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Medizinische Klinik, Abteilung für Endokrinologie, Stoffwechsel und Pathobiochemie, Eberhard-Karls-Universität, Tübingen, Germany
Address all correspondence and requests for reprints to: Dr. Michael Stumvoll, Medizinische Universitätsklinik, Otfried-Müller-Str. 10, D-72076 Tübingen, Germany. E-mail: Michael.Stumvoll{at}med.uni-tuebingen.de
Abstract
Disruption of the insulin receptor substrate-2 was shown to cause
type 2 diabetes in mice. This could be largely attributed to abnormal
ß-cell development. In humans, a prevalent polymorphism in insulin
receptor substrate-2 (Gly1057Asp) was not found be associated with type
2 diabetes in linkage and association studies. We tested the hypothesis
that an extreme challenge of the ß cell might reveal subtle
abnormalities in carriers of this polymorphism undetected by
conventional insulin secretion tests. Therefore, in addition to
assessing ß-cell function by oral glucose tolerance testing (n =
318, normal glucose tolerance), we measured the secretory response to
maximal stimulation by hyperglycemia (10 mM), glucagon-like
peptide-1, and arginine administered in an additive fashion (n =
77, nondiabetic). The allelic frequency of the Asp allele was 
37%.
Neither the ß-cell function indices from the oral glucose tolerance
test nor the secretory response during the hyperglycemic clamp differed
measurably between carriers and controls. Moreover, maximal plasma
C-peptide concentrations in response to the combined glucose,
glucagon-like peptide-1, and arginine stimulus was not different
between Gly/Gly (10,745 ± 1,186 pmol/liter) and X/Asp
(10,800 ± 490 pmol/liter, P = 0.99). In
conclusion, our findings strongly suggest that the Gly1057Asp
polymorphism in insulin receptor substrate-2 is not associated with
ß-cell dysfunction. The normal maximal insulin secretory response
makes it unlikely that this common polymorphism results in abnormal
ß-cell development.
DISRUPTION OF THE insulin receptor substrate (IRS)-2 was shown to cause type 2 diabetes in mice as a result of hepatic insulin resistance and lack of compensatory ß-cell hyperplasia (1, 2). Whereas intact signaling through IRS-1 seems to be important for ß-cell function (3, 4, 5), IRS-2 signaling plays a crucial role in the development of ß cells (6) and regulation of ß-cell mass (2).
In humans, a number of genetic variants of the IRS-2 gene were identified. Only one of the resulting amino acid polymorphisms (Gly1057Asp), however, reached a sufficiently high prevalence to be attributed a candidate role for common type 2 diabetes. This polymorphism was found not to be associated with type 2 diabetes in association (7) and linkage studies (8, 9). Nevertheless, in an Italian population obesity seemed to modify the effect of the Asp allele on the risk of type 2 diabetes: a decreased risk in lean and an increased risk in obese subjects (10). In glucose-tolerant subjects no consistent evidence for an effect of this polymorphism on insulin sensitivity or ß-cell function was reported (11).
Although it is difficult to predict a possible effect of the Gly1057Asp polymorphism on the function of IRS-2, it is possible that it contributes to the biological variation in ß-cell development. We specifically addressed the question whether an extreme challenge of the ß cell will reveal abnormalities in carriers of this polymorphism that are too subtle to be picked up by conventional insulin secretion tests. We, therefore, performed elaborate phenotyping of ß-cell function using a modified hyperglycemic clamp with the additional secretagogues glucagon-like-peptide-1 and arginine administered in an additive fashion in nondiabetic individuals. In addition, ß-cell function was estimated in normal glucose-tolerant (NGT) subjects using recently validated indices from the oral glucose tolerance test (OGTT).
Materials and Methods
Subjects
We studied 77 nondiabetic subjects [NGT, n = 59; impaired
glucose tolerant, n = 18, WHO criteria (12)] by a
modified hyperglycemic clamp (13) and 318 healthy, NGT
volunteers by OGTT after local ethical committee approval and obtaining
consent. The subjects (characteristics are shown in Table 1
) were unrelated, had tested negative
for glutamate decarboxylase antibodies, and their genotype was
unknown at the time of recruitment. The Gly1057Asp polymorphism in
IRS-2 was determined by PCR and sequenced by dye terminator sequencing
using ABI PRISM 310 genetic Analyzer (PE Applied Biosystems, Foster City, CA). The nucleotide substitution at
codon 1057 was detected by the following primers: sense
5'-CCGACTACATGAACCTCGAC-3' and antisense 5'-GAGGAGAAGGTCTCGGAACT-3'.
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2 test). The rare allelic frequency in the
subjects undergoing the hyperglycemic clamp was not different in NGT
and impaired glucose-tolerant subjects (P = 0.3,
2-test). Hyperglycemic clamp
After an overnight fast and after baseline samples had been obtained, a hyperglycemic clamp was performed as described previously (4, 13). An iv bolus of 20% glucose over 1 min was given to instantaneously raise blood glucose to 10 mM [bolus dose (mg) = body weight (kg) · desired increase in blood glucose (mg/dl) · 1.5). Subsequently, a glucose infusion was adjusted to maintain blood glucose at 10 mmol/liter. After 120 min, glucagon-like peptide-1 (GLP-1) [human GLP-1 (7–36) amide; Poly Peptide, Wolfenbüttel, Germany] was given as a primed, continuous infusion (0.6 pmol/kg; 1.5 pmol·kg-1·min-1) during the next 80 min. At 180 min a bolus of 5 g arginine hydrochloride (Pharmacia & Upjohn, Erlangen Germany) was injected over 45 sec while the GLP-1 infusion was continued.
Calculations
The following published estimates of ß-cell function were calculated from parameters obtained during the OGTT estimated first phase (14); the ratio of the area under the curve (AUC) of insulin over the AUC of glucose (14); the corrected insulin response (CIR30) as Ins30/(Gluc30 x (Gluc30 - 70) using conventional units (15). Insulin sensitivity [insulin sensitivity index (ISI)] from the hyperglycemic clamp was determined by relating the glucose infusion rate to the plasma insulin concentration during the second hour (13). Insulin sensitivity from the OGTT was estimated according to the index proposed by Matsuda and DeFronzo (16).
Phases of insulin secretion based on insulin and C-peptide concentrations during the hyperglycemic clamp were calculated as: first phase, mean of 2.5–10 min; second phase, mean of 80–120 min; first GLP phase, mean of 125 and 130 min; GLP plateau, mean of 160–180 min; maximal insulin secretion, mean of 182.5–190 min (13). Insulin secretion rates during the hyperglycemic clamp were calculated by deconvolution from C-peptide concentrations using standard kinetic parameters from the literature as described previously (17, 18).
Statistics
For statistical comparisons with the wild-type (Gly/Gly) subjects heterozygous (Gly/Asp) and homozygous (Asp/Asp) for the mutation were combined and referred to as X/Asp. Data shown in tables and figures are unadjusted. For statistical analysis the secretion indices of both clamp and OGTT were log transformed and linearly adjusted for insulin sensitivity, body mass index, age, and sex. Comparisons between genotypes were made on adjusted values using the t test, ANOVA, or Wilcoxon/Kruskal-Wallis rank tests where appropriate. A P value of less than 0.05 was considered to be statistically significant.
Results
Blood glucose, plasma insulin, and C-peptide concentrations during the glucose tolerance test were similar in Gly/Gly and X/Arg (data not shown). The ISI calculated from the OGTT was not significantly different between Gly/Gly and X/Asp. Estimated first phase was not significantly reduced in Gly/Asp (1051 ± 43 pM) or Asp/Asp (1014 ± 83 pM) compared with Gly/Gly (1216 ± 62 pM, P ANOVA = 0.31) when adjusted for insulin sensitivity and age. Similarly, neither the ratio of AUC of insulin over the AUC of glucose nor corrected insulin response were statistically different in carriers of the Asp allele (data not shown). Moreover, in the subjects homozygous for the Asp allele none of the insulin secretion parameters were different from those in the wild-type subjects.
As shown in Table 2 and Fig. 1, insulin
secretion during the modified
hyperglycemic clamp was also not
different between carriers of the polymorphism and controls. In
particular, plasma C-peptide concentrations after the combined glucose,
GLP-1 and arginine stimulus, the best parameter for
maximal insulin secretion, was not different between Gly/Gly
(10,745 ± 1,186 pmol/liter) and X/Asp (10,800 ± 490
pmol/liter, P = 0.99) (Fig. 2). Based on the variation of this
parameter in our population, we had the statistical power to detect a
10% difference (
= 0.05, ß = 0.80).
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In the present studies, we found no evidence for a contribution of the Gly1057Asp polymorphism in IRS-2 to the biological variation of ß-cell function. Neither ß-cell function indices from the OGTT nor the secretory response during the hyperglycemic clamp differed measurably between carriers and controls. The lack of an effect of this polymorphism on ß-cell function is in agreement with previously published data from a healthy Danish population using an iv glucose tolerance test (11).
Our use of the hyperglycemic clamp was previously shown to produce a greater insulin secretory response than any other in vivo assessment of insulin secretion (13). This test may, thus, come closest to an in vivo measurement of functional ß-cell mass. Our data, therefore, strongly suggest that residual ß-cell capacity is not affected by the presence of this polymorphism. Because the Gly1057Asp polymorphism is by far the most common genetic variant of IRS-2 in humans, based on the literature and our data this protein seems unlikely to harbor genetic variants contributing in a major way to reduced insulin secretion. Therefore, if a human genetic equivalent to the IRS-2 knockout paradigm with dysfunction and developmental dysregulation of the ß cell exists, it would have to be either a very rare and hitherto unidentified genetic variant in IRS-2, a variant in an intronic region or, alternatively, a mutation in other proteins specific for the IRS-2 pathway. It is necessary to point out that our study population was young and normal glucose tolerant. We can, therefore, not exclude an interaction effect of the IRS-2 variant with age or impaired (and diabetic) glucose tolerance.
In conclusion, our results from the OGTT and the hyperglycemic clamp strongly suggest that the Gly1057Asp polymorphism in IRS-2 alone is not associated with ß-cell dysfunction. Moreover, the normal maximal insulin secretory response makes it unlikely that this common polymorphism results in reduced functional ß-cell mass.
Acknowledgments
We thank all the research volunteers for their participation. We gratefully acknowledge the excellent technical assistance Isolde Riedlinger and Sabine Obermüller. We are also indebted to Elke Hardt who helped with many experiments.
Footnotes
This study was supported in part by a research grant from the European Community (QLRT-1999-00674), the Deutsche Forschungsgemeinschaft (Fr 1561/1-1), and Roche Diagnostics (Mannheim, Germany).
Abbreviations: AUC, Area under the curve; GLP, glucagon-like peptide; IRS, insulin receptor substrate; ISI, insulin sensitivity index; NGT, normal glucose-tolerant; OGTT, oral glucose tolerance test.
Received March 28, 2001.
Accepted July 6, 2001.
References
Arg amino acid polymorphism in IRS-1 impairs insulin secretion
in pancreatic ß cells. J Clin Invest 104:357–364[Medline]
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