(INS VNTR) Genotype and Metabolic Syndrome in Childhood Obesity

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The Journal of Clinical Endocrinology & Metabolism 91(11):4641– 4644 Copyright © 2006 by The Endocrine Society doi: 10.1210/jc.2005-2705

Insulin Gene Variable Number of Tandem Repeats (INS VNTR) Genotype and Metabolic Syndrome in Childhood Obesity Nicola Santoro, Grazia Cirillo, Alessandra Amato, Caterina Luongo, Paolo Raimondo, Antonietta D’Aniello, Laura Perrone, and Emanuele Miraglia del Giudice Department of Pediatrics, “F. Fede” Seconda Universita` degli Studi di Napoli, 80138 Napoli, Italy Objective: The insulin variable number of tandem repeats (VNTR) polymorphism located in the insulin gene promoter (INS VNTR) has been associated with insulin levels in obese children. Hyperinsulinemia is a pivotal factor in the development of metabolic syndrome, an emerging complication in childhood obesity. With the present study, we aimed to test the associations between INS VNTR and the metabolic syndrome in juvenile-onset obesity. Subjects and Methods: We screened for the INS VNTR in 320 obese children (152 girls; mean age, 11.2 ⫾ 2.3 yr; mean z-score body mass index, 3.6 ⫾ 1.1). All of them underwent a standard oral glucose tolerance test; baseline measurements included blood pressure and plasma lipid and fasting insulin levels. By using the data derived from the oral glucose tolerance test, the whole-body insulin sensitivity and the insulinogenic index were calculated.

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HE EPIDEMIC DIFFUSION of childhood obesity makes the study of early onset obesity and its complications almost a priority. Metabolic syndrome represents an emerging complication of childhood obesity. It has been defined by Reaven (1) as a link between insulin resistance, hypertension, dyslipidemia, impaired glucose tolerance (IGT), and other metabolic abnormalities associated with an increased risk of atherosclerotic cardiovascular diseases in adults. The constellation of metabolic abnormalities includes glucose intolerance (type 2 diabetes, IGT, or impaired fasting glycemia), insulin resistance, dyslipidemia, and hypertension. These conditions co-occur in an obese individual more often than might be expected by chance. Hyperinsulinemia consequent to the insulin resistance contributes to the pathogenesis of all these features. Pathogenesis of insulin resistance has been studied for many years. Free fatty acid accumulation in the liver, fat cells, and, particularly, skeletal muscle of obese patients, interfering with the normal insulin signaling, appears to be the primary determinant of insulin resistance (2). As a consequence of insulin resistance, the pancreas needs to increase its insulin production to maintain normal values of glycemia. Under these conditions, First Published Online July 25, 2006 Abbreviations: BMI, Body mass index; DBP, diastolic blood pressure; HDL, high-density lipoprotein; IGI, insulinogenic index; IGT, impaired glucose tolerance; OGTT, oral glucose tolerance test; SBP, systolic blood pressure; VNTR, variable number of tandem repeats; WBISI, wholebody insulin sensitivity index. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Results: The prevalence of metabolic syndrome reached 39%. No differences in INS VNTR genotype distribution were observed between obese subjects and 200 lean, age- and sex-matched children (P ⫽ 0.7). Among obese subjects, the prevalence of the metabolic syndrome was significantly higher in subjects with the I/I genotype (P ⫽ 0.006); the risk for developing the metabolic syndrome was significantly higher in subjects carrying the I/I genotype (odds ratio, 2.5; 95% confidence interval, 1.5–3.9). Obese subjects homozygous for the class I allele showed higher insulin levels and insulinogenic index but lower whole-body insulin sensitivity. Conclusions: We conclude that the I variant of the insulin promoter, when expressed in homozygotes, can predispose obese children to develop the metabolic syndrome. (J Clin Endocrinol Metab 91: 4641– 4644, 2006)

hyperinsulinemia turns the liver into a fat-producing factory with all of its negative downstream effects (2, 3). Recent studies have shown that the prevalence of metabolic syndrome among obese children oscillates from 20 –50%, according to the studied populations and the diagnostic criteria (4 – 6). The effect of insulin resistance in the development of early-onset metabolic syndrome has been described by Weiss et al. (6), who observed that the prevalence of metabolic syndrome among obese children increases with increasing insulin resistance. Considering this interplay, the possibility exists that genetic factors involved in insulin-glucose homeostasis and implicated in the regulation of insulin secretion may be crucial in predisposing obese children to develop the metabolic syndrome. Hypothetically, all the genes coding for the proteins involved in the regulation of insulin secretion and action, when mutated, may contribute to an increase in insulin resistance and to the development of metabolic syndrome. The gene variants may be localized in the coding region or in the regulatory regions, such as the promoter (7). Because of its role in the insulin secretion, a promising gene that may be involved in the development of metabolic syndrome is the gene codifying for insulin itself (INS). In particular, the insulin variable number of tandem repeats (VNTR) polymorphism located in the insulin gene promoter (INS VNTR) has been largely studied in cohorts of children and adolescents (8). Two classes of INS VNTR are observed in Caucasians, the short class I (26 – 63 repeats) and the long class III (141–209 repeats), whereas class II alleles are rare (9, 10). Studies on cadaver adult and fetal pancreas have demonstrated differ-

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J Clin Endocrinol Metab, November 2006, 91(11):4641– 4644

ences in steady-state levels of INS mRNA between class I and class III alleles, with lower transcript levels for the class III allele (11, 12). Association studies carried out on children agreed in defining the class I allele as a risk factor for hyperinsulinemia in obese but not in lean children and adolescents (12). With the present study, we aimed 1) to verify the prevalence of metabolic syndrome in a group of 320 Italian obese children and adolescents, 2) to test the genotype distribution of INS VNTR in this group of patients compared with nonobese controls, 3) to evaluate the effect of different INS VNTR genotypes on insulin levels and secretion, and 4) to verify whether the INS VNTR I/I genotype might predispose this kind of patient to develop metabolic syndrome. Subjects and Methods Three hundred twenty Caucasian obese children and adolescents, referred to our ward (childhood obesity service) since 1999, have been enrolled. Eligible subjects were between 2 and 16 yr of age and with a BMI exceeding the 97th percentile for their age and sex. Subjects with known presence of diabetes or using medications that alter blood pressure or glucose or lipid metabolism were excluded. Moreover, all subjects missing the complete data set needed for the analysis were excluded. The ethical committee of the Second University of Study of Naples approved the study. Informed consent was obtained from parents and, where appropriate, from children. Of the 320 subjects enrolled, 152 were girls. This sample was representative of the 1792 children referred to our ward from 1999 –2003; in fact, no differences in mean age, sex distribution, and pubertal stage were observed between the study sample and the sample of excluded subjects. Weight and height were measured, and BMI was calculated. The sd scores for BMI were calculated by using the LMS method (13). The population mean age was 11.2 ⫾ 2.3 yr; the mean z-score BMI was 3.6 ⫾ 1.1. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured three times while the subjects were seated, and the two last measurements were averaged for the analysis (14). Pubertal stage was assessed using Tanner criteria (15). Less than one third (28%) of children included in the study were pubertal (47 girls). Waist circumference was measured with an elastic tape, the subject being in a standing position; the tape was applied horizontally midway between the lowest rib margin and the iliac crest. The z-score of waist circumference was calculated according to references values (16). Triglycerides and high-density lipoprotein (HDL) were measured. To undergo an oral glucose tolerance test (OGTT) by assuming 1.75 g glucose/kg body weight, subjects were evaluated at 0800 h after an overnight fast; they consumed a diet containing at least 250 g carbohydrates/d for 3 d before the study and refrained from vigorous physical

Santoro et al. • INS VNTR and Metabolic Syndrome in Obese Children

activity; insulin and glucose levels were measured during the OGTT at baseline and later every 30 min for 120 min. To diagnose the metabolic syndrome, three of the following criteria needed to be present: BMI exceeding the 97th percentile, SBP and/or DBP exceeding the 95th percentile, triglyceride levels higher than 110 mg/dl, HDL cholesterol lower than 40 mg/dl for males and 50 mg/dl for females, and IGT (glucose level greater than 140 mg/dl but less than 200 mg/dl after 2 h from the beginning of the OGTT). Insulinogenic index (IGI), which reflects the early phase of insulin secretion, was calculated as the ratio of the 30-min insulin (in picomoles per liter) increment to the 30-min glucose concentration (in millimoles per liter). The degree of insulin sensitivity was assessed by using the whole-body insulin sensitivity index (WBISI). The composite WBISI is based on values of insulin and glucose obtained from the OGTT and the corresponding fasting values, as originally described (17). It represents a good estimate for clamp-derived insulin sensitivity, and it has been demonstrated to be correlated with intramyocellular lipid content (18). It was obtained according to the following formula: 10.000/公[(fasting insulin ⫻ fasting glycemia) ⫻ (mean insulin concentration during OGTT) ⫻ (mean glycemia during OGTT)] (18). All patients were genotyped for the INS VNTR. For genotyping the INS VNTR, the T/A polymorphism at the ⫺23 Hph1 locus was used; in fact, in Europeans, the Hph1 polymorphism T is in complete linkage disequilibrium with class III alleles of the neighboring VNTR, whereas the A polymorphism is in complete linkage disequilibrium with class I alleles (12). The following primers were used to perform the PCR: INSVNTR forward, 5⬘-TCCAGGACAGGCTGCATCAG-3⬘, and INS-VNTR reverse, 5⬘-AGCAATGGGCGGTTGGCTCA-3⬘. The amplified PCR products were digested with 1 U of the appropriate enzyme, and the digested samples were separated by electrophoresis through an agarose gel and visualized by staining with ethidium bromide. To test the INS VNTR allelic distribution, a group of controls, composed of 200 nonobese age- and sex-matched children, was recruited as previously described (19). The ␹2 test was used to verify whether the genotypes were in HardyWeinberg equilibrium and to compare allele frequencies between obese and nonobese subjects and metabolic syndrome prevalence between the different INS VNTR genotypes. A general linear model was used to evaluate the differences between groups of genotypes. When necessary, the variables were adjusted for age, sex, BMI, and pubertal stage. A logistic regression was generated to calculate the odds of developing the metabolic syndrome for subjects carrying the I/I genotype. Although raw values are shown, nonnormally distributed variables were log-transformed before performing the analysis. P values ⬍ 0.05 were considered statistically significant.

Results

One hundred twenty-eight patients (39%; 75 girls) met the criteria to make a diagnosis of metabolic syndrome, 15 sub-

TABLE 1. Clinical features of obese subjects according to the occurrence of metabolic syndrome Presence of metabolic syndrome (n ⫽ 128)

Absence of metabolic syndrome (n ⫽ 192)

P

10.9 ⫾ 2.0 32.7 ⫾ 12 3.8 ⫾ 0.9 94 ⫾ 11 2.8 ⫾ 0.9 223 ⫾ 107 126 ⫾ 30 2.8 ⫾ 2.1 39 ⫾ 6.7 131 ⫾ 51 123 ⫾ 14 2.6 ⫾ 1.7 70 ⫾ 11 2.7 ⫾ 1.0

11.4 ⫾ 2.6 31.8 ⫾ 9 3.4 ⫾ 1.3 86 ⫾ 12 1.3 ⫾ 1.1 122 ⫾ 64 104 ⫾ 32 3.7 ⫾ 2.3 51 ⫾ 9.3 80 ⫾ 41 114 ⫾ 14 1.8 ⫾ 0.5 64 ⫾ 9 1.1 ⫾ 0.8

0.2 0.4 0.03 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001 ⬍0.001

Age (yr) BMI (kg/m2) z-score BMI Waist (cm) z-score waist Insulin (pmol/liter)a IGI (pmol/mmol)a WBISIa HDL (mg/dl)a Triglycerides (mg/dl)a SBP (mm Hg)b z-score SBP DBP (mm Hg)b z-score DBP a b

Adjusted for age, sex, and pubertal stage. Adjusted for age, sex, and height.


jects (4.7%) had IGT, but none showed type 2 diabetes. Subjects with metabolic syndrome had higher insulin levels, IGI, triglycerides, z-score SBP, and z-score DBP and lower WBISI and HDL cholesterol levels than subjects who did not meet the criteria for the diagnosis of metabolic syndrome (Table 1). The INS VNTR genotype distributions were in Hardy-Weinberg equilibrium both in obese subjects and in 200 controls and were not statistically different between the two groups (␹2 ⫽ 0.9; P ⫽ 0.7). Genotype distribution among the obese subjects was as follows: 36% were homozygous for the I allele, 47% were heterozygous, and 12.8% were homozygous for the III allele. In the control population, 35% of subjects were I/I homozygotes, 55% were heterozygotes, and 10% were III/III homozygotes. No significant differences in sex or pubertal status distribution were observed within genotype groups (␹2 ⫽ 0.8 and P ⫽ 0.7, and ␹2 ⫽ 2.1 and P ⫽ 0.1, respectively). A significantly higher prevalence of the metabolic syndrome in the group of patients carrying the I/I genotype was observed. Fifty-seven percent of obese children with I/I genotype showed the metabolic syndrome, whereas it was present in only 31% of subjects carrying the other two genotypes (␹2 ⫽ 7.4; P ⫽ 0.006). The risk of developing the metabolic syndrome for obese children with the I/I genotype was significantly higher compared with the risk for the group of patients with the other genotypes (odds ratio ⫽ 2.5; 95% confidence interval ⫽ 1.5–3.9). In these analyses, we pooled the patients carrying the III/III and III/I genotypes, considering the dominant-negative effect suggested for the III allele (12). Furthermore, class I homozygotes compared with the patients with I/III and III/III INS genotypes showed higher IGI and insulin levels and lower WBISI. The differences persisted after adjustment for age, sex, BMI, waist circumference, and pubertal stage (Table 2). The genotype groups were compared for OGTT insulin and glucose levels (Fig. 1). Although no differences concerning the glucose levels during the OGTT were observed, the I allele homozygotes showed at every time significantly higher insulin levels. No difference in IGT prevalence was observed between the genotypes (seven subjects with IGT were homozygotes for the I allele; ␹2 ⫽ 0.7; P ⫽ 0.8) or between subjects with and without metabolic syndrome (nine subjects with metabolic syndrome TABLE 2. Clinical features of obese subjects according to the INS VNTR genotypes

Age (yr) BMI z-score BMI Waist z-score waist Insulin (pmol/liter)a IGI (pmol/mmol)a WBISIa HDL (mg/dl)a Triglycerides (mg/dl)a SBP (mm Hg)b z-score SBP DBP (mm Hg)b z-score DBP a b

I/I (n ⫽ 109)

I/III III/III (n ⫽ 211)

P

11.0 ⫾ 2.3 32.4 ⫾ 16 3.8 ⫾ 0.8 91 ⫾ 14 2.2 ⫾ 0.7 194 ⫾ 72 130 ⫾ 35 2.5 ⫾ 2.0 44 ⫾ 9 104 ⫾ 50 118 ⫾ 14 2.3 ⫾ 1.1 65 ⫾ 13 2.1 ⫾ 1.0

11.3 ⫾ 2.3 32.0 ⫾ 13 3.4 ⫾ 1.4 89 ⫾ 10 1.9 ⫾ 1.3 151 ⫾ 100 99 ⫾ 27 3.9 ⫾ 2.4 46 ⫾ 7 96 ⫾ 42 119 ⫾ 15 2.1 ⫾ 1.7 63 ⫾ 11 1.7 ⫾ 1.5

0.3 0.8 0.07 0.1 0.07 ⬍0.001 ⬍0.001 0.005 0.2 0.4 0.5 0.4 0.1 0.1

Adjusted for age, sex, and pubertal stage. Adjusted for age, sex, and height.


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FIG. 1. Insulin and glucose levels comparison according to the INS VNTR genotypes during an OGTT. Insulin and glucose means and SD are reported for each point of the OGTT. *, P ⬍ 0.001. f, I/I homozygotes; ⽧, subjects carrying the III allele.

had IGT; ␹2 ⫽ 1.3; P ⫽ 0.2). Differences in fasting insulin levels according to genotype were not observed in the control group (data not shown). Discussion

The effect of INS VNTR on insulin levels has been largely investigated. Preceding studies showed that I/I genotype is associated with increased insulin levels in obese children, corroborating data showing that class I INS VNTR alleles are associated with increased transcriptional activity of the insulin gene in adult and fetal pancreas as well as of reporter genes in transfected rodent cell lines (11, 12). We report data showing a clear, statistically significant effect of INS VNTR on insulin levels and secretion in a group of obese children and adolescents from southern Italy. Particularly, in agreement with Dos Santos et al. (20), we observed that although children that are I/I have an insulin secretion pattern similar to those carrying the III allele, they show, at every time of OGTT, significantly higher plasma insulin levels, whereas no differences were present for fasting or OGTT glucose levels. Moreover, we observed an association between the I/I genotype and the higher prevalence of metabolic syndrome in obese children and adolescents. In fact, compared with the patients carrying the other genotypes, subjects homozygous for the class I allele more than doubled their risk to develop the

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metabolic syndrome. Metabolic syndrome arises as a consequence of obesity and increased insulin resistance. When insulin resistance increases, insulin secretion must increase for glucose tolerance to remain normal, and the consequent hyperinsulinemia is associated with dyslipidemia, cardiovascular issues, and other complications of obesity. In other words, metabolic syndrome represents not a disease but, as mentioned above, a cluster of several conditions, in part, a consequence of the hyperinsulinemia (1). The link between the INS VNTR polymorphisms and the predisposition to develop the metabolic syndrome is likely represented by the higher z-score BMI observed in patients with the I/I genotype (Table 1). In fact, the increased fatness observed in subjects carrying the I/I genotype, contributing to further increase the insulin secretion, favors peripheral fat deposition (i.e. free fatty acid accumulation in liver, adipose tissue, and skeletal muscle), worsening the insulin resistance and thus perpetuating a vicious circle that promotes the development of metabolic syndrome. This is in agreement with the observation of Le Stunff et al. (12), showing that the VNTR I/I genotype stimulates the propensity of obese children to gain weight during late childhood and adolescence. No differences in prevalence of IGT between groups of genotypes were observed, suggesting that the I/I genotype is not involved in development of childhood-onset IGT. Previous studies assessed an association between class III INS VNTR and type 2 diabetes in adulthood (21, 22). Class III alleles have been hypothesized to lead to type 2 diabetes by inducing a chronic progressive failure of pancreatic ␤-cells to maintain adequate insulin secretion in the presence of peripheral insulin resistance and unsuppressed hepatic glucose output (22). Together with our findings, these observations suggest that INS VNTR is a lifelong active polymorphism with different effects in different stages of life. In other words, it may predispose obese children to develop the metabolic syndrome (class I) and obese adults to develop type 2 diabetes (class III). In conclusion, although the I/I genotype is not a primary determinant of early-onset obesity, it could be an important enhancer of weight gain and insulin resistance among obese children and adolescents, making, for this reason, the appearance of metabolic syndrome in this kind of patient easier. Acknowledgments Received December 12, 2005. Accepted July 13, 2006. Address all correspondence and requests for reprints to: Dr. Emanuele Miraglia del Giudice, Dipartimento di Pediatria, Seconda Universita` di Napoli, Via Luigi De Crecchio No. 2, 80138 Napoli, Italy. E-mail: [email protected].

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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.