INS VNTR is a QTL for the insulin response to oral glucose in obese children
Christine Dos Santos1,
Daniele Fallin2,
Catherine Le Stunff1,
Sophie LeFur1 and
Pierre Bougnères1
1 Department of Pediatric Endocrinology and Unité 561 Institut National de la Santé et de la Recherche Médicale, Hôpital Saint Vincent de Paul, René Descartes University, 75014 Paris, France
2 Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21025
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ABSTRACT
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Dos Santos, Christine, Daniele Fallin, Catherine Le Stunff, Sophie LeFur, and Pierre Bougnères. INS VNTR is a QTL for the insulin response to oral glucose in obese children. Physiol Genomics 16: 309-313, 2004. First published December 2, 2003; 10.1152/ physiolgenomics.00024.2003. We performed a genotype-phenotype association study to examine whether the insulin VNTR (INS VNTR) polymorphism located in the insulin gene promoter was associated with changes in insulin response to oral glucose. Two classes of INS VNTR alleles are observed in Caucasians, the "short" class I and the "long" class III. Plasma insulin and glucose concentrations and indices of insulin secretion (IGI) and sensitivity (ISI) were measured using an oral glucose tolerance test (OGTT) in 387 obese children aged 12 ± 0.1 yr with a mean body mass index (BMI) of 30.6 kg/m2 (161% of the normal mean). During OGTT, plasma insulin and IGI were 2030% higher in I/I obese children vs. III carriers (P < 0.01). A general linear model adjusting for age, sex, and puberty was also used to evaluate the influence of the VNTR genotype on the BMI-IGI (P = 0.07) and the BMI-ISI (P < 0.006) relationships. The INS VNTR can therefore be considered a quantitative trait locus influencing glucose-stimulated insulin physiology in obese juveniles.
insulin gene; variable number of tandem repeats; quantitative trait locus; insulin secretion; obesity
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INTRODUCTION
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WHEN NONOBESE OR OBESE HUMANS ingest glucose, their insulin concentrations spread over a large range of values (37), while plasma glucose levels remain within a much narrower range. Insulin response to oral glucose varies with age, nutrition habits, diet, and physical activity, but genetic factors are also likely to play a role, as they do for fasting insulin values (7, 31). Ingestion of glucose is signaled to the endocrine pancreas through increased portal glucose, secretion of incretins, and the autonomic nervous system, followed by the processing and integration of these signals within the ß-cells. The level of expression of the insulin gene, the biosynthesis, storage, and exocytosis of insulin, as well as the clearance of insulin from plasma, are also likely to depend on individual genetic factors.
Among the multiple genetic candidates suspected to affect insulin response to glucose, we elected to study the insulin gene promoter region, which harbors a VNTR ("variable number of tandem repeats") that is highly polymorphic among humans (4). We have shown previously that the INS VNTR polymorphism is associated with variations in fasting insulin levels (20). Experiments in transfected ß-cell lines have documented that various (genomic or artificially synthesized) VNTR alleles affect the expression of the insulin gene (22, 33) or of a reporter gene (17), and increased insulin gene transcription was consistently associated with class I alleles in human pancreata (33). In addition, associations of the INS VNTR polymorphism with type 2 diabetes (T2D) (14), early obesity (21), polycystic ovaries (35), as well as type 1 diabetes (4), have been reported.
Since nonobese humans have a narrow range of insulin response to oral glucose tolerance test (OGTT) (9, 37), this limited variability would make it almost impossible to test a genotype-phenotype association in the normal population, since this would require thousands of normal controls to undergo OGTT in controlled nutritional conditions. Patients with long-term obesity would also not be appropriate for test of a genotype-insulin relationship, since their insulin response can be impaired by various degrees of pancreatic dysfunction and drug effects. Obese juveniles, by contrast, can allow examination of primary genetic associations across a wide range of insulin responses, long before impaired glucose tolerance (IGT) or T2D affect insulin-glucose homeostasis (12, 16). Thus this is an attractive sampling scheme to address this hypothesis.
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PATIENTS AND METHODS
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Patients.
We recruited a new sample of 387 Caucasian obese children (Table 1) according to previous criteria (20): a body mass index (BMI) having exceeded the 85th percentile before the age of 6 yr, a monotonic weight curve since birth excluding weight reduction prior to the study. None of the children had Asian or African recent ancestry. The average BMI in our obese sample is 3031 kg/m2. This should be considered in the context of the normal average BMI in young ages, which has been recently measured to be 19 kg/m2 (99th centile 25 kg/m2) at 1213 yr, vs. 23 kg/m2 in 20-yr-old adults (99th centile 31 kg/m2) (8). A separate set of 38 Caucasian children of normal weight and height were studied for comparison (Table 1). All children were in good health. Puberty was staged by experienced endocrinologists according to Tanner. Written consent was obtained according to the French Bioethics law and our Institutional Review Board.
Procedures.
Obese children were given a weight-maintenance diet containing at least 250 g of carbohydrates/day for 7 days before admission to the hospital. Once admitted, children received a standardized diet (20) for 3 days. On the 11th day, and after 12 h of overnight fasting, OGTT took place in unstressed conditions (an intravenous microcatheter was inserted in a peripheral vein 24 h before sampling). OGTT consisted of the ingestion of 1.75 g/kg body wt (75 g maximum) as a 25% dextrose solution flavored with lemon. This was followed by venous blood sampling at 0, 30, 60, 90, and 120 min. Insulin was measured in each sample in duplicate (20). Replication of OGTT on following days in 20 obese children showed an intra-individual variation of 1318% of insulin measurements at various time points. IGT was defined as a 2-h plasma glucose level of 140200 mg/dl and a fasting level of less than 126 mg/dl; T2D was defined as a fasting glucose level of 126 mg/dl or higher or a 2-h level greater than 200 mg/dl.
Nonobese children followed a comparable protocol, except that they were given a smaller dose of oral glucose in proportion to their body weight.
Genotyping.
We genotyped patients at the -23 Hph1 polymorphism, located within the insulin gene promoter, according to published RFLP methods (20). In Caucasians, Hph1 "+" alleles (A) are in near complete LD with class I alleles of the neighboring VNTR, with only 0.20% recombinants, and "-" alleles (T) with class III alleles (4). Therefore, we tested the VNTR by using -23 Hph1 as a surrogate (25). Hph1 allele frequencies were in Hardy-Weinberg equilibrium and were comparable in our patients and in 568 lean Caucasians (20).
Calculations and statistical methods.
The insulin and glucose values during OGTT were used to calculate the "insulinogenic index" (IGI) reflecting the early phase of insulin secretion, as the ratio of the 30 min insulin increment (in pmol/l) to the 30 min glucose concentration (in mmol/l) (32, 34). The composite insulin sensitivity index (ISIcomp, called ISI in this article) was calculated according to the formula: 10,000/square root of [(fasting insulin x fasting glycemia) x (mean insulin concentration during OGTT) x (mean glycemia during OGTT)] (24).
Mean values and standard errors for all continuous measures were compared using t-test and ANOVA procedures. Although raw values are reported in Table 2 for ease of interpretation, IGI and ISI were highly skewed and natural-log transformed to approach normality for all statistical comparisons. Simultaneous effects of all factors on IGI or ISI were estimated via general linear models, and P values for estimates were based on Wald statistics. Genotype was coded as I/III or III/III genotypes vs. I/I, and pubertal stage was included as an ordinal variable (from 1 to 5). All variables and tested interactions were entered simultaneously. All calculations were performed in SAS version 8 for UNIX.
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Table 2. Plasma insulin and glucose concentrations during OGTT in the studied children, with indices of insulin secretion and of insulin sensitivity
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RESULTS
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None of the obese children met criteria of T2D. Only 6/387 (1.5%) had IGT. The distribution of genotypes in these IGT children was unremarkable: I/I (n = 2), I/III (n = 3), and III/III (n = 1). The mean insulin and glucose values in obese and control children are presented in Table 2. As expected, obese children secreted more insulin and were more insulin resistant than the lean controls (P < 0.01, Table 2). Because all values of interest were similar for I/III and III/III carriers and following previous modeling of this locus, we collapsed these two genotypes in a single "class III carrier" category. Within the VNTR genotypic groups, we observed marked differences in insulin secretion (Table 2). Replicating almost exactly our previous observation (20) in an independent sample, plasma insulin levels in the fasting state were higher in children with I/I VNTR genotypes (P < 0.05, Table 2). The novel finding of the present study was that, during OGTT, obese patients with class I/I VNTR alleles showed 2030% higher insulin concentrations (P < 0.01) and IGI (P < 0.01) than obese children with I/III or III/III INS VNTR genotypes (Table 2).
In the overall sample of obese children, IGI correlated with BMI (R = 0.30, P < 0.0001), as expected. VNTR I/I homozygotes showed a steeper slope of correlation between IGI and BMI than the other genotypes (Fig. 1). The significance of this difference in slope based on VNTR genotype was tested as an interaction term in a general linear model while adjusting for age, sex, and puberty (Table 3). This effect is reflected by the non-zero regression coefficient for the BMI and VNTR genotype term in the general linear model (Table 3), although this effect only achieved marginal significance (P = 0.07).

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Fig. 1. Relationship between indices of insulin secretion (IGI) and sensitivity (ISI) in the two INS VNTR genotypic groups of obese children. A: the relationship BMI-IGI is described by the equation y = 6.2x - 89 (R = 0.40, P < 0.0001) in the obese children with I/I INS VNTR genotype, and y = 1.8x + 19 (R = 0.20, P < 0.005) in the other genotypic group. B: the relationship BMI-ISI is described by the equation y = -0.18x +8.3 (R = 0.44, P < 0.0001) in the obese children with I/I INS VNTR genotype, and y = -0.12x + 6.8 (R = 0.33, P < 0.0001) in the other genotypic group. Note that the genotypic differences become significant only for BMI >30 kg/m2, where I/I obese children secrete more insulin and are less sensitive than the others. VNTR, "variable number of tandem repeats"; BMI, body mass index.
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The conjunction of comparable glucose and higher insulin values in INS VNTR I/I patients (Table 2) initially suggested to us a greater degree of insulin resistance than in patients with I/III or III/III genotypes. The index of insulin sensitivity, ISI, was not significantly different between genotypic groups in the unadjusted analyses (Table 2). However, once BMI, age, sex, and puberty were considered, VNTR did have an effect on ISI. In addition, the correlation between ISI and BMI was modified by VNTR genotype, with class III carriers being more insulin sensitive than I/I for those obese children having the higher BMI (Fig. 1 and Table 3).
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DISCUSSION
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Obese children like those studied here represent nearly 5% of the general pediatric population in Europe (30) and the United States (27), among whom 20% will be affected by T2D in middle adulthood, and more than half will develop IGT (18, 25). The occurrence of these complications depends largely on the pancreatic ability to compensate for insulin resistance in the long term (12). Our approach to evaluating mechanisms leading obese patients to T2D is to search for genetic factors involved in the ß-cell adaptation to fat accumulation. During the dynamic phase of juvenile obesity, basal and postalimentary insulin secretion compensates the insulin resistance that develops in liver and muscles (6, 19). As in adults with normal glucose tolerance (5, 16), insulin sensitivity and insulin secretion in obese children are related to each other in a hyperbolic manner (S. Kahn and P. Bougnères, unpublished results). The efficacy of this homeostatic relationship is shown by the strictly normal plasma glucose levels of our patients, both in the fasting state and during OGTT (except for 6/387 with IGT).
The current study confirms our previously reported VNTR association with fasting insulin among young obese patients (20) in a newly recruited sample. In addition, the results document that VNTR alleles influence the secretory response of ß-cells to glucose ingestion. Within our sample of 387 obese children, those whose INS VNTR genotype is I/I have an increase of
2030% of their insulin response to OGTT, compared with other genotypes. Contrasting with these results and the trend observed in Fig. 1 for the BMI-IGI relationship, the lack of significance (P = 0.07) of the BMI x VNTR interaction in the general linear regression analysis could be due to the sensitivity of such a model to the presence of several outliers. According to these observations, the INS VNTR could thus be one of the first quantitative trait loci (QTLs) significantly associated with insulin secretory response to glucose ingestion in humans, with the only other known potential QTL being the Glu23Lys variant of the ß-cell K+ channel molecule KIR6.2 (13, 26). Our speculation for the INS VNTR is that, as in ß-cell lines in vitro (22) and in postmortem pancreata (33), short class I VNTR alleles are associated with increased basal insulin biosynthesis in vivo, allowing a greater preformed insulin release to take place following glucose ingestion. Complementary to our results, the studies which examined steady-state insulin mRNA in adult (3) and fetal (17) human pancreas showed that mRNA derived from VNTR class I chromosomes is 20% higher than that from class III, a result recapitulated almost precisely by the present study.
An intriguing observation is that I/I VNTR obese children, although secreting more insulin, have similar glucose levels. Among the limitations of our study, due to ethical and practical reasons, we used indices derived from OGTT, rather than euglycemic or hyperglycemic clamps, to determine insulin secretion and sensitivity. Although these do not have the same precision and physiological meaning as do clamps, these indices, i.e., IGI and composite ISI, have been validated across against clamps in subjects with various degrees of glucose tolerance, including normoglycemic insulin-resistant individuals (23, 24). Another possibility is that VNTR alleles affect insulin pulsatility (1), since more insulin does not necessarily mean more insulin effect if pulsatility is disturbed. Multivariate regression analysis found that ISI is dependent on BMI and on the VNTR, and the VNTR effect is different for different levels of BMI (Table 3). In class I/I obese children, decreased insulin sensitivity could be a secondary consequence of chronic insulin hypersecretion (11, 28, 36). It may also be, as seen in patients with "maturity onset diabetes of the youth" (MODY), that the reduced insulin secretion in I/III and III/III patients is matched by improved insulin sensitivity to maintain normoglycemia. Not only the amount but also the timing of insulin secretion is important to the effective suppression of hepatic glucose production (2).
Abnormal glucose metabolism is common in first degree relatives of patients with T2D (15), due to both insulin resistance and impaired ß-cell function, with the latter being more important in determining glucose disposal (15). Unfortunately, we do not know of data that can predict T2D among obese juveniles or predict loss of glucose homeostasis based on insulin response to glucose among obese juveniles (10). A meta-analysis of the INS VNTR literature (4) including pioneering studies by Rotwein et al. (29) support that class III alleles carry an increased risk of T2D. The INS VNTR alleles may therefore not only be a QTL for insulin physiology but also a genetic marker of future ß-cell dysfunction and later risk of IGT and T2D in young obese patients. Until it is studied specifically, however, this remains a speculation.
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ACKNOWLEDGMENTS
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We thank A. Dermane for the daily care of the patients.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: P. Bougnères, Endocrinology Dept., Hôpital Saint Vincent de Paul, René Descartes Univ., 82 Ave. Denfert Rochereau, 75014 Paris, France (E-mail: pierre.bougneres{at}wanadoo.fr)
10.1152/physiolgenomics.00024.2003.
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