The IL6 Gene G174C Polymorphism Related to ... - Wiley Online Library

Brief Genetic Analysis

The IL-6 Gene G-174C Polymorphism Related to Health Indices in Greek Primary School Children George V.Z. Dedoussis,* Yannis Manios,* Despoina M. Choumerianou,* Nikos Yiannakouris,† Demosthenes B. Panagiotakos,* Katerina Skenderi,* and Antonis Zampelas*

Abstract DEDOUSSIS, GEORGE V.Z., YANNIS MANIOS, DESPOINA M. CHOUMERIANOU, NIKOS YIANNAKOURIS, DEMOSTHENES B. PANAGIOTAKOS, KATERINA SKENDERI, AND ANTONIS ZAMPELAS. The IL-6 gene G-174C polymorphism related to health indices in Greek primary school children. Obes Res. 2004; 12:1037–1041. Interleukin-6 (IL-6) is a pleiotropic cytokine expressed in many tissues. A polymorphism in the IL-6 gene, associated with differences in IL-6 transcription rate, has been recently described. Subjects with the ⫺174GG genotype are prone to lipid abnormalities. We investigated the effect of the G-174C IL-6 polymorphism on health indices and lipid values of 184 Greek primary school children. The genotype distribution of the polymorphism was 37.5% for GG and 52.2% and 10.3% for GC and CC, respectively. No differences were observed between genotype distribution and gender (p ⫽ 0.37). Boys homozygous for the G allele showed higher triglyceride levels than boys carrying the C allele (86 ⫾ 28 vs. 74 ⫾ 20 mg/dL, p ⫽ 0.02) and lower mid-upper arm muscle circumference (17.46 ⫾ 1.86 vs. 18.91 ⫾ 2.53 cm, p ⫽ 0.013). In addition, girls homozygous for the G allele had higher values for suprailiac skinfolds compared with those bearing the C allele (21.28 ⫾ 12.56 vs. 17.09 ⫾ 13.36 mm, p ⫽ 0.06). These findings were confirmed by multiple linear regression analysis, after controlling for age, sex, BMI, energy and total fat intake, and

Received for review October 20, 2003. Accepted in final form May 17, 2004. The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Departments of *Nutrition and Dietetics and †Home Economics and Ecology, Harokopio University of Athens, Athens, Greece. Address correspondence to George Dedoussis, Laboratory of Molecular Genetics, Department of Nutrition and Dietetics, Harokopio University of Athens, 70 El. Venizelou str, 17671 Kallithea-Athens, Greece. E-mail: [email protected] Copyright © 2004 NAASO

weekly physical activity. From the results of the present study, we concluded that individuals homozygous for G allele on the IL-6 gene have higher values in some parameters associated with obesity. Key words: genotyping, inflammatory, interleukins, triglycerides, arm muscle circumference Interleukin-6 (IL-6)1 is a pleiotropic cytokine involved in the regulation of the acute phase response (1). It is produced by many different cell types, including immune cells and adipose tissue, that mediate inflammatory responses (2). Elevated serum concentrations of IL-6 have been detected in patients with various inflammatory states, including obesity or lipid abnormalities (3,4). It has been calculated that one-third of total circulating concentrations of IL-6 originates from adipose tissue (5). In IL-6 null mice, the lack of circulating IL-6 has been associated with obesity and low energy expenditure (6). A common polymorphism of the promoter sequence of the IL-6 gene exists, comprising a G-to-C substitution at position ⫺174 from the start of transcription (7). This variant is functional in vitro (7,8) expressed in HeLa cells, the G allele construct has greater promoter strength when stimulated with lipopolysaccharide and IL-1 to mimic the acute phase response. This has been confirmed in vivo, with the G allele in healthy volunteers associated with higher mean plasma levels of IL-6 (8). Childhood obesity is an epidemic of developed countries, and ⬃22 million children under 5 years of age are overweight across the world. Obesity in childhood and adolescence can work as a key predictor for obesity in adulthood (9). This is very important because morbidity and mortality in adults are increased in individuals who were overweight as adolescents. In various studies, gene polymorphisms have been associated with

1 Nonstandard abbreviations: IL-6, interleukin-6; WHR, waist-to-hip ratio; MMC, midupper arm muscle circumference; GLU, glucose; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; WPA, weekly physical activity; MET, metabolic equivalent.

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G-174C Promoter Polymorphism of the IL-6 Gene, Dedoussis et al.

Figure 1: z scores for height and weight in boys (left) and girls (right)

obesity in children (10,11). Given the findings that the ⫺174 G⬎C polymorphism has been correlated with lipid abnormalities and low energy expenditure, we aimed to investigate the effect of IL-6 genotypes on health indices associated with lipid abnormalities, energy expenditure, and obesity in a cohort of Greek primary school children. The frequency of the GG genotype was 38% (n ⫽ 68), the frequency of the GC genotype was 52% (n ⫽ 96), and the frequency of the CC genotype was 10% (n ⫽ 20). Genotype distribution did not differ from that expected by the HardyWeinberg equation, and there was no difference between sexes regarding the genotype frequencies (p ⫽ 0.37). Girls were taller than boys (p ⫽ 0.025), but both sexes had similar body and weight distribution (p ⫽ 0.21) (Figure 1). The subjects were divided into two groups on the basis of the IL-6 genotype. The individuals bearing the C allele (GC heterozygotes and CC homozygotes) were considered as one group against the GG homozygotes. Anthropometric and biochemical characteristics of boys and girls are shown in Table 1. Boys. Higher values for mid-upper arm muscle circumference (MMC), which is an indicator of skeletal muscle mass, were detected in boys with the C allele compared with the GG homozygotes (18.91 ⫾ 2.53 vs. 17.46 ⫾ 1.86 cm, respectively; p ⫽ 0.013). In addition, boys with the GG genotype had higher triglyceride (TG) levels compared with the GC/CC group (86.33 ⫾ 28.11 vs. 73.87 ⫾ 20.48 mg/dL, p ⫽ 0.03). No differences in respect to BMI, waist-to-hip ratio (WHR), high density lipoprotein-cholesterol (HDL-C), 1038


low density lipoprotein-cholesterol (LDL-C), triceps, biceps, subscapular, and sum of skinfolds were observed (Table 1). Moreover, using the Bonferroni correction, only the differences among MMCs reached statistical significance (at p ⬍ 0.05). Girls. Participants with the GG genotype were found to have higher values in suprailiac skinfolds compared with girls with the GC/CC genotype (21.18 ⫾ 12.56 vs. 17.09 ⫾ 13.36 mm, p ⫽ 0.05). Girl homozygotes for the G allele showed a tendency toward higher plasma glucose (GLU) levels compared with those bearing the C allele (94.18 ⫾ 10.37 vs. 90.78 ⫾ 8.7 mM, p ⫽ 0.084) (Table 1). Regarding the rest of biochemical indices, no differences were detected between the two groups. Using the Bonferroni correction, none of the differences reached statistical significance (at p ⬍ 0.05). Figure 2 illustrates the z scores for MMC in boys and suprailiac skinfolds in girls by IL-6 genotypes. Regression analysis showed that BMI (standardized ␤ coefficient ⫽ 0.53, p ⬍ 0.001) and IL-6 genotype (standardized ␤ coefficient ⫽ ⫺0.35, p ⫽ 0.001) were significantly associated with TG levels in boys, after adjusting for age, energy and total fat intake, and weekly physical activity (WPA) (adjusted R2 ⫽ 30%). Moreover, BMI (standardized ␤ coefficient ⫽ 0.25, p ⫽ 0.033) was associated with TG in girls, after adjusting for age, energy and total fat intake, and WPA (adjusted R2 ⫽ 2%). In addition, BMI (standardized ␤ coefficient ⫽ 0.39, p ⫽ 0.001) and the IL-6 genotype (standardized ␤ coefficient ⫽ 0.27, p ⫽ 0.17) were independently associated with MMC in boys, after adjusting for age, energy and total fat intake, and WPA (adjusted R2 ⫽ 26%). However, only BMI levels (standardized ␤ coefficient ⫽ 0.45, p ⫽ 0.001), but not IL-6 genotype (standardized ␤ coefficient ⫽ 0.12, p ⫽ 0.29), were independently associated with MMC in girls, after adjusting for age, energy and total fat intake, and WPA (adjusted R2 ⫽ 14%). Regarding suprailiac skinfolds, BMI (standardized ␤ coefficient ⫽ 0.75, p ⫽ 0.001), the IL-6 genotype (standardized ␤ coefficient ⫽ ⫺0.18, p ⫽ 0.17), and energy intake (standardized ␤ coefficient ⫽ ⫺0.39, p ⫽ 0.019) were found to be independent explanatory variables in boys, after adjusting for age, total fat intake, and WPA (adjusted R2 ⫽ 66%). When we focused on girls, only BMI levels (standardized ␤ coefficient ⫽ 0.41, p ⫽ 0.001) were independently associated with suprailiac skinfolds, after adjusting for the same set of covariates (adjusted R2 ⫽ 22%). The novel finding in this study is that the G-174C IL-6 genotype seems to be associated with obesity indices among prepubertal children. It has been previously shown that the polymorphism ⫺174 in the IL-6 gene is associated with lipid abnormalities (3). Many tissues, including adipose tissue, secrete IL-6, and IL-6 levels have been found to correlate with BMI (5) or BMI and fat mass in 12-year-old children (12). Contrary to the above, in the majority of our


Table 1. Anthropometric values and biochemical variables of the study subjects FEMALES

IL-6 genotype (n) Height (m) BMI (kg/m2) WHR GLU (mM) TG (mg/dL)* Total serum cholesterol (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Suprailiac skinfolds (mm)* Triceps skinfolds (mm) Biceps skinfold (mm)* Subscapular skinfold (mm)* Sum of skinfolds (mm)* MMC (cm)* Energy intake (kcal) Total fat intake (g) Saturated fat (g) MVPA (min)*

MALES

GG

GC/CC

38 1.48 ⫾ 0.7 20.8 ⫾ 3.3 0.78 ⫾ 0.07 94 ⫾ 10 91 ⫾ 28 192 ⫾ 36 46 ⫾ 8 127.7 ⫾ 30.9 21.1 ⫾ 12.5 20.0 ⫾ 9.9 12.3 ⫾ 5.7 13.1 ⫾ 7.5 66.7 ⫾ 32.5 18.1 ⫾ 2.9 2201 ⫾ 753 101 ⫾ 48 38 ⫾ 23 458 ⫾ 335

60 1.5 ⫾ 0.6 20.3 ⫾ 3.9 0.8 ⫾ 0.06 90 ⫾ 10 92 ⫾ 31 193 ⫾ 27 46 ⫾ 10 129.5 ⫾ 23.5 17.0 ⫾ 13.3 17.1 ⫾ 9.03 11.5 ⫾ 5.6 12.0 ⫾ 5.2 57.9 ⫾ 30.54 18.5 ⫾ 3.5 2296 ⫾ 760 2296 ⫾ 46 41 ⫾ 17 367 ⫾ 299

p

GG

GC/CC

p

NS NS NS 0.08 NS NS NS NS 0.05 NS NS NS NS NS NS NS NS NS

31 1.5 ⫾ 0.8 19.4 ⫾ 2.7 0.85 ⫾ 0.04 94 ⫾ 10 86 ⫾ 28.11 186 ⫾ 26 47 ⫾ 10 120.3 ⫾ 21.7 16.7 ⫾ 10.6 18.4 ⫾ 7.20 11.2 ⫾ 5.4 12.1 ⫾ 7.1 58.5 ⫾ 27.3 17.4 ⫾ 1.8 1731 ⫾ 509 83 ⫾ 33 33 ⫾ 14 145 ⫾ 182

55 1.5 ⫾ 0.8 20.7 ⫾ 3.7 0.84 ⫾ 0.09 92 ⫾ 11 74 ⫾ 21 188 ⫾ 27 46 ⫾ 6 126 ⫾ 23 18.8 ⫾ 16.0 16.7 ⫾ 8.7 11.5 ⫾ 6.1 11.61 ⫾ 7.0 58.8 ⫾ 35.2 18.9 ⫾ 2.5 1801 ⫾ 482 87 ⫾ 28 33 ⫾ 12 100 ⫾ 253

NS NS NS NS 0.03 NS NS NS NS NS NS NS NS 0.011 NS NS NS NS

NS, not significant; MVPA, moderate to vigorous physical activity. * Parameter was log transformed.

studied population that consisted of 184 schoolchildren, plasma IL-6 was not detectable, possibly due to the absence of inflammation at this age range and the limitations of the assay. Moreover, mean measurements of BMI were not different between the two IL-6 genotype groups in both boys and girls. This is, perhaps, due to the difference in the sample number. However, tissue-specific expression may be more important; thus, the measurement of circulating IL-6 level may not reflect biological significance at tissue level. The results of a recent study suggest that the G allele is associated with higher IL-6 secretion and that subjects with the G allele are prone to lipid abnormalities (3). In our study, the TG levels were elevated in boy homozygotes for the G allele, thus being in accordance with the above study; furthermore, the same group had lower MMC. Trying to estimate the impact of different variables on TG and MMC in boys, we applied the general linear regression model. Subjects’ TG and MMC measurements were used as dependent variables, whereas the IL-6 genotype (GG and GC/CC) was used as a fixed variable. Regarding the model applied for TG, BMI and genotype were found to affect TG, with p ⫽ 0.0001 and 0.002, respectively. Another model

was applied for MMC, where p values for BMI, WPA, and IL-6 genotype were p ⫽ 0.001, 0.04, and 0.01, respectively. In a recent study (13), the authors identified predictors of longitudinal changes in fat-free mass in very old community-living subjects. IL-6 played a catabolic role in women but not in men. In addition, it was shown that increased cellular production of IL-6 was a significant predictor of sarcopenia in women. It was also suggested that the risk associated with increased concentrations of circulating IL-6 was particularly high in women because of its additional contribution to osteoporosis. The higher MMC values were found in GC/CC boys; thus, in the lower IL-6-producing genotype, the values are in accordance with those data. No evidence is available in the literature supporting the association of IL-6 with muscle development in children, although it has been demonstrated (14) that inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-␬B. Additionally, for girl subjects, the general linear model revealed that the parameter affecting the suprailiac skinfolds is BMI and that there is a tendency for the IL-6 genotype, with p ⫽ 0.0001 and 0.06, respectively. Because at the age OBESITY RESEARCH Vol. 12 No. 7 July 2004

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Anthropometric Measurements Body weight was measured by a digital scale (Seca, Hanover, MD) with an accuracy of ⫾100 grams. BMI was calculated and used for children’s classification according to the previously proposed cut-off points (16). The height and weight varied within normal values for their age (Figure 1). Left triceps, biceps, subscapular, and suprailiac skinfold thicknesses were measured with a Lange skinfold caliper, while the subject was standing with the upper extremities relaxed at the sides of the body. Anthropometric measurements included WHR. The subjects’ waists were measured with a soft tape midway between the lowest rib and iliac crest. The hip circumference was measured at the widest part of the gluteal region. MMC was calculated using the following equation: Mid upper arm muscle circumference (C2) ⫽ mid upper arm circumference (C1) ⫺ (triceps skinfold).

Figure 2: z scores for MMC (upper figures) in boys and suprailiac skinfolds (lower figures) in girls by IL-6 genotypes.

of 12 years, factors such as smoking, alcohol intake, or anxiety, which induce inflammation, are absent, IL-6 genotype impact on the parameters discussed is evident. Sexdependent effect could be attributed to sexual dimorphism in fat distribution. Our results are in accordance with previous published results where it has been shown that pro-inflammatory genotypes are involved in the pathogenesis of obesity and common metabolic disorders (15). In future experiments, we aim to collect adipose tissue from girls of the different IL-6 genotype groups and compare IL-6 m-RNA levels. The limitation of the study was the lack of detailed registration of the pubertal status. However, because girls with menstrual cycle were excluded from the study and the z scores for height and weight do not indicate major discrepancies in physical development, it could be presumed that there were no major differences in pubertal stage.

Research Methods and Procedures Study Population The population of the study group was comprised of 184 healthy children (98 girls and 86 boys) 11 to 12 years old, registered in the sixth grade of 14 randomly selected elementary schools in the area of Volos, Greece. Girls with menstrual cycle were excluded from the study. Inclusion of subjects in the study was on a voluntary basis; before acceptance, children’s parents or guardians were fully informed about the objectives and methods of the study and signed an informed consent. 1040


Dietary Assessment Two-day 24-hour recalls, taken 2 weeks apart, were used to collect information regarding children’s dietary intake. All interviewers were rigorously trained to minimize interviewer effects. Food intake data were analyzed using the Nutritionist V diet analysis software (First Databank Inc., San Bruno, CA), extensively amended to include traditional Greek recipes, as described in Food Composition Tables and Composition of Greek Cooked Food and Dishes (17). Furthermore the database was enriched with nutritional analysis information on chemically analyzed foods, coming from either independent bodies or the industry and the fast food chains, which provide ⬎95% of fat in the diet of contemporary Greeks. Biochemical Measurements Early morning blood samples were taken from each child for biochemical screening tests after a 12-hour overnight fast. GLU levels, total serum cholesterol, TG, and HDL-C were measured in duplicate enzymatically. LDL-C was estimated with the aid of the Friedward formula (18). Plasma IL-6 concentrations were measured by using a standard commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Estimated Physical Activity WPA was estimated with the use of a questionnaire completed by the subjects. The subjects had to record all of the activities in which they were involved at school on a weekly basis. Activities were classified according to their energy expenditure, independent of body size, using the ratio between work metabolic rate and basal metabolic rate [metabolic equivalent (MET)]. The three categories are: sedentary (⬍4 METs), light (4 to 7 METs), and moderate to vigorous (moderate to vigorous physical activity ⬎ 7 METs) physical activity (19). The total weekly time devoted to moderate to vigorous phys-


ical activities has been used for the needs of the current study as an estimation of leisure time WPA. 3.

IL-6 ⴚ174 G>C Polymorphism Genotyping DNA was extracted from cellular blood components by the salting-out method. Polymerase chain reaction was used to detect the IL-6 Sfa NI restriction fragment-length polymorphism (7). Sfa NI restriction fragment-length polymorphism yielded DNA fragments of 140 and 58 bp (GG), 198/140/58 bp (G/C), and 198 bp for (CC), which were visualized using a 2.5% agarose gel stained with ethidium bromide. Genotypes were determined and confirmed by two experienced technicians blinded to all study data. Statistical Analysis Continuous variables are presented as the mean ⫾ SD. Age/sex-specific z scores for all body composition variables were calculated to provide information about how far and in what direction that variable deviated from its distribution’s mean, expressed in units of its distribution’s SD. Alleles were estimated by gene counting and presented as absolute and relative frequencies. The ␹2 criterion was used to compare the observed numbers of each genotype with those expected for a population in Hardy-Weinberg equilibrium. Plasma GLU, TGs, suprailiac, subscapular, and sum of skinfolds, and MMC values were not normally distributed; thus, data were logarithmically transformed. Differences between genotypes were assessed by ANOVA. Due to multiple significance tests performed and the inflation of type I error, the Bonferroni criterion was applied in all post hoc analyses. Moreover, relationships between the investigated variables and IL-6 genotypes were also assessed by sex-specific multiple linear regression analyses, after adjusting for age, BMI, kilocalories, total fat intake, and organized physical activity. All reported p values are based on two-sided tests and compared with a significance level of 5%. The SPSS/PC statistical program (version 11.05 for Windows; SPSS, Inc., Chicago, IL) was used for all of the statistical calculations.

Acknowledgment We would like to thank Kellogg’s company for funding the presented study.

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