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Gene on Estimates of the Pancreatic ß-Cell Function in Caucasian Glucose-Tolerant First-Degree Relatives of Type 2 Diabetic Patients1
Steno Diabetes Center and Hagedorn Research Institute (S.A.U., A.M.M., C.T.E., T.H., O.P.), Gentofte, Denmark; Department of Medicine M (Endocrinology and Diabetes) (B.N., O.S.), Kommunehospitalet, University Hospital of Aarhus, Copenhagen, Denmark; University Institute of Medical Biochemistry and Genetics (H.E.), Department of Medical Genetics, University of Copenhagen, Copenhagen, Denmark; Center of Preventive Medicine (J.O.C.), Glostrup University Hospital, Copenhagen, Denmark
Address all correspondence and requests for reprints to: Søren Urhammer, M.D., Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Copenhagen, Denmark.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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gene were associated with alterations in
glucose-induced serum C-peptide and serum insulin responses among
glucose-tolerant first-degree relatives of type 2 diabetic patients.
The study comprised 2 independent Danish cohorts. Among 74 unrelated
type 2 diabetic relatives, 12 homozygous carriers of the Ile/Leu27
polymorphism had a 32% decrease in the 30-min serum C-peptide level
(P = 0.01), as well as a 39% decrease in the
30-min serum insulin level (P = 0.02) during an
oral glucose tolerance test. Ten homozygous carriers of the
Ile/Leu27 variant did, however, not differ from wild-type carriers,
with respect to the acute circulating insulin and serum C-peptide
responses during an iv glucose tolerance test in the same study cohort.
In a larger (more than 3-fold) study group of 230 glucose tolerant
offspring of 62 type 2 diabetic probands, 33 homozygous carriers of the
Ile/Leu27 variant did not differ, with respect to either serum insulin
and serum C-peptide levels during an oral glucose tolerance test or
acute serum insulin and serum C-peptide responses during an iv glucose
tolerance test. We therefore consider the former positive finding as a
statistical type I error. There were no differences in the above
mentioned variables between carriers of the Ser/Asn487 polymorphism and
wild-type carriers within any of the 2 study populations. Nor did
carriers of combined genotypes, i.e. carriers of both
the Ile/Leu27 and the Ser/Asn487 variants, show any associations with
the examined variables. In conclusion, the Ile/Leu27 and Ser/Asn487
polymorphisms of the hepatocyte nuclear factor-1 gene have
apparently no major impact on the pancreatic ß-cell function, after
an oral and iv glucose challenge, in Caucasian first-degree relatives
of type 2 diabetic patients. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(HNF-1), cause MODY3, which phenotypically is characterized by an
early age of onset and an impaired insulin secretion (1, 2). In
addition, it has been suggested that the MODY3 locus is implicated in
subsets of late-onset type 2 diabetes (3). During the initial screening
of the HNF-1 gene for MODY-specific mutations, amino acid
polymorphisms of the gene were detected, including two frequent
variants, Ile/Leu27 and Ser/Asn487 (1). We have previously assessed the
contribution of these variants to type 2 diabetes susceptibility in
random samples of type 2 diabetic patients and healthy individuals (4).
No associations were observed between these variants and diabetes
per se. Nor were we able to detect any impact of the
polymorphisms on insulin secretion assessed, during an oral glucose
tolerance test (OGTT) and an iv glucose tolerance test (IVGTT), among
randomly sampled healthy individuals (5). However, because of genetic
interaction, it is possible that genetic defects, showing no (or only
minor) effect when expressed alone, might actually influence the
phenotype when present in combination with other, yet unidentified
genetic risk factors. This could be the case in subjects at high risk
of developing type 2 diabetes, i.e. first-degree relatives
of type 2 diabetic patients, which presumably harbor several diabetes
susceptibility genes. Hence, the objectives of the present study,
consisting of two independent study samples of glucose-tolerant
first-degree relatives, were to evaluate the potential impact of the
Ile/Leu27 and Ser/Asn487 polymorphisms of the HNF-1 gene on serum
insulin and C-peptide responses during an OGTT and an IVGTT. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unrelated type 2 diabetic relatives (study A). A total of 99 unrelated, glucose-tolerant first-degree relatives of type 2 diabetic patients participated in this study. The relatives were recruited either via their type 2 diabetic parents attending the outpatient clinic, Department of Endocrinology, the University Hospital of Aarhus (all these participants underwent a standard 75-g OGTT, with measurements of plasma glucose, serum insulin, and serum C-peptide at 0, 30, 60, 90, and 120 min during the test and, in addition, 30 of the subjects underwent an IVGTT), or from a population screening of young individuals in Copenhagen (6) (28 relatives who underwent an IVGTT).
Glucose-tolerant offspring of type 2 diabetic probands (study B). Two hundred thirty offspring of 62 type 2 diabetic probands from 62 families were studied. The families were recruited either from the Danish family resource bank at the Department of Human Genetics, University of Copenhagen (18 families), or from the outpatient clinic at Steno Diabetes Center (44 families). The families were collected through one type 2 diabetic proband. All nondiabetic participants underwent a 2-day examination with anthropometric and biochemical measurements, including a 75-g OGTT with measurements of plasma glucose, serum insulin, and serum C-peptide and a frequently sampled IVGTT. Type 2 diabetes was diagnosed in accordance with World Health Organization (1985) criteria.
All study subjects were Danish Caucasians by self-identification. Before the participation, informed consent was obtained for all studied subjects. The studies were approved by the Regional Ethical Committees and were in accordance with the principles of the Declaration of Helsinki II.
Biochemical variables
The concentration of plasma glucose was measured using standard methods. Serum insulin was determined by enzyme-linked immunoadsorbent assay with a narrow specificity excluding des(31, 32)- and intact proinsulin (7). Serum C-peptide was determined by RIA (8), employing the polyclonal antibody M1230 (9, 10). At the University Hospital of Aarhus, serum C-peptide was measured using a commercial kit (Dako Corp. Diagnostics Ltd. Cambridgeshire, UK).
IVGTT
After a 12-h overnight fasting period, an IVGTT was performed. Baseline values of serum insulin, serum C-peptide, and plasma glucose were taken in duplicate with 5-min intervals. Glucose was injected in an antecubital vein over a period of 1 min (0.5 g/kg body weight of 50% glucose in subjects examined at the University Hospital of Aarhus, otherwise 0.3 g/kg). The glucose-induced acute serum insulin and C-peptide responses (0–8 min), as well as the acute plasma glucose response, were calculated by means of the trapezoidal rule, as the incremental values at 2, 4, 6, and 8 min during the test (area under the curve, when expressed above basal values).
Screening for the amino acid polymorphisms in the HNF-1
gene
The Ile/Leu27 and Ser/Asn487 polymorphisms were detected using PCR-RFLP (restriction fragment length polymorphism), as described (4).
Statistical analysis
Differences in continuous variables between groups of subjects were tested with one-way ANOVA, Student’s t test, or Kruskal-Wallis test, when appropriate. In study A, multiple-regression analysis was applied, to adjust for confounding variables such as age, body mass index (BMI), and gender. In study B, the variables were analyzed using a variance component model (random-effects model) (11), where an extra source of variation is allowed to account for the fact that individuals from the same family might be correlated. The variant of interest and gender were included as fixed variables, age and BMI as covariates, and family as a random effect. Data are means (SD), except for nonnormally distributed variables, which are expressed as medians (interquartile ranges). A P-value less than 0.05 (two-tailed) was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, comprising 99 unrelated relatives of type 2
diabetic subjects, the allelic frequencies of the Ile/Leu27 and
Ser/Asn487 polymorphisms were 36.4% (95% confidence interval:
29.7–43.1%) and 32.8% (26.8–38.8%), respectively. The observed
genotype frequencies were in Hardy-Weinberg equilibrium. Age, BMI,
gender distribution, and fasting levels of serum C-peptide, serum
insulin, and plasma glucose did not differ between carriers of the
three genotypes, whereas the ANOVA showed significant differences
between groups, with respect to serum insulin and serum C-peptide
levels at 30 min during the OGTT (Table 1
). Comparing Leu/Leu homozygotes with
subjects carrying the wild-type, the insulin and C-peptide values at 30
min were significantly decreased (P = 0.007 and
P = 0.004, respectively). When controlling for BMI,
age, and gender in a multiple-regression analysis, the levels were
still significantly lower (Table 1). Heterozygous carriers did not
differ from wild-type carriers, regarding these variables. Neither were
there any differences between genotype groups, with respect to plasma
glucose, serum insulin, and serum C-peptide levels at other time points
(60, 90, and 120 min) during the OGTT. Leu/Leu homozygotes had
decreased acute serum insulin and serum C-peptide responses during the
IVGTT, compared with wild-type carriers. However, when adjusting for
age, BMI, and gender in a multiple-regression analysis, the
significance disappeared, with respect to both variables (Table 1). No
differences in any of the above mentioned variables were observed
between carriers of the Ser/Asn487 polymorphism and wild-type carriers.
Neither did combinations of the genotypes, i.e. carriers of
both Ile/Leu27 and Ser/Asn487 polymorphisms, show any alterations in
the glucose-stimulated serum insulin or C-peptide responses during the
IVGTT or OGTT, as compared with the analyses of the Ile/Leu27 variant
(data not shown).
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In this study, 33 homozygous carriers of the Ile/Leu27
polymorphism and 102 subjects carrying the variant in its heterozygous
form were identified. Whereas no differences were observed with respect
to age and BMI, there were significant differences in gender
distribution among the three genotype groups (Table 2). This is, however, believed to be an
incidental finding. The variance component model (11) (which allows us
to model the familiarity correlation among sibs from the same family
and, at the same time, to adjust for differences in confounding
variables such as age, BMI, and gender revealed significant differences
among the 3 groups, with respect to the 30-min value of serum C-peptide
(P = 0.02) (Table 2). Homozygous mutant carriers,
however, did not differ from the combined group, wild-type/heterozygous
carriers; whereas heterozygous carriers had a higher serum C-peptide
level, compared with wild-type/homozygous carriers (P =
0.005). This is considered a spurious finding. No differences were
observed among the 3 genotype groups, with respect to the acute serum
insulin and C-peptide responses during the IVGTT or levels of serum
insulin, serum C-peptide, and plasma glucose at other time points (60,
90, and 120 min) during the OGTT. Twenty-six homozygotes and 105
heterozygotes of the codon 487 amino acid substitution were identified.
No differences were observed between carriers of this variant and
wild-type carriers, either with respect to age, BMI, and gender, or
with respect to the estimates of the ß-cell function mentioned above
(data not shown). Nor did carriers of the combined genotype (carriers
of both the Ile/Leu27 and the Ser/Asn487 variant) show any associations
with the estimates of the ß-cell function (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene, and the Ile is conserved among several
species, indicating a potential functional importance of this residue
(12). Because our recent study in random samples of middle-aged and
young healthy individuals did not reveal any impact of this amino acid
replacement on insulin secretion, we wanted to clarify whether the
variant, in combination with other genetic risk factors for type 2
diabetes, influences the glucose-induced ß-cell response. In study A,
the Ile/Leu27 variant, in its homozygous form, indeed was associated
with an approximately 30% and 40% reduction in serum C-peptide and
serum insulin responses, respectively, at 30 min during the OGTT.
Because it has been shown that serum insulin levels obtained at 30 min
during an OGTT are positively correlated with the acute insulin
response after an iv glucose load (r = 0.55, P <
0.0003) (13), we also examined ß-cell responses during an IVGTT.
However, the results obtained from the IVGTT in this study cohort could
not confirm the OGTT findings after adjustments were made for
differences between genotypes in age, BMI, and gender distribution.
Neither were we able to show any impact of the variant in the more than
3-fold larger study sample of 230 glucose-tolerant offspring of type 2
diabetic probands either during the OGTT or during the IVGTT. There is
no obvious explanation for this discrepancy, because the 2 study
samples are comparable, with regard to age, BMI, and gender
distribution (Tables 1
and 2). Also the allelic frequencies are similar
and are in accordance with allelic frequencies found previously (1, 4).
The power, in the study of 230 offspring, to detect an effect of 30%
and 40% on the serum C-peptide and serum insulin levels at 30 min
during the OGTT, as demonstrated in study A, is 90% and 98%,
respectively at a 5% significance level. Hence, we have to consider
the initial findings of decreased serum insulin and C-peptide levels in
12 Leu/Leu27 carriers, in the study of type 2 diabetic relatives, as a
statistical type I error. These data illustrate clearly the potential
pitfalls in case control comparisons and point to the necessity for
replication studies.
From the present study, we conclude that neither of the common amino
acid polymorphisms of the HNF-1 gene (Ile/Leu27 and Ser/Asn487) has
any measurable impact on the pancreatic ß-cell function, as estimated
during OGTTs and IVGTTs in Caucasian first-degree relatives of type 2
diabetic patients. From these data and our previously reported findings
in random samples of glucose tolerant subjects and type 2 diabetic
patients, we can exclude these variants from being implicated in
susceptibility to common type 2 diabetes.
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Acknowledgments |
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Footnotes |
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Received June 11, 1998.
Revised July 17, 1998.
Accepted July 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene in Danish
Caucasians with late-onset NIDDM. Diabetologia. 40:473–475.[CrossRef][Medline]
is associated with reduced serum C-peptide and
serum insulin responses to an oral glucose challenge. Diabetes. 46:912–916.[Abstract]
. Biol Chem Hoppe Seyler. 374:419–425.[Medline]
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