J C E M
O N L I N E
Hot Topics in Translational Endocrinology—Endocrine Research
Cardiorespiratory Fitness and Visceral Fat Are Key
Determinants of Serum Fibroblast Growth Factor 21
Concentration in Japanese Men
Hirokazu Taniguchi,* Kumpei Tanisawa,* Xiaomin Sun, Zhen-Bo Cao,
Satomi Oshima, Ryuken Ise, Shizuo Sakamoto, and Mitsuru Higuchi
Graduate School of Sport Sciences (H.T., K.T., X.S., R.I.) and Faculty of Sport Sciences (Z.-B.C., S.O., S.S.,
M.H.), Waseda University, and Institute of Advanced Active Aging Research (S.S., M.H.), Tokorozawa
359 –1192, Japan
Context: Fibroblast growth factor-21 (FGF21) is an important metabolic regulator suggested to
improve glucose metabolism and prevent dyslipidemia. An FGF21-resistant state that increases
circulating FGF21 has been reported in obese patients. Although regular exercise prevents metabolic disease, the relationship of the fitness level to serum FGF21 level and body fat distribution
in humans remains poorly understood.
Objective: The objective of the study was to determine the relationship among the serum FGF21
concentration, cardiorespiratory fitness (CRF) level, and visceral fat area (VFA).
Design: Serum FGF21 was measured by an ELISA in 166 middle-aged and elderly Japanese men (aged
30 –79 y) and 25 untrained and 21 endurance-trained young men (aged 19 –29 y). CRF was assessed by
measuring the peak oxygen uptake (VO2peak) and VFA by magnetic resonance imaging.
Results: In the middle-aged and elderly subjects, the serum FGF21 level correlated with the
VO2peak (r ⫽ ⫺0.355, P ⬍ .001) and VFA (r ⫽ 0.487, P ⬍ .001). Stepwise multiple regression analysis
showed VFA to be most strongly associated with the serum FGF21 level (␤ ⫽ .360, P ⬍ .001), and
VO2peak was also an independent predictor of the serum FGF21 level (␤ ⫽ ⫺.174, P ⫽ .019).
Furthermore, the proportion of subjects with an FGF21 level below the limit of detection was
significantly higher among the endurance-trained than among the untrained young men (71.4%
vs 24.0%, P ⫽ .001), and the VO2peak and VFA were independently associated with an undetectable
FGF21 level (P ⬍ .05).
Conclusions: CRF and VFA are key determinants of the circulating FGF21 concentration. (J Clin
Endocrinol Metab 99: E1877–E1884, 2014)
ibroblast growth factor-21 (FGF21) is an important
metabolic regulator with multiple beneficial effects
on glucose homeostasis and lipid metabolism in animal
models of diabetes and obesity (1, 2). The finding that
injection of LY2405319, an FGF21 variant, for 28 days
significantly improved glucose homeostasis and alleviated
F
dyslipidemia in patients with obesity and type 2 diabetes
(3) has led to considerable recent interest in the physiological effects of FGF21 in humans.
Although the beneficial effects of circulating FGF21 are
known, several cross-sectional studies have demonstrated
a positive correlation between the circulating FGF21 level
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received March 27, 2014. Accepted July 1, 2014.
First Published Online July 11, 2014
* H.T. and K.T. contributed equally to this work.
Abbreviations: ALT, alanine aminotransferase; ApoC-III, apolipoprotein C-III; AST, aspartate aminotransferase; BMI, body mass index; CI, confidence interval; CRF, cardiorespiratory fitness; ET, endurance-trained athletes; FFA, free fatty acid; FFM, fat free mass; FGF21,
fibroblast growth factor-21; FPG, fasting plasma glucose; ␥-GTP, ␥-glutamyl transferase;
HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostasis model assessment of insulin resistance; LDL-C, low-density lipoprotein cholesterol; OR, odds ratio; SFA, sc fat area; TG, triglyceride; total-C, total cholesterol; UT,
untrained men; VFA, visceral fat area; VO2peak, peak oxygen uptake.
doi: 10.1210/jc.2014-1877
J Clin Endocrinol Metab, October 2014, 99(10):E1877–E1884
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CRF and VFA Influence Serum FGF21 Level
and body mass index (BMI) in humans (4, 5). Animal
models of obesity also exhibit elevated endogenous levels
of FGF21 (6, 7), suggesting that a high serum level of
FGF21 may not be beneficial in the obese state. To address
this discrepancy, previous animal studies have investigated the existence of FGF21 resistance in dietary-induced
obese mice (6, 7). In addition to elevated serum FGF21
levels, the dietary-induced obese mice display a lower level
of FGF21 receptors and a severely impaired signaling response to FGF21 in the liver and adipose tissue (6, 7).
Therefore, a chronically elevated serum level of FGF21
likely represents resistance to and reduction of the therapeutic effect of FGF21, which may lead to abnormal glucose and lipid metabolism. In fact, prospective studies
have revealed that an elevated level of serum FGF21 precedes the development of type 2 diabetes in both European
and Asian populations (8, 9).
On the other hand, voluntary wheel running exercise
for 36 weeks was recently reported to reduce the blood
FGF21 level in a rat model of obesity (10). In that study,
the level of FGF21 receptor expression in the liver was
significantly higher in exercised Otsuka Long-Evans
Tokushima Fatty rats than in sedentary Otsuka Long-Evans Tokushima Fatty rats, suggesting that regular exercise
prevents FGF21 resistance in target organs (10). Although
the effect of regular exercise on FGF21 resistance has not
been evaluated in humans, the ability of regular exercise to
increase cardiorespiratory fitness (CRF) and reduce the
risk for type 2 diabetes (11, 12) and metabolic syndrome
(13) is well documented. This suggests that a high CRF
level might prevent FGF21 resistance and thus the onset of
many chronic diseases. However, no association between
the CRF level and the circulating FGF21 concentration has
yet been documented. Furthermore, although visceral fat
accumulation has been convincingly shown to be an independent predictor of metabolic risk factors (14), the
contribution of visceral fat area (VFA) to the serum FGF21
level in humans is poorly understood.
Therefore, this study aimed to determine the relationship between the serum FGF21 concentration and the CRF
level. Because obesity has been suggested to induce FGF21
resistance, we also examined whether the degree of obesity
affects the relationship between the serum FGF21 concentration and the CRF level.
Materials and Methods
Subjects
The first group of study participants consisted of 166 Japanese men aged 30 –79 years (middle aged and elderly). Subjects
were originally recruited for a separate study examining the effects of aging and exercise on the relationship between genetic
J Clin Endocrinol Metab, October 2014, 99(10):E1877–E1884
factors and metabolic syndrome risk (15); samples obtained during that study were reanalyzed in the present study. The participants had no history of diabetes or cardiovascular disease and
were free of other chronic diseases such as cancer, chronic kidney
failure, nonalcoholic steatohepatitis, and autoimmune disorders. Eleven subjects (6.9%) were using lipid-lowering medication. Fourteen subjects (8.8%) with prediabetes [defined as fasting plasma glucose (FPG) levels of 110 –125 mg/dL or glycated
hemoglobin (HbA1c) levels of 5.7%– 6.4%] were included. The
current and/or former smoking status was recorded using a questionnaire. Daily alcohol intake was assessed using a brief, selfadministered diet history questionnaire.
To determine whether long-term endurance training influences the circulating FGF21 level, we also recruited a group of
men aged 19 –29 years consisting of 21 endurance-trained athletes (ET: rowers, n ⫽ 10; triathletes, n ⫽ 10; and cycle racers, n ⫽
1) and 25 healthy untrained men (UT). None of these younger
participants had any history of chronic disease or medication
use. The ET participants regularly performed 1022 ⫾ 399 minutes of endurance training per week.
All participants provided written informed consent to participate before enrolling in the study. This research project was
approved by the Ethics Committee of Waseda University.
Anthropometric characteristics
Body weight and body fat percentage (as assessed by bioelectrical impedance analysis) were measured using an electronic
scale (Inner Scan BC-600; Tanita, Inc), and height was measured
with a stadiometer (YL-65; YAGAMI, Inc). BMI and fat free
mass (FFM) were calculated from the measurements of body
weight, body fat percentage, and height. The VFA and sc fat area
(SFA) were measured by magnetic resonance imaging (Signa 1.5
T; General Electric, Inc) as described previously (15). The imaging conditions included a T1-weighted spin-echo and axialplane sequence with a slice thickness of 10 mm, a repetition time
of 140 milliseconds, and an echo time of 12.3 milliseconds. The
cross-sectional area of the VFA and SFA at the umbilical level
was determined using image-analysis software (Slice-o-matic 4.3
for Windows; Tomovision). The coefficient of variation for the
cross-sectional area at the umbilical level was 0.4%.
Cardiorespiratory fitness
CRF was assessed via a maximal graded exercise test using a
cycle ergometer (Ergomedic 828E; Monark; or Aerobike
75XLII; Combi) and quantified as the peak oxygen uptake
(VO2peak). The graded cycle exercise began at a workload of
45–90 W, which was then increased by 15 W/min until the participant could no longer maintain a pedaling frequency of 60
rpm. The heart rate and rating of perceived exertion were monitored each minute during exercise. During the incremental portion of the exercise test, the participant’s expired gas was collected and the O2 and CO2 concentrations measured and
averaged over 30-second intervals using an automated gas analyzing system (Aeromonitor AE-300; Minato Medical Science).
The highest value of VO2 recorded during the exercise test was
considered the VO2peak (milliliters per kilogram⫺1 per minute⫺1). Middle-aged and elderly subjects were subsequently divided into low- and high-CRF groups according to the median
VO2peak value of each age group, as follows (in milliliters per
kilogram⫺1 per minute⫺1): 30 –39 years, 41.8; 40 – 49 years,
doi: 10.1210/jc.2014-1877
36.3; 50 –59 years, 38.7; 60 – 64 years, 32.7; 65– 69 years, 30.4;
and 70 –79 years, 28.4.
Collection and analysis of blood samples
The participants were instructed not to engage in any intensive exercise on the day previous to blood sampling. Blood samples were collected between 8:30 and 11:00 AM after a 12-hour
overnight fast and then centrifuged at 3000 ⫻ g at 4°C for 15
minutes. The serum and plasma were collected and stored at
⫺80°C until analysis. The serum enzymatic activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT),
and ␥-glutamyl transferase (␥-GTP), and the concentrations of
total cholesterol (total-C), low-density lipoprotein cholesterol
(LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), free fatty acids (FFAs), apolipoprotein C-III (ApoCIII), fasting plasma glucose (FPG), insulin, and HbA1c were determined by BML, Inc. The homeostasis model assessment of
insulin resistance (HOMA-IR) value was calculated from the
fasting concentrations of plasma glucose and serum insulin as
follows:
HOMA-IR ⫽ 关fasting glucose (milligrams per deciliter)]
⫻[fasting insulin (microunits per milliliter)]/405
The serum FGF21 concentration was determined using a commercially available ELISA kit (DF2100; R&D Systems, Inc) according to the manufacturer’s instructions. The detectable range
of the assay was 17.0 –2410.9 pg/mL. The intra- and interassay
coefficients of variation reported by the manufacturer were
2.9%–3.9% and 5.2%–10.9%, respectively.
Statistical analysis
All statistical analyses were performed using SPSS version
21.0 (SPSS, Inc). The Kolmogorov-Smirnov test was performed
to assess the normality of data distribution, and several variables
were log or square root transformed prior to analysis to obtain
a normal distribution of values. A Student’s t test (for normally
distributed data), the Mann-Whitney U test (for nonnormally
distributed data) and the ␹2 test (for categorical data) were used
to evaluate the differences between the low- and high-CRF
groups of middle-aged and elderly subjects and between the ET
and UT groups of young subjects. Associations among the variables were detected using Pearson’s correlation coefficients. Partial correlation analysis adjusted for age and BMI was also
performed.
We performed stepwise multiple linear regression analyses to
identify the factors influencing FGF21 in the middle-aged and
elderly subjects. Age, alcohol consumption, current or former
smoking status, use of lipid-lowering medication, and the variables that showed significant partial correlation with FGF21
after adjustment for age and BMI were included in the linear
regression model. Because a considerable number of the young
subjects had a serum FGF21 level below the limit of detection of
the assay, a multiple logistic regression analysis was performed
to assess the odds ratio (OR) and 95% confidence interval (CI)
for undetectable serum FGF21 with respect to the VO2peak and
VFA values. All measurements and calculated values are presented as the mean ⫾ SD (for normally distributed variables) or
median (interquartile ratio) (for nonnormally distributed variables), and the level of statistical significance was set at P ⬍ .05.
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Results
Comparison of the characteristics of the middleaged and elderly participants between the lowand high-CRF groups
After exclusion of subjects with a serum FGF21 level
below the limit of detection of the ELISA, as well as two
subjects with an extremely high FGF21 level (1913.8 and
2410.9 pg/mL), a total of 160 participants were analyzed.
The serum FGF21 level among these 160 subjects ranged
from 17.0 to 628.9 pg/mL. Because the Kolmogorov-Smirnov test showed that the serum FGF21 level was normally
distributed (P ⫽ .075) whereas the log-transformed
FGF21 level was not normally distributed (P ⬍ .001), we
used the raw FGF21 values in subsequent analyses.
VO2peak and the HDL-C level were higher in the highCRF than in the low-CRF group, whereas BMI, body fat
percentage, VFA, SFA, the TG, HbA1c, and insulin levels
and HOMA-IR were lower in the high-CRF group than in
the low-CRF group (Table 1, P ⬍ .05). The serum FGF21
level was also significantly lower in the high-CRF group
than in the low-CRF group (Table 1, P ⫽ .007).
Associations of the serum FGF21 level with CRF,
body composition, and metabolic parameters in
middle-aged and elderly men
Table 2 presents the correlations of the serum FGF21
level with other variables. Our cross-sectional study
showed that the serum FGF21 level correlated negatively
with VO2peak (r ⫽ ⫺0.355, P ⬍ .001) (Figure 1A). This
correlation remained significant after adjustment for age
and BMI (r ⫽ ⫺0.314, P ⬍ .001).
Furthermore, the serum FGF21 level correlated positively with VFA (r ⫽ 0.487, P ⬍ .001) (Figure 1B). This
correlation remained significant after adjustment for age
and BMI (r ⫽ 0.494, P ⬍ .001). Although SFA correlated
with serum FGF21 level (r ⫽ 0.187, P ⫽ .018), this correlation was lost when adjusted for age and BMI. The
serum FGF21 level also correlated positively with the ALT
(r ⫽ 0.160, P ⬍ .044), ␥-GTP (r ⫽ 0.257, P ⫽ .001), TGs
(r ⫽ 0.352, P ⬍ .001), and ApoC-III levels (r ⫽ 0.289, P ⬍
.001), even after adjustment for age and BMI.
Stepwise multiple regression analysis was performed to
elucidate the independent predictors of the serum FGF21
level (Table 3). We included VFA, VO2peak, the ALT,
␥-GTP, TG, and ApoC-III levels, age, alcohol consumption, current or former smoking status, and use of lipidlowering medication as independent variables. In the bestfit model, VFA was most strongly associated with the
serum FGF21 level (␤ ⫽ .360, P ⬍ .001), whereas
VO2peak was an independent negative predictor of the
serum FGF21 level (␤ ⫽ ⫺.174, P ⫽ .019). The serum TG
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Table 1.
CRF and VFA Influence Serum FGF21 Level
J Clin Endocrinol Metab, October 2014, 99(10):E1877–E1884
Characteristics of the Middle-Aged and Elderly Subjects
Variable
Low CRF
High CRF
P
Valuea
n
Age, y
Height, cm
Body weight, kg
BMI, kg/m2
Body fat, %
VFA, cm2
SFA, cm2
FFM, kg
VO2peak, mL 䡠 kg⫺1 䡠 min⫺1
AST, IU/L
ALT, IU/L
␥-GTP, IU/L
Total-C, mg/dL
HDL-C, mg/dL
LDL-C, mg/dL
TGs, mg/dL
FFAs, mEq/L
ApoC-III, mg/dL
FPG, mg/dL
HbA1c, %
Insulin, ␮U/mL
HOMA-IR
FGF21, ng/mL
Alcohol consumption, g/d
Current or former smoking status, %
Lipid-lowering medication use, %
Prediabetes, %
80
62.0 ⫾ 11.5
169.8 ⫾ 6.7
69.7 ⫾ 9.6
24.1 ⫾ 2.5
21.7 ⫾ 4.6
121.9 ⫾ 40.6
125.5 ⫾ 45.5
54.3 ⫾ 6.0
28.3 ⫾ 5.2
24.0 (20.0 –27.0)
20.0 (16.0 –27.0)
29.0 (23.0 – 41.0)
214.3 ⫾ 31.5
55.0 (49.0 – 63.8)
127.0 ⫾ 28.3
101.5 (71.0 –138.0)
0.62 (0.42– 0.77)
10.6 ⫾ 3.8
96.8 ⫾ 10.0
5.05 ⫾ 0.30
6.1 (4.2– 8.2)
1.41 (0.99 –1.96)
277.5 ⫾ 118.2
14.1 (2.6 –39.1)
58.8
5.0
13.8
80
61.4 ⫾ 11.9
170.7 ⫾ 6.4
67.9 ⫾ 8.6
23.2 ⫾ 2.3
19.3 ⫾ 4.1
97.0 ⫾ 42.7
104.4 ⫾ 45.3
54.6 ⫾ 5.6
37.4 ⫾ 5.8
24.0 (21.0 –28.8)
19.0 (15.0 –24.0)
26.0 (21.0 – 45.0)
211.8 ⫾ 34.8
65.0 (55.0 –71.8)
120.4 ⫾ 28.2
78.0 (62.0 –101.0)
0.51 (0.40 – 0.72)
10.0 ⫾ 2.8
96.0 ⫾ 8.1
4.91 ⫾ 0.22
4.2 (3.2– 6.3)
1.04 (0.73–1.48)
228.6 ⫾ 107.0
23.3 (9.8 – 43.4)
38.8
8.8
3.8
.746
.351
.233
.022
.001
<.001
.004
.755
<.001
.348
.467
.354
.633
<.001
.143
<.001
.094
.230
.559
.001
<.001
.001
.007
.884
.017
.534
.025
Data are the mean ⫾ SD or median (interquartile ratio) values. Prediabetes was defined as FPG 110 –125 mg/dL or HbA1c 5.7%– 6.4%. Boldface
indicates significance.
a
Low CRF vs high CRF.
level was also positively associated with the serum FGF21
level (␤ ⫽ .206, P ⫽ .005).
Effect of endurance training on the serum FGF21
level in young men
The VO2peak was higher in ET men than in healthy UT
men (P ⬍ .001), whereas age, VFA, and SFA were lower
in the ET than in the UT group (Supplemental Table 1, P ⬍
.001). Surprisingly, 21 (45.7%) subjects in the young
group had a serum FGF21 level below the limit of detection of the assay. Therefore, we compared subjects from
the ET and UT groups for whom FGF21 was not detected.
Although measurable serum FGF21 levels did not differ
significantly between the groups (P ⫽ .106), more subjects
in the ET than in the UT group had levels of serum FGF21
that were below the limits of detection (Supplemental Table 1, P ⫽ .001).
Because VO2peak and VFA were independent predictors of the serum FGF21 level in the middle-aged and elderly men, we performed multiple logistic regression analysis to examine whether the extremely low levels of serum
FGF21 among the ET participants were due to a high
VO2peak or low VFA. After adjustment for age, VFA, and
TGs, the OR for a serum FGF21 level below the limit of
detection increased significantly by 1.114 (95% CI
1.001–1.239, P ⫽ .047) per increment of VO2peak value
(mL 䡠 kg⫺1 䡠 min⫺1) (Table 4). After adjustment for age,
VO2peak, and TGs, VFA was also independently associated with the occurrence of a serum FGF21 level below the
limit of detection (OR 0.938, 95% CI 0.882– 0.998, P ⫽
.044).
Discussion
The present study revealed that CRF is negatively associated with the serum FGF21 level. In contrast, a higher
circulating FGF21 level was associated with greater visceral fat content. This was a first investigation of the relationship of CRF on the serum FGF21 concentration in
humans.
FGF21 is reported to be expressed primarily in the liver
(16). Because hepatic expression of FGF21 is directly regulated by peroxisomal proliferator-activated receptor-␣,
which is up-regulated by fatty acids (17), serum FGF21
level in humans is increased by both overfeeding (18) and
doi: 10.1210/jc.2014-1877
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Table 2. Correlations of the Serum FGF21 Level With
the Other Variables in Middle-Aged and Elderly Subjects
FGF21 (Age and
BMI Adjusted)
FGF21
Age, y
BMI, kg/m2
Body fat, %
VFA, cm2
SFA, cm2
FFM, kg
VO2peak,
mL 䡠 kg⫺1 䡠 min⫺1
AST, IU/L
ALT, IU/L
␥-GTP, IU/L
Total-C, mg/dL
HDL-C, mg/dL
LDL-C, mg/dL
TG, mg/dL
FFA, mEq/L
ApoC-III, mg/dL
FPG, mg/dL
HbA1c, %
Insulin, ␮U/mL
HOMA-IR
r
P
Value
r
P
Value
0.144
0.133
0.146
0.487
0.187
0.019
⫺0.355
.069
.093
.065
<.001
.018
.812
<.001
0.026
0.494
0.150
⫺0.028
⫺0.314
.747
<.001
.059
.727
<.001
0.101
0.120
0.245
0.061
⫺0.143
0.085
0.364
0.090
0.303
0.131
0.134
0.038
0.048
.204
.131
.002
.441
.072
.285
<.001
.257
<.001
.102
.093
.635
.549
0.112
0.160
0.257
0.045
⫺0.137
0.067
0.352
0.076
0.289
0.092
0.083
⫺0.026
⫺0.025
.162
.044
.001
.572
.085
.402
<.001
.342
<.001
.257
.306
.743
.761
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Table 3. Stepwise Multiple Linear Regression Analysis
of FGF21 in Middle-Aged and Elderly Subjects
Dependent
Variable
Independent
Variable
FGF21
VFA, cm2
TG, mg/dL
VO2peak,
mL 䡠 kg⫺1 䡠 min⫺1
␤
P
Value
0.360
0.206
⫺0.174
<.001
.005
.019
Abbreviation: ␤, standardized coefficient. The model was adjusted for
age, alcohol consumption, current or former smoking status, and use
of lipid-lowering medication. TGs were log transformed prior to
analysis. Alcohol consumption was square root transformed prior to
analysis (model r2 ⫽ 0.327, P ⬍ .001). Boldface indicates significance.
exhibit elevated serum FGF21 accompanied by reduction
of FGF receptor expression in the liver and adipose tissue
(6, 7). A high level of circulating FGF21 has also been
documented in obese people (4, 5, 27), and elevated serum
FGF21 has been reported to be an independent predictor
of type 2 diabetes (8, 9) and metabolic syndrome (9). It is
therefore plausible that chronic disease is induced by
FGF21 resistance-mediated metabolic decline, whereas a
lower basal FGF21 concentration suggests sensitivity to
FGF21 and thus resistance to chronic diseases. Because
Data are the Pearson’s correlation coefficients. The AST, ALT, ␥-GTP,
regular exercise was reported to prevent FGF receptor
HDL-C, TG, FFA, insulin, and HOMA-IR data were log transformed
down-regulation and serum FGF21 level elevation in
prior to analysis. Boldface indicates significance.
obese model rats (10), the present study examined the
fasting (19). FGF21 signaling has been investigated at the relationship between the fitness level and serum FGF21
molecular level in vitro and in vivo and appears to act via concentration in humans. In agreement with the animal
cell surface classic FGF receptors complexed with study, the serum FGF21 level was significantly lower in the
␤-klotho (20 –23). ␤-Klotho is expressed mainly in the high than in the low fitness group (Table 1). Furthermore,
liver and adipose tissue (20), consistent with the pattern of the circulating FGF21 concentration correlated negatively
FGF21 activity. FGF21 has been shown to stimulate he- with the CRF level (Figure 1A). These results suggest that
patic fatty acid oxidation (24, 25), adipose tissue glucose regular exercise that increases aerobic capacity also imuptake (1), adipose thermogenic gene expression, and proves FGF21 sensitivity in humans as seen in rodents.
The previous animal study did not eliminate the effect
browning of white adipose tissue (26). Therefore, the therof
obesity
on FGF21 resistance because the body weight
apeutic efficacy of FGF21 is likely affected by the expresand
epididymal
fat mass were significantly lower in the
sion of tissue-specific FGF receptors.
trained
than
in
the
sedentary rats (10). However, our stepRecent animal studies have suggested the existence of
FGF21 resistance, and obese rats with FGF21 resistance wise multiple linear regression analysis showed CRF to be
a significant predictor of the serum FGF21 level independent of the VFA in middle-aged and
elderly subjects (Table 3), suggesting
that higher aerobic capacity itself is
associated with greater FGF21 sensitivity. Therefore, the benefits of
regular exercise may be due in part to
prevention of FGF21 resistance.
Although obesity is known to be
associated with elevated circulating
FGF21 level in humans (4, 5, 27), the
association between body fat distriFigure 1. Correlations of the serum FGF21 level with the VO2peak (A) and VFA (B) in middleaged and elderly subjects.
bution and the FGF21 level has not
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CRF and VFA Influence Serum FGF21 Level
J Clin Endocrinol Metab, October 2014, 99(10):E1877–E1884
Table 4. Odds Ratio for a Serum FGF21 Level Below
the Limit of Detection of the Assay With Respect to the
VFA and VO2peak in Young Subjects
VFA, cm2
VO2peak,
mL 䡠 kg⫺1 䡠 min⫺1
OR
95% CI
P
Value
0.938
1.114
0.882– 0.998
1.001–1.239
.044
.047
Data are the OR (95% CI). The model was adjusted for age and TGs.
TGs were log transformed prior to analysis. Boldface indicates
significance.
been fully elucidated. Our data clearly showed that the
serum FGF21 level was the most robustly associated with
VFA among the measures of adiposity such as BMI, body
fat percentage, and SFA (Table 2). The multiple linear
regression analyses in both older and young subjects suggested that VFA is the predominant determinant of the
serum FGF21 level, although CRF was also independently
associated with serum FGF21 (Table 3). Several studies
have reported that waist circumference and waist to hip
ratio, both indices of abdominal obesity, were no longer
associated with the circulating FGF21 level after adjustment for BMI (4, 28). However, these simple anthropometric measures do not always reflect the VFA (29). In the
present study, adjustment for BMI did not affect the association between VFA measured by magnetic resonance
imaging and the serum FGF21 level (Table 2). Therefore,
a chronically high circulating FGF21 level is probably induced by visceral rather than overall obesity. Although the
release of inflammatory adipokines and FFAs from excess
VFA is recognized as the primary mechanism linking VFA
and metabolic abnormalities (14, 30), FGF21 resistance is
another possible explanation for the important role of
VFA in the development of various chronic diseases.
To understand further the effect of CRF and VFA on the
serum FGF21 level, we recruited healthy untrained and
endurance-trained individuals. As shown in Supplemental
Table 1 and Figure 2, the proportion of participants with
a serum FGF21 level below the limit of detection was significantly higher in the ET than in the UT participants
(71.4% vs 24.0%, P ⫽ .001). Furthermore, the median
serum FGF21 concentration was higher in the middleaged and elderly subjects with high CRF than in the young
untrained subjects (231.8 pg/mL vs 118.8 pg/mL, P ⫽
.019). This can mainly be explained by an age-related increase in VFA (see Supplemental Table 2). Consistent with
the result in middle-aged and elderly participants, our
multiple logistic regression analysis in the young subjects
revealed that the CRF and VFA were independently associated with an undetectable serum FGF21 level (Table 4,
P ⬍ .05), supporting our hypothesis that the CRF level is
independently associated with FGF21 sensitivity. A pre-
Figure 2. Distribution of the serum FGF21 concentration in young,
middle-aged, and elderly subjects. Closed circles (F) indicate
detectable serum FGF21 levels, and open circles (E) indicate levels
below the limit of detection. Horizontal lines represent the median
values (including undetectable serum FGF21 levels, which were
considered zero). Levels for the middle-aged and elderly men: low CRF,
n ⫽ 84, 266.7 pg/mL; high CRF, n ⫽ 82, 231.8 pg/mL; young men:
healthy untrained, n ⫽ 25, 118.8 pg/mL; endurance trained, n ⫽ 21
(median value was below the limit of detection). The six middle-aged
and elderly subjects excluded from the statistical analyses due to an
undetectable serum FGF21 level or an extremely high FGF21 level are
included here.
vious German study also reported that there were many
undetectable levels of serum FGF21 and found a more
favorable metabolic profile in the subjects with FGF21
concentrations below the limit of detection than in those
with measurable circulating FGF21 (31). Therefore, an
undetectable level of FGF21 may be common in both European and Asian populations and may indicate greater
sensitivity to FGF21.
Acute exercise training or a short-term supervised physical activity program has been shown to increase the serum
FGF21 level (32, 33). These previous studies consistently
observed that exercise increased the circulating FFA level
and concluded that the exercise-induced increase in FFAs
might explain the induction of FGF21. However, longterm aerobic exercise increases CRF and tends to reduce
the release of FFAs from adipose tissue during submaximal exercise (34). In the present study, CRF was not actually associated with the serum FFA level after adjustment for age and BMI (Table 2, r ⫽ ⫺0.073, P ⫽ .369).
Therefore, short-term and long-term exercise seem to have
completely different effects on the serum FGF21 concentration response.
In accordance with previous studies (4, 28, 35), we
showed that the serum FGF21 level correlates with the
serum levels of TGs and hepatic enzymes, including ALT
and ␥-GTP (Table 2), that are well-accepted biochemical
markers of liver injury. Obese mice exhibit decreased hepatic FGF21 sensitivity (6), and human patients with non-
doi: 10.1210/jc.2014-1877
alcoholic fatty liver disease have high levels of circulating
FGF21 as well as elevated ALT or ␥-GTP levels (36, 37).
The elevation of serum levels of TGs and hepatic enzymes
in the FGF21-resistant state is consistent with such findings. We also revealed that the level of ApoC-III, a key
regulator of TG metabolism, correlated positively with the
serum FGF21 level (Table 2). ApoC-III decreases the clearance of circulating TGs by inhibiting lipoprotein lipase
activity (38). Interestingly, the activation of peroxisomal
proliferator-activated receptor-␣, a primary regulator of
FGF21, decreases ApoC-III expression (39). Administration of FGF21 or a variant decreased the circulating levels
of both ApoC-III and TGs in monkeys and humans (2, 3),
suggesting that a marked reduction in ApoC-III might contribute to the decrease in TGs. Therefore, the association
between the serum TGs and FGF21 levels may be mediated
in part by up-regulation of ApoC-III induced by FGF21
resistance.
The present study has several limitations. First, our
sample size was relatively small, which might have led to
type 2 error. Second, we did not evaluate hepatic fat content despite a previous study that showed it to be independently associated with the circulating FGF21 level
(40). The association between the CRF and serum FGF21
levels should be reexamined with hepatic fat content as a
covariate. Finally, most the participants in this study were
free from chronic diseases. Further investigation is needed
to clarify whether a high CRF level is also associated with
a low serum FGF21 level, even in patients with chronic
diseases such as type 2 diabetes and cardiovascular
disease.
In conclusion, the present study revealed that CRF is
negatively associated with the serum FGF21 level in middle-aged and elderly Japanese men. We also demonstrated
that young ET men have extremely low levels of serum
FGF21. These findings provide novel evidence that regular
aerobic exercise and a high CRF level improve health by
combatting an underlying resistance to FGF21.
Acknowledgments
We thank Hiroshi Kawano, Yuko Gando, Ryoko Kawakami,
Takafumi Ando, Tomoko Ito, and Taishi Susa for their
assistance.
Address all correspondence and requests for reprints to: Mitsuru Higuchi, PhD, Faculty of Sport Sciences, Waseda University, 2–579-15 Mikajima, Tokorozawa, Saitama 359 –1192, Japan. E-mail: [email protected].
This work was supported by a Grant-in-Aid for the Global
COE (“Sport sciences for the promotion of active life,” to
Waseda University) from the Ministry of Education, Culture,
Sports, Science, and Technology and by a grant for strategic
jcem.endojournals.org
E1883
research initiatives (“Paradigm shifts in a superaged society”)
from Waseda University.
Disclosure Summary: The authors have nothing to declare.
References
1. Kharitonenkov A, Shiyanova TL, Koester A, et al. FGF-21 as a novel
metabolic regulator. J Clin Invest. 2005;115:1627–1635.
2. Kharitonenkov A, Wroblewski VJ, Koester A, et al. The metabolic
state of diabetic monkeys is regulated by fibroblast growth factor21. Endocrinology. 2007;148:774 –781.
3. Gaich G, Chien JY, Fu H, et al. The effects of LY2405319, an FGF21
analog, in obese human subjects with type 2 diabetes. Cell Metab.
2013;18:333–340.
4. Zhang X, Yeung DC, Karpisek M, et al. Serum FGF21 levels are
increased in obesity and are independently associated with the metabolic syndrome in humans. Diabetes. 2008;57:1246 –1253.
5. Dostalova I, Kavalkova P, Haluzikova D, et al. Plasma concentrations of fibroblast growth factors 19 and 21 in patients with anorexia
nervosa. J Clin Endocrinol Metab. 2008;93:3627–3632.
6. Fisher FM, Chui PC, Antonellis PJ, et al. Obesity is a fibroblast
growth factor 21 (FGF21)-resistant state. Diabetes. 2010;59:2781–
2789.
7. Hale C, Chen MM, Stanislaus S, et al. Lack of overt FGF21 resistance in two mouse models of obesity and insulin resistance. Endocrinology. 2012;153:69 – 80.
8. Chen C, Cheung BM, Tso AW, et al. High plasma level of fibroblast
growth factor 21 is an independent predictor of type 2 diabetes: a
5.4-year population-based prospective study in Chinese subjects.
Diabetes Care. 2011;34:2113–2115.
9. Bobbert T, Schwarz F, Fischer-Rosinsky A, et al. Fibroblast growth
factor 21 predicts the metabolic syndrome and type 2 diabetes in
Caucasians. Diabetes Care. 2013;36:145–149.
10. Fletcher JA, Meers GM, Laughlin MH, Ibdah JA, Thyfault JP, Rector RS. Modulating fibroblast growth factor 21 in hyperphagic
OLETF rats with daily exercise and caloric restriction. Appl Physiol
Nutr Metab. 2012;37:1054 –1062.
11. Wei M, Gibbons LW, Mitchell TL, Kampert JB, Lee CD, Blair SN.
The association between cardiorespiratory fitness and impaired fasting glucose and type 2 diabetes mellitus in men. Ann Intern Med.
1999;130:89 –96.
12. Sawada SS, Lee IM, Muto T, Matuszaki K, Blair SN. Cardiorespiratory fitness and the incidence of type 2 diabetes: prospective study
of Japanese men. Diabetes Care. 2003;26:2918 –2922.
13. LaMonte MJ, Barlow CE, Jurca R, Kampert JB, Church TS, Blair
SN. Cardiorespiratory fitness is inversely associated with the incidence of metabolic syndrome: a prospective study of men and
women. Circulation. 2005;112:505–512.
14. Fox CS, Massaro JM, Hoffmann U, Pou KM, et al. Abdominal
visceral and subcutaneous adipose tissue compartments: association
with metabolic risk factors in the Framingham Heart Study. Circulation. 2007;116:39 – 48.
15. Tanisawa K, Ito T, Sun X, et al. High cardiorespiratory fitness can
reduce glycated hemoglobin levels regardless of polygenic risk for
type 2 diabetes mellitus in non-diabetic Japanese men. Physiol
Genomics. 2014;46:497–504.
16. Nishimura T, Nakatake Y, Konishi M, Itoh N. Identification of a
novel FGF, FGF-21, preferentially expressed in the liver. Biochim
Biophys Acta. 2000;1492:203–206.
17. Lundasen T, Hunt MC, Nilsson LM, et al. PPAR␣ is a key regulator
of hepatic FGF21. Biochem Biophys Res Commun. 2007;360:437–
440.
18. Heilbronn LK, Campbell LV, Xu A, Samocha-Bonet D. Metabolically protective cytokines adiponectin and fibroblast growth factor-21 are increased by acute overfeeding in healthy humans. PLoS
One. 2013;8:e78864.
E1884
Taniguchi et al
CRF and VFA Influence Serum FGF21 Level
19. Galman C, Lundasen T, Kharitonenkov A, et al. The circulating
metabolic regulator FGF21 is induced by prolonged fasting and
PPAR␣ activation in man. Cell Metab. 2008;8:169 –174.
20. Kurosu H, Choi M, Ogawa Y, et al. Tissue-specific expression of
␤Klotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21. J Biol Chem.
2007;282:26687–26695.
21. Ogawa Y, Kurosu H, Yamamoto M, et al. ␤Klotho is required for
metabolic activity of fibroblast growth factor 21. Proc Natl Acad Sci
USA. 2007;104:7432–7437.
22. Kharitonenkov A, Dunbar JD, Bina HA, et al. FGF-21/FGF-21 receptor interaction and activation is determined by ␤Klotho. J Cell
Physiol. 2008;215:1–7.
23. Suzuki M, Uehara Y, Motomura-Matsuzaka K, et al. ␤Klotho is
required for fibroblast growth factor (FGF) 21 signaling through
FGF receptor (FGFR) 1c and FGFR3c. Mol Endocrinol. 2008;22:
1006 –1014.
24. Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, MaratosFlier E. Hepatic fibroblast growth factor 21 is regulated by PPAR␣
and is a key mediator of hepatic lipid metabolism in ketotic states.
Cell Metab. 2007;5:426 – 437.
25. Inagaki T, Dutchak P, Zhao G, et al. Endocrine regulation of the
fasting response by PPAR␣-mediated induction of fibroblast growth
factor 21. Cell Metab. 2007;5:415– 425.
26. Fisher FM, Kleiner S, Douris N, et al. FGF21 regulates PGC-1␣ and
browning of white adipose tissues in adaptive thermogenesis. Genes
Dev. 2012;26:271–281.
27. Mraz M, Bartlova M, Lacinova Z, et al. Serum concentrations and
tissue expression of a novel endocrine regulator fibroblast growth
factor-21 in patients with type 2 diabetes and obesity. Clin Endocrinol (Oxf). 2009;71:369 –375.
28. Li H, Bao Y, Xu A, et al. Serum fibroblast growth factor 21 is
associated with adverse lipid profiles and ␥-glutamyltransferase but
not insulin sensitivity in Chinese subjects. J Clin Endocrinol Metab.
2009;94:2151–2156.
J Clin Endocrinol Metab, October 2014, 99(10):E1877–E1884
29. Kay SJ, Fiatarone Singh MA. The influence of physical activity on
abdominal fat: a systematic review of the literature. Obes Rev. 2006;
7:183–200.
30. Matsuzawa Y. The role of fat topology in the risk of disease. Int J
Obes (Lond). 2008;32(suppl 7):S83–S92.
31. Kralisch S, Tonjes A, Krause K, et al. Fibroblast growth factor-21
serum concentrations are associated with metabolic and hepatic
markers in humans. J Endocrinol. 2013;216:135–143.
32. Cuevas-Ramos D, Almeda-Valdes P, Meza-Arana CE, et al. Exercise
increases serum fibroblast growth factor 21 (FGF21) levels. PLoS
One. 2012;7:e38022.
33. Kim KH, Kim SH, Min YK, Yang HM, Lee JB, Lee MS. Acute
exercise induces FGF21 expression in mice and in healthy humans.
PLoS One. 2013;8:e63517.
34. Martin WH 3rd, Dalsky GP, Hurley BF, et al. Effect of endurance
training on plasma free fatty acid turnover and oxidation during
exercise. Am J Physiol. 1993;265:E708 –E714.
35. Lin Z, Wu Z, Yin X, et al. Serum levels of FGF-21 are increased in
coronary heart disease patients and are independently associated
with adverse lipid profile. PLoS One. 2010;5:e15534.
36. Dushay J, Chui PC, Gopalakrishnan GS, et al. Increased fibroblast
growth factor 21 in obesity and nonalcoholic fatty liver disease.
Gastroenterology. 2010;139:456 – 463.
37. Li H, Fang Q, Gao F, et al. Fibroblast growth factor 21 levels are
increased in nonalcoholic fatty liver disease patients and are correlated with hepatic triglyceride. J Hepatol. 2010;53:934 –940.
38. Ooi EM, Barrett PH, Chan DC, Watts GF. Apolipoprotein C-III:
understanding an emerging cardiovascular risk factor. Clin Sci
(Lond). 2008;114:611– 624.
39. Staels B, Vu-Dac N, Kosykh VA, et al. Fibrates downregulate apolipoprotein C-III expression independent of induction of peroxisomal acyl coenzyme A oxidase. A potential mechanism for the
hypolipidemic action of fibrates. J Clin Invest. 1995;95:705–712.
40. Yan H, Xia M, Chang X, et al. Circulating fibroblast growth factor
21 levels are closely associated with hepatic fat content: a crosssectional study. PLoS One. 2011;6:e24895.