Cigarette smoking is a risk factor for various diseases and is one of the most critical coronary risk factors.[1–3] Smoking cessation reduces the risk of cardiovascular disease and improves clinical outcomes.[4,5] The risk of acute myocardial infarction (AMI) has been reported to be substantially reduced 1 year after smoking cessation. We previously reported that the spread of a nonsmoking policy significantly decreased the in-hospital onset of AMI in our hospital. Thus, smoking cessation is being actively promoted worldwide.
Klotho is a well-known antiaging molecule.[8–12] Overexpression of klotho in transgenic mice significantly extended their life span compared to that of wild-type mice. Higher klotho levels have been associated with a lower prevalence of cardiovascular disease. Low serum klotho levels have been associated with increased carotid artery intima-media thickness and epicardial fat thickness and with decreased flow-mediated dilation of the brachial artery, indicating that a low serum klotho level is a predictor of atherosclerosis. On the contrary, in another study, serum levels of α-klotho in never-smokers were low, and smokers showed highly increased serum levels of α-klotho.
The life expectancy of smokers is at least one decade less than that of subjects who have never smoked. Smoking cessation before the age of 40 years reduces the risk of death associated with continued smoking by about 90%. Smokers may exhibit low-grade inflammation and inflammation in blood vessels may be improved by smoking cessation.[14,15] The serum levels of the inflammation-related cytokine interleukin-6 were significantly higher in smokers than in never-smokers. In addition, serum levels of α-klotho were correlated with interleukin-6 in middle-aged never-smokers, but not in smokers. However, little is known about the association between smoking cessation and this antiaging molecule. Thus, we believe that it is important to study this association in Japanese smokers. In this study, we hypothesized that smoking cessation could lead to a compensatory response in α-klotho as an antiaging molecule, and therefore we evaluated α-klotho in participants before and after smoking cessation.
2.1 Study design
Tsukahara et al previously reported a randomized controlled open comparative trial of varenicline versus nicotine patch in adult smokers: efficacy, safety, and withdrawal symptoms (the VN-SEESAW study). Briefly, 32 Japanese adult smokers were enrolled for treatment at Fukuoka University Hospital. Participants either received varenicline for 12 weeks or wore a transdermal nicotine patch on the chest for 8 weeks. Successful smoking cessation was identified by both a self-assessment and the end-expiratory carbon monoxide (CO) concentration (<8 ppm). Body weight (BW), body mass index (BMI), systolic blood pressure (SBP), diastolic BP (DBP), and pulse rate (PR) were measured. The study protocol was approved by the Independent Review Board (IRB) of Fukuoka University Hospital (#7-05 [08-27]), and all participants gave their written informed consent. Takata et al performed an additional examination of plasma lipid profiles (low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglyceride levels), high-density lipoprotein subfractions, and cholesterol efflux capacity in 28 participants (VN-SEESAW-HDL study). The VN-SEESAW-HDL study including current investigation was approved by the IRB of Fukuoka University Hospital (#14-5-13).
2.2 Measurement of plasma levels of α-klotho, fibroblast growth factor-19, fibroblast growth factor-21, and hemoglobin
We measured α-klotho, fibroblast growth factor 19 (FGF-19), FGF-21, and hemoglobin (Hb) in blood samples obtained at baseline (0 weeks) and at 12 weeks in both the varenicline and nicotine patch groups. Plasma levels of α-klotho, FGF-19, and FGF-21 were measured using commercial sandwich enzyme immunoassay kits according to the respective manufacturer's instructions (Quantikine ELISA Human FGF-19 and FGF-21 immunoassay kits were purchased from R&D Systems, Minneapolis, MN, and a Human soluble α-Klotho assay kit was purchased from Immuno-Biological Laboratories Co, Ltd, Gumma, Japan).
2.3 Statistical analysis
Data are presented as the mean ± standard deviation. Changes were calculated as the values after 12 weeks minus the values at week 0. Differences between values at baseline and after 12 weeks were evaluated with the paired t test. The associations between 2 parameters were assessed by Spearman rank correlation coefficient. Differences between the successful and unsuccessful smoking cessation groups or the varenicline and nicotine patch groups were evaluated with the unpaired t test. A P-value of <.05 was considered statistically significant. Analyses were performed using the Stat View statistical software package (Stat View 5; SAS Institute Inc, Cary, NC).
3.1 Patient characteristics and various parameters at weeks 0 and 12 in all participants
Twenty-eight participants (14 for varenicline, 14 for a nicotine patch) were analyzed (Table 1, Fig. 1). In all participants, PR, plasma levels of α-klotho (Fig. 1A), Hb, and CO concentration were significantly decreased after 12 weeks, whereas there were no changes in FGF-19 or FGF-21 (Fig. 1B, C). On the contrary, there were significant increases in BW and BMI after 12 weeks.
3.2 Patient characteristics and various parameters at weeks 0 and 12 in the successful and unsuccessful smoking cessation groups
Seven of the 28 participants belong to the unsuccessful smoking cessation group (Table 2). The proportions of participants in the group were not significantly different between those who received varenicline (4 participants) and a nicotine patch (3 participants). There were no statistically significant differences in BW, BMI, α-klotho, FGF-19, FGF-21, CO concentration, or Hb between the successful and unsuccessful groups at week 0. After 12 weeks, both groups showed significant reductions in α-klotho (Fig. 2A), Hb, and CO concentration. PR and SBP in the successful group, but not the unsuccessful group, significantly decreased between baseline and 12 weeks, whereas there were significant increases in BW and BMI after 12 weeks in the successful group.
3.3 Patient characteristics and various parameters at weeks 0 and 12 in the varenicline and nicotine patch groups
We also analyzed the differences in characteristics of the participants and various parameters between the varenicline and nicotine patch groups (Table 3). There were no statistically significant differences in BW, BMI, α-klotho, FGF-19, FGF-21, CO concentration, or Hb between the groups at week 0. After 12 weeks, both groups showed significant reductions in α-klotho (Fig. 2B), CO concentration, and Hb. BW and BMI in both groups significantly increased after 12 weeks.
3.4 Associations between α-klotho and Hb or CO concentration
Figure 3 shows the associations between α-klotho and Hb or CO concentration. The change in the plasma level of α-klotho from week 0 to week 12 (Δ = the value at week 12 − the value at week 0) was negatively associated with α-klotho at week 0 and positively associated with ΔHb (Fig. 3A, B). On the contrary, there were no associations between ΔCO and Δα-klotho or ΔHb (Fig. 3C, D).
In this study, smoking cessation significantly decreased serum levels of the antiaging molecule α-klotho, which suggests that an increase in α-klotho may be a compensatory response to smoking stress.
We hypothesized that smoking cessation could lead to a compensatory response in α-klotho as an antiaging molecule. We found that smoking cessation significantly decreased plasma levels of α-klotho. Higher klotho levels have been associated with a reduced risk of cardiovascular disease, and lower levels were associated with the progression of atherosclerosis.[10,11] Since all of the participants in this study were smokers, we did not analyze α-klotho levels before they began smoking. Nakanishi et al reported that α-klotho levels in never-smokers were low, while those in smokers were very high. In addition, serum α-klotho was positively correlated with the proinflammatory molecule interleukin-6, and has been reported to act as an antiinflammatory molecule. The positive correlation suggested that the increase in serum levels of α-klotho might be a compensatory response to smoking stress. In this study, Δα-klotho was negatively associated with α-klotho at week 0. Thus, the α-klotho levels in our participants were upregulated by smoking, and smoking cessation may produce a compensatory decrease in these levels. Our results were in agreement with those in a previous report.
The levels of FGF-19 and FGF-21 were not significantly decreased after 12 weeks in this study. β-Klotho, which is a homolog of α-klotho, interacts with FGF-19, which is a known regulator of bile acid homeostasis, and with FGF-21, which regulates the metabolism. Although smoking cessation should reduce FGF-21 levels, since smoking increased serum levels of FGF-21, these levels did not change and BMI significantly increased after smoking cessation in this study. Serum levels of FGF-21 have been shown to increase in subjects with metabolic syndrome, and this could explain why FGF-21 levels did not change after smoking cessation. Since no prior studies have examined the influence of smoking cessation on changes in FGF-19 and FGF-21, further studies are needed to clarify these changes.
Another important result in this study was that PR, Hb, and CO concentration significantly decreased after 12 weeks in all patients. The successful smoking cessation group showed significant reductions in PR, SBP, Hb, and CO concentration, and all of these reductions in this group were reasonable. A meta-analysis supported an association between heavy smoking and a high resting heart rate. Moreover, heavy smoking is associated with a persistent rise in BP and with an increase in BP variability. Since smokers had significantly higher levels of Hb, the level of Hb may significantly decrease after smoking cessation, with a reduction of the CO concentration. Δα-klotho was positively associated with ΔHb. Decreased α-klotho levels are associated with decreased Hb in patients with chronic kidney disease. Under hypoxic conditions, erythropoietin stimulates the differentiation of erythroid progenitor cells and normoblasts to increase the amount of red blood cells. Erythropoietin promotes the expression of α-klotho and an increase in α-klotho suppresses the production of erythropoietin.[26,27] After smoking cessation, hypoxic conditions improve and erythropoietin may not be needed to stimulate differentiation to increase red blood cells, and consequently erythropoietin may not promote the expression of α-klotho.
This study has several limitations. For example, the sample size was small, which limits our ability to generalize the results. Furthermore, this study assessed α-klotho levels only over the short term. The long-term effect of smoking cessation on α-klotho will need to be elucidated in the future.
Our results agree with those of a previous report that an increase in α-klotho might be a compensatory response to smoking stress.
Conceptualization: Yoko Kamizono, Satoshi Imaizumi.
Data curation: Yoko Kamizono, Yuhei Shiga, Yasunori Suematsu, Hitomi Tsukahara.
Formal analysis: Yoko Kamizono, Yasunori Suematsu, Hitomi Tsukahara.
Investigation: Yoko Kamizono, Kanta Fujimi.
Methodology: Yoko Kamizono, Yuhei Shiga, Satoshi Imaizumi, Takashi Kuwano.
Project administration: Yasunori Suematsu, Takashi Kuwano.
Supervision: Keita Noda, Keijiro Saku, Shin-ichiro Miura.
Validation: Keita Noda, Keijiro Saku.
Writing – original draft: Shin-ichiro Miura.
Writing – review & editing: Shin-ichiro Miura.
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