Serum FGF21 levels are altered with various factors including lifestyle behaviors

Fibroblast growth factor (FGF) 21 has various functions, including glucose and lipid metabolism, yet the biology of FGF21 remains unclear. This study aimed to investigate specic conditions that might affect the functions of FGF21. Subjects included 398 healthy men who underwent health examinations to obtain information on physical and biochemical parameters and lifestyle behaviors. Associations of serum FGF21 levels with each parameter were assessed in the study. FGF21 levels correlated with age, body mass index, waist circumference (WC), systolic blood pressure (SBP), diastolic blood pressure, aspartate aminotransferase, alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (γ-GTP), uric acid, total cholesterol (TC), triglycerides, high-density lipoprotein cholesterol, fasting plasma glucose, and HbA1c. Moreover, FGF21 levels were signicantly associated with lifestyle behaviors, including smoking status and breakfast and alcohol consumption frequency. Multivariable regression analysis showed that age, ALT, γ-GTP, smoking status, and breakfast and alcohol consumption frequency were independent variables for FGF21 levels. Assessment among the non-obese and obese groups showed that FGF21 levels correlated with WC, SBP, and TC only in the non-obese group. Thus, serum FGF21 levels were affected by several factors, including lifestyle behaviors, age, and liver function. To assess the functions of FGF21 in individuals, considering these factors would be essential. pressure (DBP), aspartate aminotransferase (AST), alanine aminotransferase gamma-glutamyl transpeptidase (γ-GTP), uric acid total cholesterol triglycerides high-density lipoprotein cholesterol fasting plasma glucose HbA1c.


Introduction
Fibroblast growth factor (FGF) 21 is predominantly derived from the liver and is a member of the FGF19 subfamily, which includes FGF15/19 and FGF23. Unlike other FGF subfamilies, members of the FGF19 subfamily exhibit hormone-like functions and require Klotho proteins, namely αKlotho and βKlotho, as cofactors to increase their binding a nity to FGF receptors 1 . FGF21 interacts with βKlotho and plays an important role in glucose and lipid metabolism. It regulates glucose uptake in adipocytes, enhances fatty acid oxidation in the liver, and inhibits lipogenesis 2,3 .
FGF21 administration in obese animal models has shown an improvement in insulin sensitivity, a decrease in triglyceride and cholesterol levels, and a reduction in adiposity; therefore, FGF21 is expected to be a potential new therapy for obesity and obesity-related diseases, including type 2 diabetes and nonalcoholic steatohepatitis 4,5 . Paradoxically, serum levels of FGF21 increase in individuals with obesity, type 2 diabetes, and metabolic syndrome 6,7 . While this increase in FGF21 levels is regarded as a compensatory response or the FGF21-resistant state, the precise underlying mechanism for this increase remains unclear 8,9 . We previously reported that FGF21 levels were high in smokers and negatively correlated with the metabolic syndrome-related cytokine, adiponectin. Interestingly, FGF21 levels were differentially associated with liver function and total cholesterol among smokers and never-smokers, suggesting that smoking stress affects the relationship between FGF21 and metabolic parameters 10 . Moreover, we demonstrated a sex-based difference in the relationship between FGF21 levels and metabolic parameters 11 . These results suggest that there may be some conditions that affect FGF21 functions.
Since FGF21 has various functions in multiple target organs, FGF21 biology is complicated and several uncertainties remain 12 . Therefore, in the present study, we focused on evaluating speci c conditions that might affect the functions of FGF21 by assessing the association of serum levels of FGF21 with physical parameters, biochemical parameters, and lifestyle behaviors using cross-sectional data of healthy subjects.

Association of FGF21 levels with each parameter
The characteristics of the study participants are presented in Table 1. The median age and FGF21 levels were 42 (37-49) years and 165 (106-256) pg/mL, respectively.    Abbreviations are as in Table 1.

Lifestyle behaviors affect serum levels of FGF21
In line with our previous report that serum levels of FGF21 were upregulated in smokers 10,11 , FGF21 levels in the current study were signi cantly higher in smokers [210 (124-353) pg/mL] than in never smokers [147 (101-224) pg/mL, p < 0.0001] and correlated with smoking status (τ = 0.14, p < 0.0001). Moreover, serum levels of FGF21 signi cantly correlated with breakfast consumption frequency (τ = −0.12, p < 0.0001), alcohol consumption frequency (τ = 0.15, p < 0.0001), and daily alcohol intake (τ = 0.08, p = 0.0237). We further assessed the associations between FGF21 levels and lifestyle behaviors, as shown in Fig. 1. Serum levels of FGF21 were signi cantly increased in subjects whose breakfast consumption frequency was low (Fig. 1A). In addition, FGF21 levels were signi cantly increased in subjects whose alcohol consumption frequency was high (Fig. 1B), with marginal increase in subjects whose daily alcohol intake was high (Fig. 1C). These results suggest that lifestyle behaviors, including breakfast consumption and alcohol consumption affected the serum levels of FGF21 as smoking status.
Age strongly associates with serum levels of FGF21 Multivariable regression analysis showed that age, ALT, γ-GTP, smoking status, and breakfast and alcohol consumption frequency were independent variables for FGF21 levels (Table 3). Correlation and regression analyses showed that age is a strong variable for FGF21 levels; hence, we assessed FGF21 levels in each age group. The median serum levels of FGF21 in participants of age 30-39 years (n = 129), 40-49 years (n = 175), and ≥ 50 years (n = 94) were 119 (81-201) pg/mL, 181 (117-271) pg/mL, 214 (150-359) pg/mL, respectively. Signi cant differences were observed among age groups (p = 0.0001). Relationship between FGF21 and each parameter in nonobese and obese group FGF21 levels show a strong association obesity, whereby FGF21 therapy has been identi ed as a possible treatment for obesity and obesity-related diseases in recent years 4 . We categorized the study participants into non-obese (BMI < 25 kg/m 2 ) and obese (BMI ≥ 25 kg/m 2 ) groups and assessed the relationship between FGF21 levels and each parameter in both groups. The characteristics of each group are shown in Table 4. Serum levels of FGF21 in the non-obese and obese groups were 145 (96-225) pg/mL and 189 (124-300) pg/mL, respectively. Abbreviations are as in Table 1.

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The correlations between FGF21 levels and each parameter among the non-obese and obese groups are summarized in Table 5. Serum levels of FGF21 signi cantly correlated with WC, SBP, and TC only in the non-obese group, while the correlation between FGF21 and HbA1c was observed only in the obese group. Thus, it can be suggested that FGF21 effects may differ depending on the BMI of each subject. Abbreviations are as in Table 1.

Discussion
FGF21 has multiple functions, and recent studies have shown its various protective effects. Our previous study showed that FGF21 levels correlated with adiponectin, a metabolic syndrome-related cytokine that also has protective effects, including anti-in ammatory, anti-atherogenic, and anti-diabetic effects 10,13 . Since FGF21 regulates oxidative stress and attenuates in ammation, it is considered a stress-response factor 14,15 . In the present study, we analyzed the association of FGF21 levels with metabolic parameters and lifestyle behaviors. We previously reported that smoking status affects the serum levels of FGF21 10 .
In this study, we demonstrated that not only smoking, but also breakfast and alcohol consumption frequency affected the FGF21 levels. Breakfast is considered the most important meal of the day and affects physical and mental health [16][17][18] . Skipping breakfast has been reported to be associated with in ammation and cardiovascular disease risk, related to an increase in WC and low-density lipoprotein cholesterol [19][20][21][22] . We found FGF21 levels to be increased in subjects with a low breakfast consumption frequency and a high alcohol consumption frequency. FGF21 has a protective effect against liver damage induced by alcohol. In animal models, FGF21 administration reduces alcohol intake via the central nervous system, which interacts with βKlotho 14,23 . An increase in FGF21 levels in a stressed condition, including smoking, skipping breakfast, and alcohol ingestion, is suggested to be a protective response of FGF21.
While serum levels of FGF21 were related to many parameters, we found that age was a strong factor affecting FGF21 levels. Evaluation of serum levels of FGF21 in each age group showed that FGF21 levels were signi cantly increased along with aging. Aging is associated with various diseases, including cardiovascular diseases, cancer, and pulmonary diseases, which are known to be the leading causes of death worldwide 24 . Moreover, obesity and obesity-related diseases are associated with aging 25 . Previous studies have reported that FGF21 extended the lifespan of mice and showed a protective effect on agerelated changes 26,27 . Since FGF21 can ameliorate both aging and metabolism, FGF21 is regarded as a key factor linking aging and metabolism 28 . In the present study, although the subjects were healthy individuals who did not present any signs of disease, FGF21 levels were upregulated in the aged groups. Considering the anti-aging effects of FGF21, these increased FGF21 levels might be a compensatory response toward progress in aging and age-related changes.
Individuals with obesity, in whom protective effects are not seen with increased serum levels of FGF21, are considered FGF21-resistant 6,8 . To evaluate differential functions of FGF21 between non-obese and obese subjects, we assessed the association of FGF21 with metabolic parameters among both groups. As previously reported, FGF21 levels were signi cantly high in the obese group. Serum levels of FGF21 correlated with more variables in the non-obese group than in the obese group. Moreover, WC, SBP, and TC correlated with FGF21 only in the non-obese group. WC is a known parameter for the de nition of metabolic syndrome and is strongly associated with visceral fat accumulation 29 . Although both WC and FGF21 levels were high in obese subjects, there was no correlation between WC and FGF21. This disrupted relationship between FGF21 and WC suggests that the protective effects of FGF21 are attenuated in obese subjects. We previously reported that serum levels of FGF21 correlated with metabolic parameters differently among smokers and never-smokers 10 . We also con rmed that there were sex differences in the relationship between FGF21 levels and metabolic parameters 11 . These results show a variable relationship between FGF21 levels and metabolic parameters and that the effect of FGF21 may vary depending on the individual background. Since FGF21 is expected as a new therapy for obesity and obesity-related diseases 4 , effective conditions are needed to be evaluated.
FGF21 is reported to induce angiotensin-converting enzyme 2, which has recently been focused on as a key mechanism against SARS-coronavirus 2 (SARS-CoV-2) infection, and prevent hypertension and vascular damage in mice 30,31 . Since FGF21 upregulating factors, including smoking, type 2 diabetes, and obesity, are also known risk factors for SARS-CoV-2-related mortality 32 , it is intriguing to speculate the protective effects of FGF21 on SARS-CoV-2 infection.
FGF21 levels have been reported to be altered in various diseases 6,7,33 . However, as demonstrated in the present study, the serum levels of FGF21 are affected by several factors. Therefore, to assess the functions of FGF21 in subjects with such diseases, considering these factors and matching the conditions among the subjects is essential. Furthermore, as the mechanisms of change in FGF21 levels are still unclear, we believe that our ndings might help elucidate the complicated biology of FGF21.
In conclusion, we evaluated the parameters and lifestyle behaviors that may affect FGF21 functions in the present study. Serum levels of FGF21 were affected by several lifestyle behaviors, including smoking status, breakfast consumption, and alcohol consumption. Moreover, in the FGF21 relating parameters, age was strongly associated with FGF21 levels. Serum levels of FGF21 are associated with metabolic parameters differently in non-obese and obese subjects.

Study subjects
This study included cross-sectional data obtained from employees at Osaka University. The subjects were randomly selected from among those who underwent an annual health checkup at the Osaka University Health and Counseling Center. A total of 398 healthy Japanese men who had not taken any chronic or frequent medication for at least 1 year before their health checkup and had not experienced any acute illness within the previous 2 weeks were enrolled in the study. This study was carried out in accordance with the Declaration of Helsinki and the Ethics Guidelines for Clinical Research from the Ministry of Health, Labour and Welfare and the Ministry of Education, Culture, Sports, Science and Technology. All experimental protocols in this study were approved by the Ethics Committee of Health and Counseling Center, Osaka University, and written informed consent was obtained from all subjects prior to participation in the study.
Physical and biochemical parameters BMI, WC at the umbilical level, SBP, DBP were measured as physical parameters.
Serum was collected from subjects between 9 and 11 AM after an overnight fast and stored at ≤ − 20°C until assayed. Serum levels of FGF21 were measured using a sandwich enzyme-linked immunoassay system according to the manufacturer's instructions (R&D Systems Inc., Minneapolis, USA).

Lifestyle assessments
Information on the medical history, current treatments, smoking status, and lifestyle behaviors of the study participants were obtained via questionnaires. Each piece of information was recon rmed through expert interviews by trained nurses. Smoking status was semi-quanti ed as 0 = never smoker and 1 = smoker. Lifestyle behaviors, including breakfast and alcohol consumption frequency, and daily alcohol intake, were asked as follows, and each answer was semi-quanti ed using the following scales. Breakfast consumption frequency: "How many days a week do you eat breakfast?" on three scales: 1 = 0-2 days a week, 2 = 3-6 days a week, 3 = everyday; alcohol consumption frequency: "How many days a week do you drink alcohol?" on three scales: 1 = 0-2 days a week, 2 = 3-6 days a week, 3 = everyday; daily alcohol intake: "How many amounts of pure alcohol do you have on a typical day when you are drinking?" on three scales: 1 = < 20 g, 2 = 20-40 g, 3 = ≥ 40 g.

Statistical analyses
All statistical analyses were performed using STATA 14 (STATA Corp LLC, College Station, TX, USA). The distribution of continuous variables was tested using the Shapiro-Wilk test. Normally distributed variables are presented as means ± standard deviation; non-normally distributed variables are reported as medians with interquartile ranges. Student's t-test or the Mann-Whitney U test was used to compare differences between the two groups. Kendall's rank correlation coe cient and multiple regression analysis were used to analyze the variables. For multi-group comparisons, the Kruskal-Wallis test with Dunn's post-hoc test was used. Statistical signi cance was set at P < 0.05.

Declarations Data Availability Statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.