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Association of exonic variants of Klotho with metabolic syndrome in Asian Indians
Clinica Chimica Acta 412 (2011) 1116–1121
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Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Association of exonic variants of Klotho with metabolic syndrome in Asian Indians Vijaya Majumdar, Rita Christopher ⁎ Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore-560029, India
a r t i c l e
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Article history: Received 10 December 2010 Received in revised form 25 February 2011 Accepted 26 February 2011 Available online 3 March 2011 Keywords: Metabolic syndrome Insulin resistance Asian Indians Klotho Klotho KL-VS Klotho C1818T
a b s t r a c t Background: Klotho, an anti-aging gene, is a functional candidate for metabolic syndrome. We conducted a cross-sectional study to evaluate the association of the genetic variants of Klotho with metabolic syndrome and surrogates of insulin resistance in Asian Indians. Methods: We recruited 428 clinically normal subjects for the study. Genotyping was done by polymerase chain reaction and restriction fragment length polymorphism. Results: Significant and borderline associations of the KL-VS (OR = 15.88 [95%CI, 2.56–98.70], p = 0.003) and C1818T (OR = 0.28 [95%CI, 0.07–1.07], p = 0.063) variants of the Klotho gene, respectively, were observed with metabolic syndrome. The association of the KL-VS variant with metabolic syndrome could be linked to its observed influence on high blood glucose (OR = 6.92 [95% CI = 1.75–27.44], p = 0.006), high blood pressure (OR = 5.21 [95%CI = 1.00–38.43], p = 0.046), insulin resistance (OR = 3.59, [95%CI = 1.01–12.79], p = 0.048) and trend towards its association with hypertriglyceridemia (OR = 3.69 [95%CI = 0.92–14.77], p = 0.065). Conclusions: The genetic variants of Klotho might predict risk for metabolic syndrome and insulin resistance in Asian Indians. However, larger studies in other ethnic populations are warranted to determine the role of these gene variants in the etiology of metabolic syndrome. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Klotho is an aging suppressor gene that extends life span when over-expressed and accelerates aging when disrupted. Klotho-deficient mice exhibit a syndrome resembling accelerated human aging in conjunction with extensive arteriosclerosis [1]. Klotho has been demonstrated to ameliorate vascular endothelial dysfunction, increase nitric oxide production, reduce elevated blood pressure, and prevent medial hypertrophy and perivascular fibrosis in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of metabolic syndrome (MetS) [2,3]. Further, renal expression of Klotho is reduced in OLETF rats and its expression is found to be augmented by treatment with troglitazone, an insulin sensitizer and agonist of PPARγ [2]. A role of Klotho in in-vitro vascular inflammation has also been described by Maekawa and co-workers who demonstrated that Klotho reduces the
Abbreviations: OLETF, Otsuka Long–Evans Tokushima Fatty; PPAR, Peroxisome proliferator-activated receptor; TNF, tumor necrosis factor; HDL, high density lipoprotein; SBP, systolic blood pressure; DBP, diastolic blood pressure; MetS, metabolic syndrome; LD, linkage disequilibrium; CAD, coronary artery disease; WC, waist circumference; HOMA, homeostasis model assessment of insulin resistance; NCEP/ ATP III, National Cholesterol Education Program/Adult Treatment Panel III; IR, insulin resistance. ⁎ Corresponding author at: Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences (NIMHANS), Hosur Road, Bangalore 560029, India. Tel.: + 91 80 26995162; fax: + 91 80 26564830. E-mail addresses: [email protected], [email protected] (R. Christopher). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.034
expression of adhesion molecules as well as recovers the suppression of endothelial nitric oxide synthase induced by the immunomodulator, tumor necrosis factor (TNFα) [4]. Polymorphisms in the human Klotho gene are associated with longevity, various cardiovascular events like stroke, coronary artery disease as well as the cardiovascular risk factors like reduced high density lipoprotein-cholesterol (HDL) levels and elevated systolic blood pressure (SBP) [5–8]. All these observations are suggestive of the candidature of Klotho in metabolic syndrome (MetS) which is a combination of risk determinants of atherogenic cardiovascular disease. Interestingly, Klotho has also been shown to be involved in the manifestation of insulin resistance [9] which appears to be at variance with its beneficial role in longevity and protection against vascular pathologies. Unger had proposed that Klotho-induced insulin resistance prevents cellular lipid overload by reducing insulin-stimulated availability of the lipogenic substrate, glucose [10]. The reduction in intracellular lipid content could raise the apoptotic threshold and extend the life of the cells. On the contrary, a recent report by Lorenzi et al. presents evidence against a direct involvement of Klotho in insulin signaling [11]. In view of these contradictory findings we undertook a study to test the association of Klotho gene variants with MetS and the surrogates of insulin resistance. The KL-VS variant of Klotho consists of six sequence variants in perfect linkage disequilibrium (LD), two of which result in amino acid substitutions, F352V and C370S [5]. Due to presence of complete LD across the SNPs, the single variant, F352V, has been used to tag the KL-VS haplotype. The presence of phenylalanine at the position 352 in the
V. Majumdar, R. Christopher / Clinica Chimica Acta 412 (2011) 1116–1121 Table 1 Clinical characteristics stratified by presence or absence of MetS. Clinical characteristics
All subjects (n = 428)
Yes (n = 127)
MetS No (n = 301)
p value
Age (years) Sex (M/F) Smokers, n (%) Hypertension, n (%) Diabetes, n (%) Dyslipidemia, n (%)
39.7 ± 12.7 235/193 54 (12.7) 100 (23.4) 34 (7.9) 240 (56.1)
47.2 ± 8.3 60/67 25 (19.7) 60 (47.2) 27 (21.3) 95 (74.8)
36.4 ± 13.0 175/126 29 (9.6) 40 (13.3) 7 (2.3) 145 (48.2)
b 0.001 0.043 0.006 b 0.001 b 0.001 b 0.001
Data are mean ± SD unless otherwise indicated. Abbreviations: MetS, metabolic syndrome.
human Klotho gene is highly conserved and its substitution to valine has been demonstrated to alter the in vitro excretion and activity of the protein. Arking and co-workers reported a positive association between the functional KL-VS variant of Klotho and early-onset occult coronary artery disease (CAD) [6]. They also found positive association of Klotho's KL-VS variant with incidence of stroke in a cohort ascertained for the analysis of longevity [7]. Another widely studied variant of Klotho gene is the synonymous C1818T variant (rs564481) located in the fourth exon. Being a silent mutation the variant is not likely to be functionally relevant. However, there are reports which demonstrate the clinical relevance of the variant which has been studied for association with cardiovascular risk factors, fasting glucose, lipid levels and blood pressure in Asian population [12,13]. An association between CAD and the C1818T variant has been reported in Koreans [14].
2. Subjects This study was approved by the ethics committee of our Institute and conformed to the principals outlined in the declaration of Helsinki. Written informed consent was obtained from all the participants. A total of 428 unrelated subjects with self-reported South Indian ancestry, were recruited between the period of August 2007 and October 2009. Study participants were asymptomatic and reported no major illness. They were all employed and were attending to their regular duties at the time of recruitment. Demographic and anthropometric analyses included the following variables: age, sex, smoking status, height, weight and waist circumference (WC) and blood pressure. Clinical laboratory measurements included serum levels of total cholesterol, LDL-cholesterol, HDLcholesterol, triglycerides, and glucose. Serum glucose, triglycerides, total cholesterol, and HDL-cholesterol concentrations were determined enzymatically using commercially available kits and auto analyzer (Olympus AU640) and plasma LDL-cholesterol concentration was calculated using Friedewald's formula [15]. Fasting serum insulin was measured by ELISA
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with a commercial kit [Human Insulin specific, Calbiotech, Inc, San Diego, CA]. The intra- and inter-assay CVs were 5.5% and 7.8%, respectively. Insulin sensitivity was assessed with fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR). Homeostasis model assessment estimates of insulin resistance (HOMA-IR) and β-cell function (HOMA-B) were calculated using fasting glucose and insulin measurements [16]. Subjects were classified as having metabolic syndrome according to the NCEP/ATP III (National Cholesterol Education Program/ Adult Treatment Panel III) definition [blood pressure of ≥130/85 mm Hg; triglycerides ≥150 mg/dl (1.69 mmol/l); HDL-cholesterol b40 mg/dl (1.03 mmol/l) and b50 mg/dl (1.29 mmol/l) in men and women, respectively; and fasting blood glucose ≥110 mg/dl (6.05 mmol/l)] with modified WC cut off values (males N90 cm, females N80 cm) appropriate for Indians [17,18]. Presence of three or more parameters was considered for defining MetS positive subjects. Insulin resistance was defined as HOMA-IR N4.65 [19]. The diagnosis of hypertension was based on seated blood pressure above a defined cut-off value [140 mm Hg for systolic blood pressure (SBP); and 90 mm Hg for diastolic blood pressure (DBP)] and/or, if the subjects were on antihypertensive therapies. Diabetes mellitus was defined as fasting blood glucose of ≥7 mmol/l or 126 mg/dl, the use of insulin or oral hypoglycemic agents. Dyslipidemia was defined as elevated total cholesterol N6.2 mmol/l (240 mg/dl), and/or decreased HDL-cholesterol b0.9 mmol/l (35 mg/dl), and/or increased triglycerides N2.3 mmol/l (200 mg/dl). The cut-offs applied are based on cut-offs for high risk for cardiovascular disorders in the NCEP guidelines [17]. 3. Materials and methods 3.1. Klotho KL-VS genotyping The KL-VS (rs9536314) variant was typed using a published method [5]. Sample DNA was amplified by PCR; sense primer 5′-GCC AAA GTC TGG CAT CTC TA-3′; antisense primer 5′-TTC CAT GAT GAA CTT TTT GAG G-3′, under the following conditions: 95 °C for 5 min, followed by 32 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min, followed by a 10 min 72 °C final extension. PCR products were then digested with MaeIII (Roche Molecular, Pleasanton, CA) at 55 °C for 4 h. The F352 allele has a single MaeIII restriction site in the amplicon; substitution of F by V at the position 352 generates another MaeIII site. Thus the amplicon from wild type homozygotes (352FF) was digested into 2 bands (319 bp and 186 bp), whereas heterozygotes (352FV) had 3 bands (319 bp, 265 bp, and 186 bp), and the 352VV homozygotes had 2 bands (265 bp and 186 bp). 3.2. Klotho C1818T genotyping C1818T variant (rs564481) was also genotyped using PCR-restriction fragment length methodology. DNA containing the polymorphic
Table 2 Clinical and biochemical parameters by Klotho KL-VS and C1818T genotypes in the MetS group. Variable
FF (n = 91)
FV (n = 27)
VV (n = 9)
p value
CC (n = 71)
CT (n = 51)
TT (n = 4)
P value
Sex (M/F) Age, years BMI, kg/m2 WC, cm SBP, mm Hg DBP, mm Hg TC, mmol/l TG, mmol/l HDL, mmol/l Fasting glucose, mmol/l Insulin, μU/ml HOMA-IR
43/48 39.4 ± 12.6 29.2 ± 6.1 94.5 ± 8.3 126.9 ± 18.5 85.9 ± 12.0 5.2 ± 1.2 2.5 ± 1.1 0.99 ± 0.22 6.0 ± 3.0 16.0 ± 14.5 4.0 ± 3.9
13/14 40.5 ± 13.2 28.3 ± 4.1 94.9 ± 9.5 131.3 ± 8.8 87.4 ± 9.0 5.8 ± 0.7 2.3 ± 1.4 0.98 ± 0.19 5.7 ± 2.6 14.4 ± 9.7 3.6 ± 3.0
4/5 40.2 ± 10.7 26.5 ± 2.7 93.7 ± 7.6 141.5 ± 20.4 89.1 ± 11.5 5.3 ± 1.1 2.3 ± 0.8 1.00 ± 0.23 6.0 ± 1.4 24.5 ± 18.9 6.8 ± 6.8
NS NS NS NS *0.025 NS NS NS NS NS NS NS
32/39 40.0 ± 13.2 28.2 ± 4.7 87.9 ± 10.7 130.8 ± 13.1 87.7 ± 11.9 5.0 ± 1.0 2.4 ± 1.2 0.98 ± 0.22 6.0 ± 3.0 14.8 ± 10.4 3.9 ± 3.9
26/25 38.9 ± 12.1 27.9 ± 4.0 88.5 ± 9.6 126.9 ± 11.3 85.2 ± 11.0 5.0 ± 1.1 2.5 ± 1.2 1.00 ± 0.21 6.1 ± 2.8 18.7 ± 18.1 4.5 ± 4.4
1/3 43.7 ± 11.4 28.6 ± 4.3 91.8 ± 8.5 130.0 ± 11.5 82.5 ± 5.0 5.3 ± 1.2 1.7 ± 0.5 1.03 ± 0.24 4.5 ± 0.5 7.3 ± 3.7 1.4 ± 0.8
NS NS NS NS NS NS NS NS NS NS NS NS
Values represent mean ± SD. Abbreviations: MetS, metabolic syndrome; BMI, body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; HDL, high-density lipoprotein-cholesterol; TG, triglycerides; HOMA-IR, Homeostasis model assessment estimates of insulin resistance.
205 138 18 140 101 8
41 15 4 NS 31 26 2 NS 178 67 8
264 97 7
+ ve (N = 60) − ve (N = 253)
127 45 3 NS 96 63 12 NS 107 67 8
184 59 2
The differences in the anthropometric and biochemical parameters were compared using t-test or Mann–Whitney test. Violation of Hardy– Weinberg equilibrium (HWE) was tested by Pearson's χ2 test, used to analyze the associations between categorical variables. Association between Klotho geotypes and MetS were analyzed using different genetic models viz., general effects, dominant and recessive. For the analysis of the components of MetS viz, high blood glucose, hypertriglyceridemia etc., recessive models of inheritance were assumed for both the genetic variants. Multivariate logistic regression analysis of risk factors was also performed, with odds ratios (ORs) (95% CIs) shown. A 2-sided P b 0.05 was considered statistically significant. Linkage disequilibrium was estimated between the 2 genetic variants in our study subjects using Haploview software, (http://www.broad.mit.edu/mpg/haploview/contact. php).
158 113 13 165 113 16 p value C1818T CC CT TT p value
91 27 9 0.0003 71 51 4 NS FF FV VV KL-VS
4. Results
p value; Chi-square/Fisher's exact test. Abbreviations: MetS, metabolic syndrome; BP, blood pressure; HDL, high density lipoprotein-cholesterol.
209 147 17
138 93 14
172 76 3
98 34 5 NS 78 51 7 NS 214 85 2
207 78 6
270 104 6 0.0006 27 17 3 NS
35 8 5
133 36 8 0.011 98 71 6 NS
121 53 9 0.011 129 97 12 NS
+ ve (N = 175) + ve (N = 183) − ve (N = 251) + ve (N = 177) − ve (N = 48) + ve (N = 380) + ve (N = 137)
− ve (N = 291)
site was amplified using the following primers (sense primer 5′-TGT CTC AGT TTA CCG ACC TGA ATG T-3′ and anti-sense 5′-ATT CAT CGT TAT CCA AAG CTT GAC G-3′) and PCR amplification conditions of 95 °C for 5 min, followed by 32 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min, followed by a 10 min 72 °C final extension. PCR products were restriction digested with Mph1103I (Fermentas) at 37 °C for 16 h overnight. The DNA segment from TT homozygotes was digested into 272 and 180 bps. For the undigested CC wild homozygotes, a single band of 452 bp was observed. Genotyping failed for 8 samples thereby there was a reduction in the sample size from the original dataset. We dealt with mis-genotyping errors by retyping 10% of our samples, with identical results.
3.3. Statistical analysis
− ve (N = 301) + ve (N = 127)
Hyperglycemia Abdominal obesity Genotypes MetS Klotho
Table 3 Distribution of Klotho KL-VS and C1818T genotypes across MetS, components of MetS and insulin resistance.
Hypertriglyceridemia
High BP
− ve (N = 245)
Low HDL
− ve (N = 368)
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Insulin resistance
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Metabolic syndrome was present in 29.4% of all the subjects and it was more prevalent in females (34.4%) compared to males (25.7%) (p = 0.043). There was a significant difference in the distribution of the clinical parameters between subjects with and without MetS (Table 1). Subjects with MetS were older (47.2 ± 8.3 vs. 36.4 ± 13.0 y; p b 0.001) and more frequent smokers (19.7 vs. 9.6%; p = 0.006) compared to those without MetS. Frequency of hypertension, diabetes and dyslipidemia (p b 0.001) were also high in subjects with MetS compared to those without MetS. Stratification of the clinical and biochemical parameters by the genotypes of the Klotho variants in MetS positive subjects is presented in Table 2. There was a significant difference in the levels of SBP among the individuals with KL-VS genotypes (p = 0.025), subjects with VV genotype had elevated SBP compared to those with FF and FV genotypes. No significant association could be found for the other parameters with the variant. C1818T variant was also not found to have any influence on any of the clinical or biochemical variables (Table 2).
4.1. KL-VS and MetS The genotype distribution of the KL-VS variant in the total population was FF 305 (71.3%), FV 112 (26.1%) and VV 11 (2.6%) and was in Hardy–Weinberg equilibrium (p = 0.997). There was a significant difference in the distribution of the KL-VS genotypes between MetS positive and negative subjects (p = 0.0003, Table 3). When examined using a recessive model, individuals with VV genotype were found to have ~16 fold higher risk of MetS than the F allele carriers (OR = 15.88 [95%CI, 2.56–98.70], p = 0.003) (Table 4). Further, stratification by sex did not indicate any gender specific influence of the variant on MetS (Table 4).
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Table 4 Estimate of the effects of the Klotho variants on MetS risk modeled with logistic regression. Combined
Men
Women
Variant
Adjusted OR (95% CI)
p value
Adjusted OR (95% CI)
p value
Adjusted OR (95% CI)
p value
KL-VS FF FV VV
1.00 [ref] 1.44 (0.77–2.71) 14.00 (2.24–87.30)
NS 0.005
1.00 [ref] 1.56 (0.75–3.28) 5.96 (0.99–35.65)
NS 0.050
1.00 [ref] 1.31 (0.56–3.06) 9.03 (0.86–94.52)
NS NS
C1818T CC CT TT
1.00[ref] 1.20 (0.68–2.13) 0.25 (0.05–1.20)
NS NS
1.00[ref] 1.26 (0.67–2.38) 0.44 (0.05–3.70)
NS NS
1.00[ref] 1.28 (0.61–2.71) 0.32 (0.07–1.47)
NS NS
Recessive models KL-VS ( VV vs. FF + FV) C1818T ( TT vs. CC + CT)
15.88 (2.56–98.70) 0.28 (0.07–1.07)
0.003 0.063
6.90 (1.11–98.70) 0.48 (0.05–4.41)
0.038 NS
11.47 (1.17–112.55) 0.28 (0.06–1.25)
0.036 NS
Dominant models KL-VS ( FV + VV vs. FF) C1818T (CT + TT vs. CC)
1.01 (0.57–1.80) 1.06 (0.62–1.79)
NS NS
0.83 (0.42–1.64) 1.16 (0.62–2.17)
NS NS
1.15 (0.53–2.53) 1.02(0.50–2.06)
NS NS
p values were adjusted on age, sex, and smoking, in the combined sample or on age and smoking in men and women separately.
4.2. KL-VS and components of MetS There was no significant association found between the KL-VS genotype and abdominal obesity (p = 0.590), or depression in HDL levels (p = 0.626) (Table 3). KL-VS variant was found to be associated with high fasting plasma glucose levels (p= 0.0006) (Table 3). Multivariate logistic regression analysis revealed a significant association between the KL-VS variant and high fasting plasma glucose levels (OR =6.92 [95% CI= 1.75–27.44], p =0.006); the other predictors being age (OR= 1.1, 95% CI =1.03–1.10, p =0.001) and triglycerides (OR =1.01, 95% CI= 1.00–1.01, p =0.006). However, individuals with VV genotype were not found to have significantly different mean fasting glucose levels compared to individuals with FF and FV genotypes (p = 0.937 and 0.836, respectively). After dichotomization, a significant association of the KL-VS genotypes with high BP (p = 0.011) was also observed (Table 3). Furthermore, multivariate logistic regression analysis indicated that the risk factors for high BP were the VV genotype (recessive model) (OR = 5.21 [95%CI = 1.00–38.43], p = 0.046), age (OR = 1.06 [95% CI = 1.03–1.08], p = 0.0001), waist circumference (OR = 1.05 [95% CI = 1.02–1.08], p = 0.002) and total cholesterol (OR = 1.01 [95% CI = 1.00–1.01], p = 0.009). Moreover, KL-VS variant was found to be associated with SBP (continuous variable), p = 0.025 (Table 2). Genotypes of KL-VS variant were significantly differentially distributed between individuals with and without hypertriglyceridemia; p = 0.011 (Table 3). Multivariate logistic regression analysis revealed a trend of association between the VV genotype and hypertriglyceridemia (OR = 3.69 [95%CI = 0.92–14.77], p = 0.065). 4.3. KL-VS variant and surrogates of IR Dichotomization based on HOMA cut off point of 4.65, revealed a borderline association of the KL-VS variant with IR (p = 0.096) (Table 3). Further evaluation using a recessive model of inheritance indicated a significant association (p = 0.031) of the variant with this trait. Multivariate logistic regression analysis indicated a significant association of IR with the VV genotype of the KL-VS variant (OR = 3.59, [95%CI = 1.01–12.79], p = 0.048). However, there was no association found between KL-VS variant and the continuous variables like HOMA-IR, fasting glucose and insulin (Table 2). 4.4. C1818T and MetS The genotype distribution of the C1818T variant was CC 236 (56.2%), CT 164 (39.0%) and TT 20 (4.8%), and complied with Hardy–
Weinberg equilibrium (p = 0.630). The genotypic distribution of the variant was not significantly different between MetS-positive and negative individuals (p = 0.592; Table 3). In general, no significant differences between MetS-positive cases and controls were observed for CT vs. CC (OR-1.20 [95% CI, 0.68–2.13], p = 0.525) as well as for TT vs. CC comparison (OR-0.25 [95% CI, 0.05–1.20], p = 0.083). We could find a borderline significant association of the variant with MetS (p b 0.10), wherein the TT genotype carriers were found to be protected compared to CC + CT genotype carriers against risk of MetS (OR = 0.28 [95% CI, 0.07–1.07], p = 0.063 compared to the C allele carriers, Table 4. However, analysis under dominant model of inheritance did not indicate any significant effect of the C1818T variant on MetS risk (OR = 1.06 [95% CI, 0.62–1.79], p = 0.842). Stratification by sex did not indicate any gender specific influence of the variant on MetS. 4.5. C1818T and components of MetS There was no significant association between the C1818T genotypes and the components of MetS viz., abdominal obesity; p = 0.889, high BP; p = 0.641, high blood glucose; p = 0.811, high triglycerides; p = 0.522 and low HDL levels; p = 0.179 (Table 3). 4.6. C1818T variant and surrogates of IR Dichotomization based on HOMA cut off point of 4.65, revealed no association of the C1818T variant genotypes with IR (p = 0.645) (Table 3). The variant was also not associated with the continuous variables like HOMA-IR, fasting glucose and insulin (Table 2). 4.7. Linkage Disequilibrium (LD) analysis Haploview was used to look for LD among the two studied polymorphisms but no significant LD was observed between the polymorphisms (D′ = 0.298; LOD = 0.14 and r2 = 0.005). 5. Discussion Metabolic syndrome is a common clinical phenotype presenting as a cluster of metabolic abnormalities including central obesity, hyperglycemia, dyslipidemia and hypertension. Although heritability is suggested to be a substantial contributor to MetS [20], the genetic basis of this complex phenotype is not yet clear. Several attempts have also been made towards deciphering the role of genetic variants in this syndrome based on candidate gene association studies [21–27].
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This is the first study to identify the KL-VS variant of Klotho to be associated with MetS. The KL-VS variant is functional and has been reproducibly associated with longevity [5,28]. In the present study, carriers of the VV genotype of the KL-VS variant were found to be at increased risk of MetS which could be related to their susceptibility to high blood pressure, high fasting glucose and trend towards hypertriglyceridemia. Along with these three markers of MetS, we could also establish an association of the variant with insulin resistance. Though assessment of insulin resistance is not included in the NCEP definition of MetS, it has been considered as one of the key underlying causes [29]. Hence, pathophysiologically, our findings indicate that Klotho has an impact on multiple aspects of MetS including blood pressure, lipid metabolism as well as insulin resistance. Klotho plays a role in the regulation of vascular tone through a homeostatic interplay between NO and the renin–angiotensin system [30]. This might explain the observed association of the Klotho with high blood pressure. A direct role of Klotho in lipid metabolism is not known. However, Klotho has been demonstrated to be a target gene for PPARγ, a lipid sensor nuclear receptor that regulates lipid metabolism through gene transcription [31,32]. Therefore, it can be speculated that the up-regulation of Klotho expression by PPARγ may contribute to its effects on lipid metabolism. We hypothesize that Klotho's link with PPARγ might underlie the association of its genetic variants with triglyceride levels. The observed association of the variant with MetS/its components might also be due to linkage disequilibrium of the KL-VS variant with another causative change in a gene near the locus. There have been contradictory views regarding the experimental evidences of Klotho's involvement in insulin resistance. Previous experimental reports indicate that Klotho causes insulin resistance by inhibiting insulin signaling pathways in rat hepatocytes and myocytes, where it inhibits insulin- and IGF-1-induced glucose transport and tyrosine phosphorylation of the insulin receptor (IR) and IRS-1 [9]. Further, Klotho-deficient mice exhibit increased insulin sensitivity, adipose tissue atrophy, and hypoglycemia, whereas Klotho transgenic mice with extended longevity are insulin resistant. However, in a recent study, Lorenzi and colleagues found no direct involvement of Klotho in insulin resistance and suggested the previous finding of Kurosu and coworkers to be a purification artifact [11]. Given the fact that insulin resistance causes diabetes, a major cause of mortality, coexistence of longevity and insulin resistance in Klotho transgenic mice is surprising. Moreover, all these experimental evidences are based on animal models and hence the results cannot be directly extrapolated to humans. In the present study, we found an association of the VV genotype of the KL-VS variant with insulin resistance. The same variant has been found to be underrepresented in elderly individuals compared to newborns (three independent populations: p =0.05–0.08; combined analysis (n= 2416): P= 0.0023) [5]. This indicates that the aging suppressing action of Klotho might involve increased insulin sensitivity rather than resistance. Our finding is consistent with the notion that enhanced insulin sensitivity is a characteristic of extended longevity [33]. Functional link with PPAR-γ could also explain the observed association between the Klotho KL-VS variant and insulin resistance. In contrast to our findings, Freathy et al., found no association of Klotho KL-VS variant with insulin resistance and type 2 diabetes mellitus in a large Caucasian cohort [34]. With regard to the C1818T variant, a borderline association could be established between the variant and MetS, p = 0.063, where the TT genotype was found to be protective compared to CC + TT genotype carriers. However, no significant association was observed between the C1818T genotypes and the components of MetS or insulin resistance. In summary, we report significant and borderline associations of the KL-VS and C1818T variants of Klotho, respectively, with MetS in South Indian population. Larger studies in other ethnic populations are warranted to determine the role of these gene variants in the etiology of MetS.
Acknowledgement The authors gratefully acknowledge the Indian Council of Medical Research, New Delhi, India, for providing financial assistance to carry out this study.
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Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Association of exonic variants of Klotho with metabolic syndrome in Asian Indians Vijaya Majumdar, Rita Christopher ⁎ Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences, Bangalore-560029, India
a r t i c l e
i n f o
Article history: Received 10 December 2010 Received in revised form 25 February 2011 Accepted 26 February 2011 Available online 3 March 2011 Keywords: Metabolic syndrome Insulin resistance Asian Indians Klotho Klotho KL-VS Klotho C1818T
a b s t r a c t Background: Klotho, an anti-aging gene, is a functional candidate for metabolic syndrome. We conducted a cross-sectional study to evaluate the association of the genetic variants of Klotho with metabolic syndrome and surrogates of insulin resistance in Asian Indians. Methods: We recruited 428 clinically normal subjects for the study. Genotyping was done by polymerase chain reaction and restriction fragment length polymorphism. Results: Significant and borderline associations of the KL-VS (OR = 15.88 [95%CI, 2.56–98.70], p = 0.003) and C1818T (OR = 0.28 [95%CI, 0.07–1.07], p = 0.063) variants of the Klotho gene, respectively, were observed with metabolic syndrome. The association of the KL-VS variant with metabolic syndrome could be linked to its observed influence on high blood glucose (OR = 6.92 [95% CI = 1.75–27.44], p = 0.006), high blood pressure (OR = 5.21 [95%CI = 1.00–38.43], p = 0.046), insulin resistance (OR = 3.59, [95%CI = 1.01–12.79], p = 0.048) and trend towards its association with hypertriglyceridemia (OR = 3.69 [95%CI = 0.92–14.77], p = 0.065). Conclusions: The genetic variants of Klotho might predict risk for metabolic syndrome and insulin resistance in Asian Indians. However, larger studies in other ethnic populations are warranted to determine the role of these gene variants in the etiology of metabolic syndrome. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Klotho is an aging suppressor gene that extends life span when over-expressed and accelerates aging when disrupted. Klotho-deficient mice exhibit a syndrome resembling accelerated human aging in conjunction with extensive arteriosclerosis [1]. Klotho has been demonstrated to ameliorate vascular endothelial dysfunction, increase nitric oxide production, reduce elevated blood pressure, and prevent medial hypertrophy and perivascular fibrosis in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of metabolic syndrome (MetS) [2,3]. Further, renal expression of Klotho is reduced in OLETF rats and its expression is found to be augmented by treatment with troglitazone, an insulin sensitizer and agonist of PPARγ [2]. A role of Klotho in in-vitro vascular inflammation has also been described by Maekawa and co-workers who demonstrated that Klotho reduces the
Abbreviations: OLETF, Otsuka Long–Evans Tokushima Fatty; PPAR, Peroxisome proliferator-activated receptor; TNF, tumor necrosis factor; HDL, high density lipoprotein; SBP, systolic blood pressure; DBP, diastolic blood pressure; MetS, metabolic syndrome; LD, linkage disequilibrium; CAD, coronary artery disease; WC, waist circumference; HOMA, homeostasis model assessment of insulin resistance; NCEP/ ATP III, National Cholesterol Education Program/Adult Treatment Panel III; IR, insulin resistance. ⁎ Corresponding author at: Department of Neurochemistry, National Institute of Mental Health and Neuro Sciences (NIMHANS), Hosur Road, Bangalore 560029, India. Tel.: + 91 80 26995162; fax: + 91 80 26564830. E-mail addresses: [email protected], [email protected] (R. Christopher). 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.02.034
expression of adhesion molecules as well as recovers the suppression of endothelial nitric oxide synthase induced by the immunomodulator, tumor necrosis factor (TNFα) [4]. Polymorphisms in the human Klotho gene are associated with longevity, various cardiovascular events like stroke, coronary artery disease as well as the cardiovascular risk factors like reduced high density lipoprotein-cholesterol (HDL) levels and elevated systolic blood pressure (SBP) [5–8]. All these observations are suggestive of the candidature of Klotho in metabolic syndrome (MetS) which is a combination of risk determinants of atherogenic cardiovascular disease. Interestingly, Klotho has also been shown to be involved in the manifestation of insulin resistance [9] which appears to be at variance with its beneficial role in longevity and protection against vascular pathologies. Unger had proposed that Klotho-induced insulin resistance prevents cellular lipid overload by reducing insulin-stimulated availability of the lipogenic substrate, glucose [10]. The reduction in intracellular lipid content could raise the apoptotic threshold and extend the life of the cells. On the contrary, a recent report by Lorenzi et al. presents evidence against a direct involvement of Klotho in insulin signaling [11]. In view of these contradictory findings we undertook a study to test the association of Klotho gene variants with MetS and the surrogates of insulin resistance. The KL-VS variant of Klotho consists of six sequence variants in perfect linkage disequilibrium (LD), two of which result in amino acid substitutions, F352V and C370S [5]. Due to presence of complete LD across the SNPs, the single variant, F352V, has been used to tag the KL-VS haplotype. The presence of phenylalanine at the position 352 in the
V. Majumdar, R. Christopher / Clinica Chimica Acta 412 (2011) 1116–1121 Table 1 Clinical characteristics stratified by presence or absence of MetS. Clinical characteristics
All subjects (n = 428)
Yes (n = 127)
MetS No (n = 301)
p value
Age (years) Sex (M/F) Smokers, n (%) Hypertension, n (%) Diabetes, n (%) Dyslipidemia, n (%)
39.7 ± 12.7 235/193 54 (12.7) 100 (23.4) 34 (7.9) 240 (56.1)
47.2 ± 8.3 60/67 25 (19.7) 60 (47.2) 27 (21.3) 95 (74.8)
36.4 ± 13.0 175/126 29 (9.6) 40 (13.3) 7 (2.3) 145 (48.2)
b 0.001 0.043 0.006 b 0.001 b 0.001 b 0.001
Data are mean ± SD unless otherwise indicated. Abbreviations: MetS, metabolic syndrome.
human Klotho gene is highly conserved and its substitution to valine has been demonstrated to alter the in vitro excretion and activity of the protein. Arking and co-workers reported a positive association between the functional KL-VS variant of Klotho and early-onset occult coronary artery disease (CAD) [6]. They also found positive association of Klotho's KL-VS variant with incidence of stroke in a cohort ascertained for the analysis of longevity [7]. Another widely studied variant of Klotho gene is the synonymous C1818T variant (rs564481) located in the fourth exon. Being a silent mutation the variant is not likely to be functionally relevant. However, there are reports which demonstrate the clinical relevance of the variant which has been studied for association with cardiovascular risk factors, fasting glucose, lipid levels and blood pressure in Asian population [12,13]. An association between CAD and the C1818T variant has been reported in Koreans [14].
2. Subjects This study was approved by the ethics committee of our Institute and conformed to the principals outlined in the declaration of Helsinki. Written informed consent was obtained from all the participants. A total of 428 unrelated subjects with self-reported South Indian ancestry, were recruited between the period of August 2007 and October 2009. Study participants were asymptomatic and reported no major illness. They were all employed and were attending to their regular duties at the time of recruitment. Demographic and anthropometric analyses included the following variables: age, sex, smoking status, height, weight and waist circumference (WC) and blood pressure. Clinical laboratory measurements included serum levels of total cholesterol, LDL-cholesterol, HDLcholesterol, triglycerides, and glucose. Serum glucose, triglycerides, total cholesterol, and HDL-cholesterol concentrations were determined enzymatically using commercially available kits and auto analyzer (Olympus AU640) and plasma LDL-cholesterol concentration was calculated using Friedewald's formula [15]. Fasting serum insulin was measured by ELISA
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with a commercial kit [Human Insulin specific, Calbiotech, Inc, San Diego, CA]. The intra- and inter-assay CVs were 5.5% and 7.8%, respectively. Insulin sensitivity was assessed with fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR). Homeostasis model assessment estimates of insulin resistance (HOMA-IR) and β-cell function (HOMA-B) were calculated using fasting glucose and insulin measurements [16]. Subjects were classified as having metabolic syndrome according to the NCEP/ATP III (National Cholesterol Education Program/ Adult Treatment Panel III) definition [blood pressure of ≥130/85 mm Hg; triglycerides ≥150 mg/dl (1.69 mmol/l); HDL-cholesterol b40 mg/dl (1.03 mmol/l) and b50 mg/dl (1.29 mmol/l) in men and women, respectively; and fasting blood glucose ≥110 mg/dl (6.05 mmol/l)] with modified WC cut off values (males N90 cm, females N80 cm) appropriate for Indians [17,18]. Presence of three or more parameters was considered for defining MetS positive subjects. Insulin resistance was defined as HOMA-IR N4.65 [19]. The diagnosis of hypertension was based on seated blood pressure above a defined cut-off value [140 mm Hg for systolic blood pressure (SBP); and 90 mm Hg for diastolic blood pressure (DBP)] and/or, if the subjects were on antihypertensive therapies. Diabetes mellitus was defined as fasting blood glucose of ≥7 mmol/l or 126 mg/dl, the use of insulin or oral hypoglycemic agents. Dyslipidemia was defined as elevated total cholesterol N6.2 mmol/l (240 mg/dl), and/or decreased HDL-cholesterol b0.9 mmol/l (35 mg/dl), and/or increased triglycerides N2.3 mmol/l (200 mg/dl). The cut-offs applied are based on cut-offs for high risk for cardiovascular disorders in the NCEP guidelines [17]. 3. Materials and methods 3.1. Klotho KL-VS genotyping The KL-VS (rs9536314) variant was typed using a published method [5]. Sample DNA was amplified by PCR; sense primer 5′-GCC AAA GTC TGG CAT CTC TA-3′; antisense primer 5′-TTC CAT GAT GAA CTT TTT GAG G-3′, under the following conditions: 95 °C for 5 min, followed by 32 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min, followed by a 10 min 72 °C final extension. PCR products were then digested with MaeIII (Roche Molecular, Pleasanton, CA) at 55 °C for 4 h. The F352 allele has a single MaeIII restriction site in the amplicon; substitution of F by V at the position 352 generates another MaeIII site. Thus the amplicon from wild type homozygotes (352FF) was digested into 2 bands (319 bp and 186 bp), whereas heterozygotes (352FV) had 3 bands (319 bp, 265 bp, and 186 bp), and the 352VV homozygotes had 2 bands (265 bp and 186 bp). 3.2. Klotho C1818T genotyping C1818T variant (rs564481) was also genotyped using PCR-restriction fragment length methodology. DNA containing the polymorphic
Table 2 Clinical and biochemical parameters by Klotho KL-VS and C1818T genotypes in the MetS group. Variable
FF (n = 91)
FV (n = 27)
VV (n = 9)
p value
CC (n = 71)
CT (n = 51)
TT (n = 4)
P value
Sex (M/F) Age, years BMI, kg/m2 WC, cm SBP, mm Hg DBP, mm Hg TC, mmol/l TG, mmol/l HDL, mmol/l Fasting glucose, mmol/l Insulin, μU/ml HOMA-IR
43/48 39.4 ± 12.6 29.2 ± 6.1 94.5 ± 8.3 126.9 ± 18.5 85.9 ± 12.0 5.2 ± 1.2 2.5 ± 1.1 0.99 ± 0.22 6.0 ± 3.0 16.0 ± 14.5 4.0 ± 3.9
13/14 40.5 ± 13.2 28.3 ± 4.1 94.9 ± 9.5 131.3 ± 8.8 87.4 ± 9.0 5.8 ± 0.7 2.3 ± 1.4 0.98 ± 0.19 5.7 ± 2.6 14.4 ± 9.7 3.6 ± 3.0
4/5 40.2 ± 10.7 26.5 ± 2.7 93.7 ± 7.6 141.5 ± 20.4 89.1 ± 11.5 5.3 ± 1.1 2.3 ± 0.8 1.00 ± 0.23 6.0 ± 1.4 24.5 ± 18.9 6.8 ± 6.8
NS NS NS NS *0.025 NS NS NS NS NS NS NS
32/39 40.0 ± 13.2 28.2 ± 4.7 87.9 ± 10.7 130.8 ± 13.1 87.7 ± 11.9 5.0 ± 1.0 2.4 ± 1.2 0.98 ± 0.22 6.0 ± 3.0 14.8 ± 10.4 3.9 ± 3.9
26/25 38.9 ± 12.1 27.9 ± 4.0 88.5 ± 9.6 126.9 ± 11.3 85.2 ± 11.0 5.0 ± 1.1 2.5 ± 1.2 1.00 ± 0.21 6.1 ± 2.8 18.7 ± 18.1 4.5 ± 4.4
1/3 43.7 ± 11.4 28.6 ± 4.3 91.8 ± 8.5 130.0 ± 11.5 82.5 ± 5.0 5.3 ± 1.2 1.7 ± 0.5 1.03 ± 0.24 4.5 ± 0.5 7.3 ± 3.7 1.4 ± 0.8
NS NS NS NS NS NS NS NS NS NS NS NS
Values represent mean ± SD. Abbreviations: MetS, metabolic syndrome; BMI, body mass index; WC, waist circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; TC, total cholesterol; HDL, high-density lipoprotein-cholesterol; TG, triglycerides; HOMA-IR, Homeostasis model assessment estimates of insulin resistance.
205 138 18 140 101 8
41 15 4 NS 31 26 2 NS 178 67 8
264 97 7
+ ve (N = 60) − ve (N = 253)
127 45 3 NS 96 63 12 NS 107 67 8
184 59 2
The differences in the anthropometric and biochemical parameters were compared using t-test or Mann–Whitney test. Violation of Hardy– Weinberg equilibrium (HWE) was tested by Pearson's χ2 test, used to analyze the associations between categorical variables. Association between Klotho geotypes and MetS were analyzed using different genetic models viz., general effects, dominant and recessive. For the analysis of the components of MetS viz, high blood glucose, hypertriglyceridemia etc., recessive models of inheritance were assumed for both the genetic variants. Multivariate logistic regression analysis of risk factors was also performed, with odds ratios (ORs) (95% CIs) shown. A 2-sided P b 0.05 was considered statistically significant. Linkage disequilibrium was estimated between the 2 genetic variants in our study subjects using Haploview software, (http://www.broad.mit.edu/mpg/haploview/contact. php).
158 113 13 165 113 16 p value C1818T CC CT TT p value
91 27 9 0.0003 71 51 4 NS FF FV VV KL-VS
4. Results
p value; Chi-square/Fisher's exact test. Abbreviations: MetS, metabolic syndrome; BP, blood pressure; HDL, high density lipoprotein-cholesterol.
209 147 17
138 93 14
172 76 3
98 34 5 NS 78 51 7 NS 214 85 2
207 78 6
270 104 6 0.0006 27 17 3 NS
35 8 5
133 36 8 0.011 98 71 6 NS
121 53 9 0.011 129 97 12 NS
+ ve (N = 175) + ve (N = 183) − ve (N = 251) + ve (N = 177) − ve (N = 48) + ve (N = 380) + ve (N = 137)
− ve (N = 291)
site was amplified using the following primers (sense primer 5′-TGT CTC AGT TTA CCG ACC TGA ATG T-3′ and anti-sense 5′-ATT CAT CGT TAT CCA AAG CTT GAC G-3′) and PCR amplification conditions of 95 °C for 5 min, followed by 32 cycles of 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 1 min, followed by a 10 min 72 °C final extension. PCR products were restriction digested with Mph1103I (Fermentas) at 37 °C for 16 h overnight. The DNA segment from TT homozygotes was digested into 272 and 180 bps. For the undigested CC wild homozygotes, a single band of 452 bp was observed. Genotyping failed for 8 samples thereby there was a reduction in the sample size from the original dataset. We dealt with mis-genotyping errors by retyping 10% of our samples, with identical results.
3.3. Statistical analysis
− ve (N = 301) + ve (N = 127)
Hyperglycemia Abdominal obesity Genotypes MetS Klotho
Table 3 Distribution of Klotho KL-VS and C1818T genotypes across MetS, components of MetS and insulin resistance.
Hypertriglyceridemia
High BP
− ve (N = 245)
Low HDL
− ve (N = 368)
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Insulin resistance
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Metabolic syndrome was present in 29.4% of all the subjects and it was more prevalent in females (34.4%) compared to males (25.7%) (p = 0.043). There was a significant difference in the distribution of the clinical parameters between subjects with and without MetS (Table 1). Subjects with MetS were older (47.2 ± 8.3 vs. 36.4 ± 13.0 y; p b 0.001) and more frequent smokers (19.7 vs. 9.6%; p = 0.006) compared to those without MetS. Frequency of hypertension, diabetes and dyslipidemia (p b 0.001) were also high in subjects with MetS compared to those without MetS. Stratification of the clinical and biochemical parameters by the genotypes of the Klotho variants in MetS positive subjects is presented in Table 2. There was a significant difference in the levels of SBP among the individuals with KL-VS genotypes (p = 0.025), subjects with VV genotype had elevated SBP compared to those with FF and FV genotypes. No significant association could be found for the other parameters with the variant. C1818T variant was also not found to have any influence on any of the clinical or biochemical variables (Table 2).
4.1. KL-VS and MetS The genotype distribution of the KL-VS variant in the total population was FF 305 (71.3%), FV 112 (26.1%) and VV 11 (2.6%) and was in Hardy–Weinberg equilibrium (p = 0.997). There was a significant difference in the distribution of the KL-VS genotypes between MetS positive and negative subjects (p = 0.0003, Table 3). When examined using a recessive model, individuals with VV genotype were found to have ~16 fold higher risk of MetS than the F allele carriers (OR = 15.88 [95%CI, 2.56–98.70], p = 0.003) (Table 4). Further, stratification by sex did not indicate any gender specific influence of the variant on MetS (Table 4).
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Table 4 Estimate of the effects of the Klotho variants on MetS risk modeled with logistic regression. Combined
Men
Women
Variant
Adjusted OR (95% CI)
p value
Adjusted OR (95% CI)
p value
Adjusted OR (95% CI)
p value
KL-VS FF FV VV
1.00 [ref] 1.44 (0.77–2.71) 14.00 (2.24–87.30)
NS 0.005
1.00 [ref] 1.56 (0.75–3.28) 5.96 (0.99–35.65)
NS 0.050
1.00 [ref] 1.31 (0.56–3.06) 9.03 (0.86–94.52)
NS NS
C1818T CC CT TT
1.00[ref] 1.20 (0.68–2.13) 0.25 (0.05–1.20)
NS NS
1.00[ref] 1.26 (0.67–2.38) 0.44 (0.05–3.70)
NS NS
1.00[ref] 1.28 (0.61–2.71) 0.32 (0.07–1.47)
NS NS
Recessive models KL-VS ( VV vs. FF + FV) C1818T ( TT vs. CC + CT)
15.88 (2.56–98.70) 0.28 (0.07–1.07)
0.003 0.063
6.90 (1.11–98.70) 0.48 (0.05–4.41)
0.038 NS
11.47 (1.17–112.55) 0.28 (0.06–1.25)
0.036 NS
Dominant models KL-VS ( FV + VV vs. FF) C1818T (CT + TT vs. CC)
1.01 (0.57–1.80) 1.06 (0.62–1.79)
NS NS
0.83 (0.42–1.64) 1.16 (0.62–2.17)
NS NS
1.15 (0.53–2.53) 1.02(0.50–2.06)
NS NS
p values were adjusted on age, sex, and smoking, in the combined sample or on age and smoking in men and women separately.
4.2. KL-VS and components of MetS There was no significant association found between the KL-VS genotype and abdominal obesity (p = 0.590), or depression in HDL levels (p = 0.626) (Table 3). KL-VS variant was found to be associated with high fasting plasma glucose levels (p= 0.0006) (Table 3). Multivariate logistic regression analysis revealed a significant association between the KL-VS variant and high fasting plasma glucose levels (OR =6.92 [95% CI= 1.75–27.44], p =0.006); the other predictors being age (OR= 1.1, 95% CI =1.03–1.10, p =0.001) and triglycerides (OR =1.01, 95% CI= 1.00–1.01, p =0.006). However, individuals with VV genotype were not found to have significantly different mean fasting glucose levels compared to individuals with FF and FV genotypes (p = 0.937 and 0.836, respectively). After dichotomization, a significant association of the KL-VS genotypes with high BP (p = 0.011) was also observed (Table 3). Furthermore, multivariate logistic regression analysis indicated that the risk factors for high BP were the VV genotype (recessive model) (OR = 5.21 [95%CI = 1.00–38.43], p = 0.046), age (OR = 1.06 [95% CI = 1.03–1.08], p = 0.0001), waist circumference (OR = 1.05 [95% CI = 1.02–1.08], p = 0.002) and total cholesterol (OR = 1.01 [95% CI = 1.00–1.01], p = 0.009). Moreover, KL-VS variant was found to be associated with SBP (continuous variable), p = 0.025 (Table 2). Genotypes of KL-VS variant were significantly differentially distributed between individuals with and without hypertriglyceridemia; p = 0.011 (Table 3). Multivariate logistic regression analysis revealed a trend of association between the VV genotype and hypertriglyceridemia (OR = 3.69 [95%CI = 0.92–14.77], p = 0.065). 4.3. KL-VS variant and surrogates of IR Dichotomization based on HOMA cut off point of 4.65, revealed a borderline association of the KL-VS variant with IR (p = 0.096) (Table 3). Further evaluation using a recessive model of inheritance indicated a significant association (p = 0.031) of the variant with this trait. Multivariate logistic regression analysis indicated a significant association of IR with the VV genotype of the KL-VS variant (OR = 3.59, [95%CI = 1.01–12.79], p = 0.048). However, there was no association found between KL-VS variant and the continuous variables like HOMA-IR, fasting glucose and insulin (Table 2). 4.4. C1818T and MetS The genotype distribution of the C1818T variant was CC 236 (56.2%), CT 164 (39.0%) and TT 20 (4.8%), and complied with Hardy–
Weinberg equilibrium (p = 0.630). The genotypic distribution of the variant was not significantly different between MetS-positive and negative individuals (p = 0.592; Table 3). In general, no significant differences between MetS-positive cases and controls were observed for CT vs. CC (OR-1.20 [95% CI, 0.68–2.13], p = 0.525) as well as for TT vs. CC comparison (OR-0.25 [95% CI, 0.05–1.20], p = 0.083). We could find a borderline significant association of the variant with MetS (p b 0.10), wherein the TT genotype carriers were found to be protected compared to CC + CT genotype carriers against risk of MetS (OR = 0.28 [95% CI, 0.07–1.07], p = 0.063 compared to the C allele carriers, Table 4. However, analysis under dominant model of inheritance did not indicate any significant effect of the C1818T variant on MetS risk (OR = 1.06 [95% CI, 0.62–1.79], p = 0.842). Stratification by sex did not indicate any gender specific influence of the variant on MetS. 4.5. C1818T and components of MetS There was no significant association between the C1818T genotypes and the components of MetS viz., abdominal obesity; p = 0.889, high BP; p = 0.641, high blood glucose; p = 0.811, high triglycerides; p = 0.522 and low HDL levels; p = 0.179 (Table 3). 4.6. C1818T variant and surrogates of IR Dichotomization based on HOMA cut off point of 4.65, revealed no association of the C1818T variant genotypes with IR (p = 0.645) (Table 3). The variant was also not associated with the continuous variables like HOMA-IR, fasting glucose and insulin (Table 2). 4.7. Linkage Disequilibrium (LD) analysis Haploview was used to look for LD among the two studied polymorphisms but no significant LD was observed between the polymorphisms (D′ = 0.298; LOD = 0.14 and r2 = 0.005). 5. Discussion Metabolic syndrome is a common clinical phenotype presenting as a cluster of metabolic abnormalities including central obesity, hyperglycemia, dyslipidemia and hypertension. Although heritability is suggested to be a substantial contributor to MetS [20], the genetic basis of this complex phenotype is not yet clear. Several attempts have also been made towards deciphering the role of genetic variants in this syndrome based on candidate gene association studies [21–27].
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This is the first study to identify the KL-VS variant of Klotho to be associated with MetS. The KL-VS variant is functional and has been reproducibly associated with longevity [5,28]. In the present study, carriers of the VV genotype of the KL-VS variant were found to be at increased risk of MetS which could be related to their susceptibility to high blood pressure, high fasting glucose and trend towards hypertriglyceridemia. Along with these three markers of MetS, we could also establish an association of the variant with insulin resistance. Though assessment of insulin resistance is not included in the NCEP definition of MetS, it has been considered as one of the key underlying causes [29]. Hence, pathophysiologically, our findings indicate that Klotho has an impact on multiple aspects of MetS including blood pressure, lipid metabolism as well as insulin resistance. Klotho plays a role in the regulation of vascular tone through a homeostatic interplay between NO and the renin–angiotensin system [30]. This might explain the observed association of the Klotho with high blood pressure. A direct role of Klotho in lipid metabolism is not known. However, Klotho has been demonstrated to be a target gene for PPARγ, a lipid sensor nuclear receptor that regulates lipid metabolism through gene transcription [31,32]. Therefore, it can be speculated that the up-regulation of Klotho expression by PPARγ may contribute to its effects on lipid metabolism. We hypothesize that Klotho's link with PPARγ might underlie the association of its genetic variants with triglyceride levels. The observed association of the variant with MetS/its components might also be due to linkage disequilibrium of the KL-VS variant with another causative change in a gene near the locus. There have been contradictory views regarding the experimental evidences of Klotho's involvement in insulin resistance. Previous experimental reports indicate that Klotho causes insulin resistance by inhibiting insulin signaling pathways in rat hepatocytes and myocytes, where it inhibits insulin- and IGF-1-induced glucose transport and tyrosine phosphorylation of the insulin receptor (IR) and IRS-1 [9]. Further, Klotho-deficient mice exhibit increased insulin sensitivity, adipose tissue atrophy, and hypoglycemia, whereas Klotho transgenic mice with extended longevity are insulin resistant. However, in a recent study, Lorenzi and colleagues found no direct involvement of Klotho in insulin resistance and suggested the previous finding of Kurosu and coworkers to be a purification artifact [11]. Given the fact that insulin resistance causes diabetes, a major cause of mortality, coexistence of longevity and insulin resistance in Klotho transgenic mice is surprising. Moreover, all these experimental evidences are based on animal models and hence the results cannot be directly extrapolated to humans. In the present study, we found an association of the VV genotype of the KL-VS variant with insulin resistance. The same variant has been found to be underrepresented in elderly individuals compared to newborns (three independent populations: p =0.05–0.08; combined analysis (n= 2416): P= 0.0023) [5]. This indicates that the aging suppressing action of Klotho might involve increased insulin sensitivity rather than resistance. Our finding is consistent with the notion that enhanced insulin sensitivity is a characteristic of extended longevity [33]. Functional link with PPAR-γ could also explain the observed association between the Klotho KL-VS variant and insulin resistance. In contrast to our findings, Freathy et al., found no association of Klotho KL-VS variant with insulin resistance and type 2 diabetes mellitus in a large Caucasian cohort [34]. With regard to the C1818T variant, a borderline association could be established between the variant and MetS, p = 0.063, where the TT genotype was found to be protective compared to CC + TT genotype carriers. However, no significant association was observed between the C1818T genotypes and the components of MetS or insulin resistance. In summary, we report significant and borderline associations of the KL-VS and C1818T variants of Klotho, respectively, with MetS in South Indian population. Larger studies in other ethnic populations are warranted to determine the role of these gene variants in the etiology of MetS.
Acknowledgement The authors gratefully acknowledge the Indian Council of Medical Research, New Delhi, India, for providing financial assistance to carry out this study.
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