Abstract
Background.
Although the “anti-aging hormone” klotho is associated with sarcopenia in mice, the relationship between klotho and muscle strength in older adults is not well known.
Methods.
Plasma klotho concentrations were measured in 2,734 older adults, aged 71–80 years, who participated in the Health, Aging and Body Composition Study, a prospective observational cohort study conducted in Memphis, TN and Pittsburgh, PA. Knee extension strength was measured using isokinetic dynamometry at baseline and follow-up 2 and 4 years later. Knee extension strength was normalized for weight.
Results.
At baseline, participants in the highest tertile of plasma klotho had higher knee extension strength (β = .72, standard error [SE] = .018, p < .0001) compared with those in the lowest tertile in a multivariable linear regression model adjusting for age, sex, race, smoking, study site, C-reactive protein, interleukin-6, and diabetes. Participants in the highest tertile of plasma klotho at baseline had less of a decline in knee strength over 4 years of follow-up (β = −.025, SE = .011, p = .02) compared with those in the lowest tertile in a multivariable linear regression model adjusting for the same covariates above.
Conclusions.
Plasma klotho concentrations were an independent predictor of changes in knee strength over time in older adults. Further studies are needed to identify the biological mechanisms by which circulating klotho could modify skeletal muscle strength.
Key Words: Aging, Klotho, Skeletal muscle strength, Sarcopenia
The “anti-aging hormone” klotho has been shown to protect against oxidative stress, sarcopenia, and atherosclerosis, and to extend longevity in mice (1). The klotho gene—named after one of the three Fates in Greek mythology, the goddess who spins the thread of life—encodes a single-pass transmembrane protein that is expressed most strongly in the distal tubule cells of the kidney, parathyroid glands, and choroid plexus of the brain. Klotho was originally identified in a mutant mouse strain that could not express klotho. The mice developed multiple disorders resembling human aging and had a shortened life span (2).
There are two forms of klotho, membrane (beta-klotho) and secreted (alpha-klotho), and each has different functions. Beta-klotho acts as an obligate co-receptor for fibroblast growth factor-23, a bone-derived hormone that induces phosphate excretion into urine (3). Membrane klotho is clipped at the plasma membrane by membrane-anchored proteases, and the extracellular domain, alpha-klotho, circulates in the blood and is involved in regulation of nitric oxide production in the endothelium (4,5), calcium balance in the kidney (6,7), and inhibition of intracellular insulin/insulin-like growth factor-I signaling (8). There is 80% homology between the klotho hormone in mouse and humans (9). In humans, alpha-klotho protein is more dominant than beta-klotho (10). Elevated plasma klotho concentrations have been associated with longer life span in humans (11).
Given the relationship between klotho and sarcopenia in mice, we hypothesized that older adults with higher plasma klotho concentrations had greater knee strength and less decline of knee strength over time compared with those with lower plasma klotho concentrations. To examine this hypothesis, we measured plasma klotho levels in a population-based study of older adults.
Materials and Methods
The study subjects were participants in the Health, Aging, and Body Composition (Health ABC) Study, a community-based prospective study of the impact of changes in weight and body composition on age-related physiological and functional changes. A total of 3,075 men and women, aged 70–79 years, were recruited between April 1997 and June 1998 from a random sample of white and black Medicare-eligible adults living in designated ZIP codes from the metropolitan areas surrounding the two field centers (Pittsburgh, PA and Memphis, TN). Eligibility criteria included (i) no difficulty walking ¼ mile, climbing 10 steps, or performing basic activities of daily living, (ii) no life-threatening illness, and (iii) no plans to leave the area for 3 years. The presence of clinical disease at baseline was ascertained by use of an algorithm based upon self-reported physician-diagnosed disease information and medication use (12). The cohort consisted of 1,491 men and 1,584 women. Of the 3,075 participants who enrolled in the Health ABC Study, 2,734 returned for follow-up 1 year later and had serum samples available in the study repository. The second year visit, or Visit 2 (1998–1999), was selected as the “baseline” visit for the present study because of a greater availability of serum samples from the repository.
Knee strength was measured at Visits 2, 4, and 6, in 2,775, 2,401, and 2,105 participants, respectively. There were 150 participants (6.2%) who were excluded from the analysis because they were taking steroid anti-inflammatory medications at baseline and/or follow-up, since these medications can have an effect on muscle strength. Participants were also excluded from knee strength testing if they had systolic blood pressure ≥200mm Hg or diastolic blood pressure ≥100mm Hg, a history of a cerebral aneurysm or cerebral bleeding, bilateral knee replacement, or severe bilateral knee pain. After the exclusions, there were a total of 1,983 participants who had plasma klotho measurements and one baseline measurement of knee strength plus at least a second measurement of knee strength during follow-up. Knee extensor strength was measured using an isokinetic dynamometer (Kin-Com dynamometer, 125 AP; Chattanooga, TN) concentrically at 60° per second. The right knee was tested unless there was joint replacement or knee pain. Maximum muscle torque was measured in Newton meters based upon the average of three reproducible and acceptable trials out of a maximum of six (13). Knee extensor strength was normalized for concurrent weight and expressed as Newton meters/kg (N-m/kg). Grip strength (kg) was measured at baseline and follow-up visits using a hand-held isometric dynamometer (Jamar, Bolingbrook, IL). The dynamometer was adjusted for hand size for each participant, and two trials were performed on each hand. Participants with severe hand pain or recent surgery were excluded. Participants were included in grip strength analyses if they had grip strength measured at Visit 2 and at least one additional follow-up visit. Of the participants included for the grip strength analyses, grip strength was measured at Visits 2, 4, 6, 8, and 10 in 1,920, 1,920, 1,708, 1,461, and 1,309 participants, respectively. Cognitive status was assessed using the Teng-modified Mini-Mental State Examination (14).
Plasma klotho was measured at Visit 2. Blood samples were collected in the morning after a 12-hour fast. Aliquots of serum and plasma were immediately obtained and stored at −80°C. Soluble α-klotho was measured in edetic acid plasma using a solid phase sandwich enzyme-linked immunosorbent assay [ELISA; Immuno-Biological Laboratories, Takasaki, Japan (15)]. The minimum level of detectability of the assay is 6.15 pg/mL. The minimum level is below the plasma concentrations that were found in our study. The inter-assay coefficients of variation were 18% for klotho measurements. The designation α-klotho is used to describe the original klotho gene and its product (1) and to distinguish it from a homolog which was named β-klotho (10). Throughout this article, the term klotho refers to α-klotho. Commercial enzymatic tests (Roche Diagnostics) were used for measuring serum total cholesterol, triglycerides, and high-density lipoprotein cholesterol concentrations. Low-density lipoprotein cholesterol was calculated by the Friedewald formula (16).
Serum 25-hydroxyvitamin D (25[OH]D) was measured using a radioimmunoassay (DiaSorin, Stillwater, MN). Intact parathyroid hormone was measured using a radioimmunometric assay (N-tact PTHSP, DiaSorin). Inter-assay coefficients of variation for serum 25(OH)D and parathyroid hormone were 6.8% and 8.6%, respectively. C-reactive protein (CRP) was measured in duplicate using ELISA and polyclonal anti-CRP antibodies (Calbiochem). Interleukin-6 (IL-6) was measured in duplicate using ELISA (R&D Systems, Minneapolis, MN). The inter-assay coefficients of variation for CRP and IL-6 were 8.0% and 10.3%, respectively.
Variables are reported as means (SD) or as percentages. Variables that were skewed were log transformed to achieve a normal distribution. Characteristics of participants were compared across tertiles of plasma klotho using Kruskal–Wallis tests or Wilcoxon rank sum tests for continuous variables and chi-squared tests for categorical variables. Variables that were significantly different across tertiles of plasma klotho at baseline were included in multivariable logistic regression models for normalized knee strength. All models were adjusted for age, sex, race, and study site. For decline in knee strength, we used a repeated-measured analysis of covariance and mixed-model approach. PROC MIXED with the command “repeat” was used in the analyses. Within-subject correlations were modeled with subject-specific random effect (17). All analyses were performed using SAS (v. 9.3, SAS Institute, Cary, NC) with a type I error of .05.
Results
The demographic and other characteristics of the participants at baseline by tertiles of plasma klotho are shown in Table 1. Participants with higher plasma klotho concentrations were significantly more likely to be female, black, and never smokers, with higher physical activity, lower markers of inflammation (CRP and IL-6), higher normalized knee extensor strength, and seen at the Pittsburgh study site. There were no significant differences in plasma klotho concentrations by age, education, body mass index, 25-hydroxyvitamin D, parathyroid hormone, calcium, phosphorus, Mini-Mental State Examination score, and chronic diseases, except for diabetes.
Table 1.
Baseline Characteristics of 1,983 Adults, Aged 70–79 Years, in the Health ABC Study, by Tertiles of Plasma Klotho
| Characteristic* | Plasma Klotho by Tertiles (pg/mL) | p Value | ||
|---|---|---|---|---|
| <536 (n = 660) | 536–747 (n = 662) | >747 (n = 661) | ||
| Age (y) | 74.5 (2.9) | 74.5 (2.8) | 74.5 (2.9) | .97 |
| Sex | ||||
| °Male | 51.2 | 52.0 | 48.1 | .33 |
| °Female | 48.8 | 48.0 | 51.9 | |
| Race | ||||
| °White | 66.7 | 63.8 | 55.5 | <.0001 |
| °Black | 33.3 | 36.2 | 44.5 | |
| Education (y) | ||||
| °>8 | 22.5 | 21.2 | 23.4 | .91 |
| °8–12 | 32.3 | 33.3 | 31.9 | |
| °>12 | 45.2 | 45.5 | 44.7 | |
| Smoking (%) | ||||
| °Never | 37.4 | 45.3 | 49.3 | .0003 |
| °Past | 53.2 | 45.3 | 41.4 | |
| °Current | 9.4 | 9.4 | 9.3 | |
| Body mass index (kg/m2) | 27.4 (4.8) | 27.1 (4.5) | 27.0 (5.0) | .16 |
| Physical activity (kcal/wk) | 42.8 (55.9) | 45.0 (68.7) | 47.1 (65.8) | .21 |
| Log C-reactive protein (µg/mL) | 1.25 (1.15) | 1.04 (1.17) | .88 (1.21) | <.0001 |
| Log interleukin-6 (pg/mL) | .95 (.72) | .93 (.75) | .85 (.71) | .02 |
| 25-hydroxyvitamin D (ng/mL) | 26.5 (10.8) | 26.0 (11.0) | 25.8 (11.7) | .31 |
| Parathyroid hormone (pg/mL) | 39.4 (25.8) | 37.1 (17.2) | 38.4 (29.3) | .29 |
| Serum calcium (mg/dL) | 8.85 (.45) | 8.84 (.49) | 8.88 (.46) | .17 |
| Serum phosphorus (mg/dL) | 3.56 (.49) | 3.52 (.46) | 3.54 (.49) | .51 |
| Modified Mini-Mental State Exam score < 79 (%) | 7.7 | 7.6 | 6.7 | .73 |
| Knee extensor strength, mean (kg) | 90.1 (32.5) | 92.0 (33.0) | 93.8 (34.9) | .26 |
| Knee extensor strength, mean/weight (N-m/kg) | 1.19 (.38) | 1.23 (.37) | 1.26 (.41) | .02 |
| Grip strength (kg) | .39 (.11) | .40 (.12) | .41 (.12) | |
| Hypertension (%) | 42.0 | 38.1 | 41.5 | .29 |
| Cardiovascular disease (%) | 20.3 | 17.4 | 20.7 | .24 |
| Coronary artery disease (%) | 17.1 | 15.0 | 18.5 | .23 |
| Heart failure (%) | .9 | 1.4 | .9 | .65 |
| Peripheral artery disease (%) | 4.9 | 4.4 | 3.5 | .45 |
| Stroke (%) | 2.6 | 1.8 | 2.7 | .51 |
| Diabetes mellitus (%) | 11.7 | 12.7 | 16.3 | .03 |
| Cancer (%) | 21.1 | 19.8 | 19.1 | .66 |
| Depression (%) | 2.3 | 1.8 | 1.8 | .78 |
| Osteoarthritis (%) | 9.2 | 8.1 | 7.8 | .64 |
| Clinic site, Memphis (%) | 55.0 | 47.4 | 47.7 | .007 |
*Data are given as mean (SD) or percentage, as indicated.
The cross-sectional relationship between tertiles of plasma klotho and normalized knee strength was examined in multivariable linear regression models (Table 2). Compared with participants in the lower tertile of plasma klotho, those in the highest tertile had higher normalized knee strength after adjusting for age, sex, race, and study site (Model 1), additionally for smoking (Model 2), and finally, with addition of CRP, IL-6, and diabetes (Model 3).
Table 2.
Multivariable Linear Regression Models for the Relationship of Plasma Klotho and Other Factors With Normalized Knee Strength at Baseline*
| Model 1 Adjusted for Age, Sex, Race, and Study Site | Model 2 Adjusted for Age, Sex, Race, Smoking, and Study Site | Model 3 Adjusted for Age, Sex, Race, Smoking, Study Site, Log CRP, Log IL-6, and Diabetes | |||||||
|---|---|---|---|---|---|---|---|---|---|
| β | SE | p Value | β | SE | p Value | β | SE | p Value | |
| Highest tertile klotho | .079 | .018 | <.0001 | .077 | .018 | <.0001 | .072 | .018 | <.0001 |
| Middle tertile klotho | .030 | .018 | .10 | .028 | .018 | .12 | .028 | .018 | .12 |
*Lowest tertile of klotho is reference.
Of 1,983 participants with klotho and knee strength measurements at baseline, 285 (14.4%) had no further follow-up, 240 (12.1%) had 2-year follow-up only, 144 (7.3%) had 4-year follow-up only, and 1,314 (66.2%) had both 2- and 4-year follow-up. The decline of normalized knee strength (N-m/kg/year) by tertile of serum klotho is shown in Figure 1. Multivariable linear regression models were used to examine the relationship between plasma klotho at baseline and the decline of normalized knee strength over time. Participants in the highest tertile of klotho had significantly lower decline of knee strength compared with those in the lowest tertile after adjusting for age, sex, race, and study site (Model 1), additionally for smoking (Model 2), and finally, with addition of CRP, IL-6, and diabetes (Model 3) (Table 3).
Figure 1.
Decline of normalized knee extension strength (N-m/kg) over 4 years by tertile of plasma klotho, adjusted by age, sex, race, study site, and baseline normalized knee extension strength.
Table 3.
Multivariable Linear Regression Models for the Relationship of Plasma Klotho and Other Factors With Decline in Normalized Knee Strength Over Follow-Up*,†
| Model 1 Adjusted for Age, Sex, Race, and Study Site | Model 2 Adjusted for Age, Sex, Race, Smoking, and Study Site | Model 3 Adjusted for Age, Sex, Race, Smoking, Study Site, Log CRP, and Log IL-6 | |||||||
|---|---|---|---|---|---|---|---|---|---|
| β | SE | p Value | β | SE | p Value | β | SE | p Value | |
| Highest tertile klotho | −.028 | .010 | .009 | −.027 | .011 | .01 | −.025 | .011 | .02 |
| Middle tertile klotho | −.011 | .011 | .31 | −.010 | .011 | .33 | −.012 | .011 | .25 |
*All models adjusted for normalized knee strength at the beginning of each interval.
†Lowest tertile of klotho is reference. A negative beta signifies less of a decline in normalized knee strength compared with the lowest tertile.
In an alternative model, we also included physical activity at baseline in addition to all covariates used in the models above. Participants in the highest tertile of klotho had significantly lower decline of knee strength compared with those in the lowest tertile after adjusting for age, sex, race, study site, smoking, CRP, IL-6, and diabetes (β = −.025, SE = .010, p = .02).
Because race was strongly associated with plasma klotho, we explored additional bivariate and multivariable models of the relationship of race with plasma klotho concentrations. White race was associated with lower plasma klotho concentrations at baseline (β = −.045, SE = .020, p = .03) in a multivariable linear regression model adjusting for age, sex, study site, smoking, CRP, IL-6, and diabetes, where plasma klotho concentrations were considered as a continuous variable.
In order to gain further insight into the relationship of klotho with knee strength, we conducted analyses stratified by gender, race, physical activity, and inflammatory cytokine level, adjusting for the same variables as in Table 2, Model 3. When comparing the highest with the lowest tertile of plasma klotho, in men and women, the βs (SE) were −.0388 (.0182) (p = .03) and −.0098 (.0122) (p = .42), respectively; in whites and blacks, the βs (SE) were −.0234 (.0119) (p = .05) and −.0311 (.0222) (p = .16), respectively; in highest, middle, and lowest tertile of physical activity, the βs (SE) were −.0320 (.01999) (p = .10), −.0097 (.0175) (p = .57), and −.0325 (.0194) (p = .09), respectively; in participants with IL-6 ≥2.5 pg/mL versus <2.5 pg/mL, the βs (SE) were −.0179 (.0151) (p = .23) and −.039 (.0156) (p = .01), respectively. There were 844 participants with IL-6 >2.5 pg/mL. Some caution should be taken in the interpretation of these results, given the reduced power in the subgroups after stratification.
We also conducted an additional analysis to determine whether the relationship of plasma klotho with knee strength is also consistent with grip strength. From the lowest to highest tertile of plasma klotho at baseline, there were 639, 641, and 640 participants who had at least one grip strength measurement at follow-up. Participants in the highest and middle tertile of plasma klotho had βs (SE) of −.0061 (.0021) (p = .005) and −.0024 (.0021) (p = .25) compared with the lowest tertile, in a multivariable linear regression model adjusting for the same covariates as in Table 2, Model 3). These results show that there is a consistent relationship between plasma klotho and decline of both knee strength and grip strength.
Discussion
The present study shows that older adults with low circulating klotho concentrations have a greater decline in knee strength over 4 years of follow-up compared with those with higher klotho levels. To our knowledge, this is the first prospective study to show that low plasma klotho concentrations are predictive of changes in skeletal muscle strength. The strengths of this study are the well-characterized study cohort, standardized collection of data, and population-based sample of older adults that included both blacks and whites.
Although klotho is associated with widespread changes in different body systems, the role of klotho in skeletal muscle is not well understood. Some recent insights have come from studies in klotho-deficient mice and in older adults. Klotho-deficient C57BL/6 mice have decreased forelimb grip strength and running endurance compared with wild type and klotho overexpressing mice (18). In C57Bl6/J mice, plasma klotho levels increased after an acute bout of exercise, and the increase from pre- to post-exercise was relatively greater in younger mice compared with that in older mice (19). Plasma klotho concentrations increased in both younger and older adults after respective exercise training. In 12 women, aged 25–45 years, after 16 weeks of progressive exercise training consisting of rowing, walking/jogging, and cycling on a stationary bicycle, mean plasma klotho levels increased by 30% (19). In seven women, aged 65–74 years, after 12 weeks of progressive exercise training consisting of a similar program of rowing, walking/jogging, and cycling, mean plasma klotho levels increased by nearly 15% (19). In a study of healthy and postmenopausal women, aged 50–76 years, 11 women participated in aerobic training for 12 weeks and were compared with a control group that had no exercise intervention. Plasma klotho levels in the exercise group increased significantly after the exercise intervention whereas there were no significant changes in controls (20). These observations from human and mice studies suggest that exercise stimulates an increase in plasma klotho concentrations. In the present study, the association between plasma klotho concentrations and decline in knee strength remained significant even after adjusting for physical activity. The difference between the present study and previous studies of exercise training may possibly be attributed to the difference between self-reported physical activity in an observational cohort versus exercise in a controlled intervention study.
There are several potential biological mechanisms by which klotho could affect skeletal muscle. Circulating klotho is known to decrease oxidative stress (21), play a role in both transforming growth factor-β1 signaling (22) and insulin/insulin-like growth factor-I signaling (1). The insulin-like growth factor-I signaling pathway plays a role in hypertrophy of skeletal muscle through activation of PI3K/Akt and downstream upregulation of targets required for protein synthesis (23). Klotho also reduces inflammation in endothelial cells by attenuation of necrosis factor-κB activation and suppression of tumor necrosis factor-α–induced expression of adhesion molecules (24). These potential mechanisms have not been directly studied in skeletal muscle.
In the present study, higher circulating klotho concentrations were inversely associated with serum CRP and IL-6 concentrations. These findings are not consistent with the idea that serum klotho is a positive acute phase reactant, an observation that has been made in a murine model (25). Another new observation from this study, to our knowledge, is that plasma klotho levels were higher in blacks than in whites. The association between race and plasma klotho concentrations remained significant after adjusting for potential confounders. The underlying reason for this association is not known.
The present study is limited in that plasma klotho concentrations were measured at baseline only. Whether plasma klotho levels changed over time in relation to knee strength is not known. We have previously conducted pilot studies to determine how much plasma klotho levels change over time. In 12 adults, aged 65 years and older in the InCHIANTI Study, the intra-class correlation coefficient for plasma klotho measured at baseline (1998), 3-year follow-up, and 6-year follow-up was .92. In 13 adults in the Women’s Health and Aging Study II, the intra-class correlation coefficient for serum klotho measured at baseline (1994), 6-year follow-up, and 9-year follow-up was .92 (R. D. Semba, personal communication). These studies suggest that plasma klotho concentrations do not show a great deal of variability over time in older community-dwelling adults. In addition, the study was limited to adults aged 71–80 years and cannot necessarily be generalized to other age groups.
In conclusion, this study showed that older adults with higher plasma klotho concentrations had less of a decline in knee strength over 4 years than those with lower klotho levels. Future studies need to address the possible biological role of klotho on the cellular level in skeletal muscle.
Funding
This work was supported by National Institutes of Health grants R01 AG27012, R01 AG028050, R01 HL094507, and R21 HL112662, National Institute on Aging contracts N01-AG-6-2101, N01-AG-6-2103, and N01-AG-6-2106, NINR grant R01 NR012459, and the Intramural Research Program of the National Institutes of Health.
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