Journals of Gerontology: Medical Sciences cite as: J Gerontol A Biol Sci Med Sci, 2016, Vol. 71, No. 1, 103–108 doi:10.1093/gerona/glv077 Advance Access publication September 9, 2015
Research Article
Low Plasma Klotho Concentrations and Decline of Knee Strength in Older Adults Richard D. Semba,1 Luigi Ferrucci,2 Kai Sun,1 Eleanor Simonsick,2 Randi Turner,1 Iva Miljkovic,3 Tamara Harris,4 Ann V. Schwartz,5 Keiko Asao,6 Stephen Kritchevsky,7 and Anne B. Newman3 for the Health ABC Study 1 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland. 2National Institute on Aging, NIH, Baltimore, Maryland. 3Department of Epidemiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania. 4Intramural Research Program, Laboratory of Epidemiology and Population Sciences, National Institute on Aging, NIH, Bethesda, Maryland. 5 Department of Epidemiology and Biostatistics, University of California San Francisco School of Medicine. 6Department of Preventive Medicine, University of Tennessee, Memphis. 7Sticht Center on Aging, Wake Forest University, Winston-Salem, North Carolina.
Address correspondence to Richard D. Semba, MD, MPH, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Smith Building, M015, 400 North Broadway, Baltimore, MD 21287. Email: [email protected] Received July 1, 2015; Accepted December 18, 2014 Decision Editor: James Goodwin, MD
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. Betaklotho 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
© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: [email protected].
103
104
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 selfreported 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 ≥200 mm Hg or diastolic blood pressure ≥100 mm 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
Journals of Gerontology: MEDICAL SCIENCES, 2016, Vol. 71, No. 1
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; ImmunoBiological 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 interassay 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,
Journals of Gerontology: MEDICAL SCIENCES, 2016, Vol. 71, No. 1
105
Table 1. Baseline Characteristics of 1,983 Adults, Aged 70–79 Years, in the Health ABC Study, by Tertiles of Plasma Klotho Characteristic*
Age (y) Sex °Male °Female Race °White °Black Education (y) °>8 °8–12 °>12 Smoking (%) °Never °Past °Current Body mass index (kg/m2) Physical activity (kcal/wk) Log C-reactive protein (µg/mL) Log interleukin-6 (pg/mL) 25-hydroxyvitamin D (ng/mL) Parathyroid hormone (pg/mL) Serum calcium (mg/dL) Serum phosphorus (mg/dL) Modified Mini-Mental State Exam score < 79 (%) Knee extensor strength, mean (kg) Knee extensor strength, mean/weight (N-m/kg) Grip strength (kg) Hypertension (%) Cardiovascular disease (%) Coronary artery disease (%) Heart failure (%) Peripheral artery disease (%) Stroke (%) Diabetes mellitus (%) Cancer (%) Depression (%) Osteoarthritis (%) Clinic site, Memphis (%)
Plasma Klotho by Tertiles (pg/mL)
p Value
747 (n = 661)
74.5 (2.9)
74.5 (2.8)
74.5 (2.9)
.97
51.2 48.8
52.0 48.0
48.1 51.9
.33
66.7 33.3
63.8 36.2
55.5 44.5
Research Article
Low Plasma Klotho Concentrations and Decline of Knee Strength in Older Adults Richard D. Semba,1 Luigi Ferrucci,2 Kai Sun,1 Eleanor Simonsick,2 Randi Turner,1 Iva Miljkovic,3 Tamara Harris,4 Ann V. Schwartz,5 Keiko Asao,6 Stephen Kritchevsky,7 and Anne B. Newman3 for the Health ABC Study 1 Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland. 2National Institute on Aging, NIH, Baltimore, Maryland. 3Department of Epidemiology, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania. 4Intramural Research Program, Laboratory of Epidemiology and Population Sciences, National Institute on Aging, NIH, Bethesda, Maryland. 5 Department of Epidemiology and Biostatistics, University of California San Francisco School of Medicine. 6Department of Preventive Medicine, University of Tennessee, Memphis. 7Sticht Center on Aging, Wake Forest University, Winston-Salem, North Carolina.
Address correspondence to Richard D. Semba, MD, MPH, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Smith Building, M015, 400 North Broadway, Baltimore, MD 21287. Email: [email protected] Received July 1, 2015; Accepted December 18, 2014 Decision Editor: James Goodwin, MD
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. Betaklotho 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
© The Author 2015. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: [email protected].
103
104
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 selfreported 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 ≥200 mm Hg or diastolic blood pressure ≥100 mm 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
Journals of Gerontology: MEDICAL SCIENCES, 2016, Vol. 71, No. 1
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; ImmunoBiological 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 interassay 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,
Journals of Gerontology: MEDICAL SCIENCES, 2016, Vol. 71, No. 1
105
Table 1. Baseline Characteristics of 1,983 Adults, Aged 70–79 Years, in the Health ABC Study, by Tertiles of Plasma Klotho Characteristic*
Age (y) Sex °Male °Female Race °White °Black Education (y) °>8 °8–12 °>12 Smoking (%) °Never °Past °Current Body mass index (kg/m2) Physical activity (kcal/wk) Log C-reactive protein (µg/mL) Log interleukin-6 (pg/mL) 25-hydroxyvitamin D (ng/mL) Parathyroid hormone (pg/mL) Serum calcium (mg/dL) Serum phosphorus (mg/dL) Modified Mini-Mental State Exam score < 79 (%) Knee extensor strength, mean (kg) Knee extensor strength, mean/weight (N-m/kg) Grip strength (kg) Hypertension (%) Cardiovascular disease (%) Coronary artery disease (%) Heart failure (%) Peripheral artery disease (%) Stroke (%) Diabetes mellitus (%) Cancer (%) Depression (%) Osteoarthritis (%) Clinic site, Memphis (%)
Plasma Klotho by Tertiles (pg/mL)
p Value
747 (n = 661)
74.5 (2.9)
74.5 (2.8)
74.5 (2.9)
.97
51.2 48.8
52.0 48.0
48.1 51.9
.33
66.7 33.3
63.8 36.2
55.5 44.5
