness and brachial blood pressure were elevated in Low-Fit individuals with the Ala/Ala genotype compared with subjects bearing a Thr-coding allele, these differences were not observed in the High-Fit subjects. The mechanism underlying the combined effects of cardiorespiratory fitness and the Ala54Thr genotype on arterial stiffness is unclear. Regular exercise improves endothelial function through increased nitric oxide (NO) production and decreased endothelin (ET)-1 levels (24). Exercise training was also shown to alter the expression levels of vasodilation-related molecules, including endothelial NO synthase, and improve arterial stiffness in the rat aorta (23). Additionally, there was the positive correlation between cardiorespiratory fitness level and flow-mediated dilation of the brachial artery, as an index of endothelial function via flow-mediated NO production, in healthy male adults (8). Therefore, habitual exercise and higher fitness level are thought to decrease the stiffness of central arteries by improving endothelial function. Based on these previous observations, in the High-Fit subjects with Ala/Ala genotype, it is speculated that exercise training induces improvement of endothelial function via another mechanism, e.g., increase in NO production and/or decrease in ET-1 levels. These results taken together would suggest that individuals may need to exercise regularly, which improves cardiorespiratory fitness, to counteract the negative effects of the Ala/Ala genotype at the FABP2 polymorphism. In fact, low V̇o2 max levels are associated with a twofold increase in the incidence of hypertension, suggesting that high levels of fitness can prevent hypertension (28). However, our study was a cross-sectional investigation. Consequently, further interventional studies are needed to examine the effect of the FABP2 polymorphism.

In this study, ccIMT, evaluated as the thickness of the carotid arterial wall, was unaffected by the relationship between FABP2 Ala54Thr genotype and fitness level in healthy Japanese subjects. A previous study showed the relationship between FABP2 Ala54Thr polymorphism and internal carotid artery stenosis in stroke patients (37). Additionally, Wanby et al. (36) reported that allele and genotype frequencies of FABP2 Ala54Thr polymorphism did not differ between patients with severe carotid stenosis and controls. The FABP2 Ala54Thr polymorphism is not associated with severe carotid stenosis. Thus, the effect of FABP2 Ala54Thr polymorphism may depend on the condition of carotid stenosis.

The 54Thr allele is linked to the promoter haplotype B (12). This haplotype is composed of two promoter variants, i.e., haplotype A and B (12). Haplotype B was associated with BMI and blood lipid profiles in individuals (3, 12). Additional studies may be needed to examine the effects of fitness on the relationships between arterial stiffness and other FABP2 genotypes. Additionally, because our study examined only an Asian population, translating our results to other populations may be difficult owing to genotypic differences among races.

We investigated associations among cardiorespiratory fitness, arterial stiffness, and genotype at the FABP2 Ala54Thr polymorphism in healthy Japanese subjects. We identified a higher arterial stiffness associated with the Ala homozygote at Ala54Thr of the FABP2 gene in cardiorespiratory Low-Fit subjects, regardless of increased atherosclerotic risk based on lipid profile parameters in the Ala homozygote. Thus, sufficient cardiovascular fitness may affect cardiovascular adaptations to molecular variations in the FABP2 gene in Japanese individuals. Further studies, however, are required to clarify the effects of fitness level on genetically determined risk of cardiovascular disease.

GRANTS

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (#23240089, M. Miyachi; #22650166 and #23680071, M. Iemitsu) and a Grant-in-Aid for Scientific Research from the Ministry of Health, Labor, and Welfare of Japan (M. Miyachi).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.F., M.I., H.M., K.S., H.K., Y.G., and R.K. analyzed data; S.F. and M.I. prepared figures; M.I. and M.M. conception and design of research; M.I., H.M., K.S., H.K., Y.G., and R.K. performed experiments; M.I., H.M., and M.M. interpreted results of experiments; M.I., H.M., and M.M. drafted manuscript.

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