ORIGINAL
ARTICLE
Incretin and Glucagon Levels in Adult Offspring Exposed to Maternal Diabetes in Pregnancy Louise Kelstrup, Tine D. Clausen, Elisabeth R. Mathiesen, Torben Hansen, Jens J. Holst, and Peter Damm Center for Pregnant Women with Diabetes (L.K., T.D.C., E.R.M., P.D.) and Departments of Obstetrics (L.K., P.D.) and Endocrinology (E.R.M.), Rigshospitalet, DK-2100 Copenhagen, Denmark; Department of Gynaecology and Obstetrics (T.D.C.), Nordsjaellands Hospital, 3400 Hillerød, Denmark; Institute of Clinical Medicine (E.R.M., P.D.), Faculty of Health Science, University of Copenhagen, 2200 Copenhagen, Denmark; Novo Nordisk Foundation (NNF) Center for Basic Metabolic Research (T.H.), Section of Metabolic Genetics, University of Copenhagen, 2200 Copenhagen, Denmark; Faculty of Health Sciences (T.H.), University of Southern Denmark, 5230 Odense M, Denmark; and NNF Center for Basic Metabolic Research and Department of Biomedical Sciences (J.J.H.), University of Copenhagen, 2200 Copenhagen, Denmark
Context: Fetal exposure to maternal diabetes is associated with increased risk of type 2 diabetes mellitus (T2DM) later in life. The pathogenesis of T2DM involves dysfunction of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), as well as hyperglucagonemia. Objective: Our aim was to investigate circulating plasma levels of GLP-1, GIP, and glucagon during the oral glucose tolerance test (OGTT) in adult offspring of women with diabetes in pregnancy. Design and Participants: We conducted a follow-up study of 567 offspring, aged 18 –27 years. We included two groups exposed to maternal diabetes in utero: offspring of women with diet-treated gestational diabetes mellitus (O-GDM; n ⫽ 163) or type 1 diabetes (O-T1DM; n ⫽ 146). Two reference groups were included: offspring of women with risk factors for GDM, but normoglycemia during pregnancy (O-NoGDM; n ⫽ 133) and offspring from the background population (O-BP; n ⫽ 125). The subjects underwent a 75-g OGTT with venous samples at 0, 30, and 120 minutes. Results: Fasting plasma levels of GLP-1 were lower in the two diabetes-exposed groups compared to O-BP (O-GDM, P ⫽ .040; O-T1DM, P ⫽ .008). Increasing maternal blood glucose during OGTT in pregnancy was associated with reduced postprandial suppression of glucagon in the offspring. Lower levels of GLP-1 and higher levels of glucagon during the OGTT were present in offspring characterized by overweight or prediabetes/T2DM at follow-up, irrespective of exposure status. Conclusion: Lower levels of fasting GLP-1 and impaired glucagon suppression in adult offspring exposed to maternal diabetes during pregnancy are diabetogenic traits that may contribute to glucose intolerance in these persons, but further investigations are needed. (J Clin Endocrinol Metab 100: 1967–1975, 2015)
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received October 31, 2014. Accepted March 11, 2015. First Published Online March 17, 2015
doi: 10.1210/jc.2014-3978
Abbreviations: AUC, area under the curve; BMI: body mass index; CI, confidence interval; GDM, gestational diabetes mellitus; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide-1; O-BP, offspring of mothers from the background Danish population without risk factors for diabetes; O-GDM, offspring of mothers with GDM; OGTT, oral glucose tolerance test; O-NoGDM, offspring of mothers with risk factors for GDM, but normoglycemic in pregnancy; O-T1DM, offspring of mothers with pregestational T1DM; SDS, birth weight SD score; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
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1967
1968
Kelstrup et al
Incretin and Glucagon Levels in Adult Offspring
etal exposure to intrauterine hyperglycemia due to maternal diabetes is associated with increased risk of obesity, metabolic syndrome, prediabetes, and type 2 diabetes mellitus (T2DM) in the offspring (1–3), as well as impaired insulin function (4). The underlying pathology behind these associations is not known, but a fuelmediated mechanism of fetal programming is hypothesized (5, 6). The pathology in T2DM involves multiple defects of different organ systems, including the loss of the incretin effect, fasting hyperglucagonemia, and impaired suppression of glucagon in the postprandial state (7– 10). The incretin effect is the steeper rise in insulin release after oral ingestion of glucose, compared to the insulin response when glucose is infused iv to similar concentrations (isoglycemia). The effect is mediated through the insulinotropic action on the -cells by the two incretin hormones: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both secreted from endocrine cells in the small intestine in response to oral intake of nutrients (8, 11, 12). The action of GLP-1, GIP, and glucagon has been investigated in different groups with high risk of T2DM (13–15), but only one study in children exposed to maternal gestational diabetes mellitus (GDM) has been published. The authors concluded that fetal exposure to maternal GDM may alter the activity of the gut hormones and that this may contribute to the increased risk of obesity (16). The aim of our study was to investigate the circulating levels of GLP-1, GIP, and glucagon in both fasting and oral glucose-stimulated conditions in adult offspring of women with either GDM or type 1 diabetes mellitus (T1DM) in pregnancy.
F
Subjects and Methods Study design A follow-up study was conducted of adult offspring of both sexes from all pregnancies complicated by either GDM or T1DM during 1978 –1985 at the Center for Pregnant Women with Diabetes, Rigshospitalet, Copenhagen, Denmark. An equal number of control subjects, randomly selected from healthy mothers giving birth at the Department of Obstetrics, Rigshospitalet, in the same period were invited to participate. Maternal baseline data, including blood glucose levels during pregnancy, and other relevant data from the period of pregnancy and the time of delivery were accessible from the mothers’ medical records at the archives of the Department of Obstetrics. Coupling between the mothers’ medical record and the adult offspring was possible through the Danish Civil Registrar System. The materials and methods have previously been described in detail (2). The study was in accordance with the Declaration of Helsinki and was approved by the regional
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
ethical committee. All participants gave written consent before inclusion.
Participants’ exposure status and genetic predisposition to diabetes (Figure 1) The offspring exposed to intrauterine hyperglycemia were subdivided according to maternal diabetes type and differences in genetic predisposition to T2DM as follows: 1. O-GDM: Offspring of women with diet-treated GDM. This group was estimated to have a high genetic predisposition to overweight and T2DM (17). 2. O-T1DM: Offspring of women with pregestational T1DM, and with an assumed low genetic predisposition to obesity and T2DM similar to the general Danish population. Likewise, the unexposed offspring were classified, according to maternal risk factors to GDM and predisposition to T2DM as follows: 3. O-NoGDM: Offspring of women without GDM, documented by a normal oral glucose tolerance test (OGTT) in pregnancy. The mothers underwent an OGTT due to the presence of risk factors for developing GDM. Based on the maternal phenotype, subjects in this group were assumed to have a high genetic predisposition to overweight and T2DM, comparable to the OGDM group. 4. O-BP: Offspring of women from the Danish background population, with an assumed low genetic predisposition to obesity and T2DM because the Danish population in general is a low-risk population.
Exclusion criteria Offspring with previously known diabetes (both T2DM and T1DM) and offspring being positive for GAD65 auto-antibodies (GAD65ab) were excluded (Figure 1).
Diabetes in pregnancy in the period 1978 –1985 In Denmark, screening for GDM has traditionally been based on risk factors (18 –20). During the period 1978 –1985, the following risk factors were used: ⱖ20% overweight before pregnancy, family history of diabetes, previous GDM, previous delivery of a child weighing ⬎4500 g, and glucosuria (18, 21). Women with risk indicators and two consecutive fasting capillary blood glucose measurements ⱖ4.1 mmol were offered a 3-hour 50-g OGTT. The OGTT was defined as abnormal if more than two of seven values during the test exceeded the mean ⫹ 3 SD for a reference group of normal-weight nonpregnant women without a family history of diabetes (20, 22). All mothers with GDM in this study were diet-treated only. Offspring of mothers with insulin-treated GDM (15% of all women with GDM) were excluded from participation to minimize the risk of misclassification among the mothers in the GDM group (eg, undiagnosed T2DM, early-stage T1DM, and MODY). Mothers of O-T1DM fulfilled three criteria: onset of diabetes at age ⱕ40 years, a classical history of hyperglycemic symptoms before disease diagnosis, and insulin treatment started ⱕ6 months after diagnosis. Mothers of O-NoGDM had risk factors qualifying for an OGTT, but the glucose values during the OGTT were all below the mean ⫹ 2 SD of the reference group.
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doi: 10.1210/jc.2014-3978
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1969
Potential eligible offspring Born between 1978-85 n=1066
Subjects exposed to intrauterine hyperglycemia in pregnancy (n=556 )
High genetic predisposition to overweight and T2DM (n=295)
N=469
N=30
Low genetic predisposition to overweight and T2DM (n=261)
Subject of mothers without diabetes (n=510)
High genetic predisposition to overweight and T2DM (n=254)
Low genetic predisposition to overweight and T2DM (n=256)
O-GDM
O-T1DM
O-NoGDM.
O-BP
Lost to follow up: n=127 (43%)
Lost to follow up: n=101 (39%)
Lost to follow up: n=113 (45%)
Lost to follow up: n=128 (50%)
O-GDM Exclusion due to: Diabetes: n=1 GADab pos.: n=4 (2%)
O-T1DM Exclusion due to: Diabetes: n=7 GADab pos.: n=7 (5%)
O-NoGDM. Exclusion due to: Diabetes: n=2 GADab pos.: n=6 (3%)
O-BP Exclusion due to Diabetes: n=0 GADab pos.: n=3 (1%)
O-GDM (participants) n=163 (55%)
O-T1DM (participants) n=146 (56%)
O-NoGDM (participants) n=133 (52%)
O-BP (participants) n=125 (49%)
Follow up in 2003-2005 Age 18-25 years Total included n=567 (53%) Figure 1. Study design. Flowchart of subjects participating in the study (n ⫽ 567), subjects lost to follow-up (n ⫽ 469; 44%), subjects excluded due to predefined criteria of previously known diabetes (both T2DM and T1DM), and offspring being positive for GAD65ab (n ⫽ 30; 3%).
Mothers of O-BP were from the local community and were routinely referred for antenatal care and delivery at Rigshospitalet, Copenhagen.
Examination of adult offspring at follow-up Venous blood samples were obtained in the fasting state, and participants without known diabetes underwent a 120-min 75-g OGTT with venous sampling at 30 and 120 minutes. Anthropometric measuresintermsof weight(kilograms)and height (meters) were obtained, andaquestionnairewithinformationonhealth,medication,diet,levels of physical activity, and socioeconomic parameters was completed.
Exposure variables Five different estimates of intrauterine hyperglycemia were used as exposure variables: 1) assignment into the four groups (O-GDM, O-T1DM, O-NoGDM, O-BP) was used as a surrogate measure of different exposures to intrauterine exposure and/or genetic predisposition to T2DM and obesity; 2) and 3) maternal OGTT data at 0 and 120 minutes, obtained from mothers of O-GDM and O-NoGDM only; 4) and 5), mean maternal blood glucose in the first and third trimesters from mothers of O-T1DM only.
Definition of variables Primary outcome variables Our main outcomes were GLP-1, GIP, and glucagon at three time points during OGTT: 0, 30, and 120 minutes. Secondary outcomes were incremental 0 –30 minutes, 0 –120 minutes, and total area under the curve (AUC) calculated by the trapezoidal method (23).
Covariates with references to the mother were: pregestational overweight (body mass index [BMI], kg/m2), family history of diabetes (unspecified diabetes in a first-degree relative on the maternal side [yes vs no]), and ethnicity (defined as Nordic Caucasian if the mother originated from Denmark, Norway, Sweden, or Iceland [yes vs no]). Covariates associ-
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1970
Kelstrup et al
Incretin and Glucagon Levels in Adult Offspring
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
ated with the offspring were: gender (male sex [yes vs no]), age (years), level of physical activity (ⱖ30 vs ⬍30 min/d), quality of diet composition (divided into a diet with a high content of vegetables and fruits [“green diet”] vs a diet rich in meat and animal fat [“high animal fat diet”]), and socioeconomic position (based on the highest occupational status of the parents at the time of follow-up and coded into family social class I–V in accordance with the standards of the Danish National Institute of Social Research, similar to the British Registrar General’s Classification I–V). We added a social class VI representing people on transfer income, including sickness benefits and disability pension, and dichotomized the variable into family social class (I–IV vs V–VI) (24). OGTTs were evaluated according to World Health Organization criteria (25). Two covariates were considered to be mediators: birth weight SD score (SDS), calculated as the deviation from the population mean value in SD units adjusted for sex and gestational age (26); and offspring BMI at follow-up.
test. Correction for multiple comparisons was done by the Bonferroni method: we multiplied P values in the post hoc test by 4, because of the four possible comparisons (O-BP compared with the three other groups, and furthermore O-GDM with O-NoGDM) due to our hypothesis of an influence of intrauterine exposure to maternal hyperglycemia as well as genetic predisposition to obesity and T2DM. To test for associations between maternal blood glucose levels in pregnancy and the levels of the GLP-1, GIP, and glucagon during OGTT and to control for the effect of confounding, multiple linear regression analyses were done. Eight covariates chosen due to previous studies and theoretical considerations were included in the regression analyses. Two other covariates considered to be mediators were included in the basic models one by one to evaluate the effect. All tests were two-tailed, and a significance level of 0.05 was chosen. Data were processed using SPSS version 18 (SPSS Inc).
Biochemical analyses
Results
GLP-1, GIP, and glucagon concentrations in plasma were measured after extraction of plasma with 70% ethanol (vol/ vol, final concentration). The plasma concentrations of GLP-1 were measured (27) against standards of synthetic GLP-1 (7– 36) amide using antiserum code no. 89390, which is specific for the amidated C terminus of GLP-1 and therefore mainly reacts with GLP-1 of intestinal origin. The assay reacts equally with intact GLP-1 and with GLP-1 (3–36) amide, the primary metabolite. For the GIP RIA (28, 29), we used the C-terminally directed antiserum no. 867, which cross-reacts fully with human GIP but not with the so-called GIP 8000, whose chemical nature and relationship to GIP secretion is uncertain. The antiserum, which is similar to the previously employed R65, reacts equally with intact GIP and GIP 3– 42, the primary metabolite. Human GIP and 125-I human GIP (70 MBq/nmol) were used for standards and tracer. Because of the rapid and intravascular conversion of both GLP-1 and GIP to their primary metabolites, it is essential to determine the sum of the intact hormone and the metabolite for estimation of the plasma level of these hormones. The glucagon RIA was directed against the C terminus of the glucagon molecule (antibody code no. 4305) and therefore mainly measures glucagon of pancreatic origin (30, 31). For all three assays, sensitivity was ⬍1 pmol/L, intra-assay coefficient of variation was below 6% at 20 pmol/L, and recovery of standard, added to plasma before extraction, was about 100% when corrected for losses inherent in the plasma extraction procedure. GAD65ab were detected by ELISA (GAD65 Autoantibody Kit; RSR Ltd) and defined as positive when ⱖ5 U/mL.
Statistical analyses Normally distributed continuous data are presented as mean (SD), whereas non-normally distributed data are presented either as median (25th-75th percentiles) or as geometric mean (95% confidence interval [CI]). Results from the linear regression analyses are presented as regression coefficient (), 95% CI, and P value. Overall differences between the groups were analyzed with one-way ANOVA, 2 test, or Mann-Whitney U test when appropriate. As a post hoc test, and according to the hypothesis, specific comparisons between the groups were carried out by independent t test or 2
Data on mothers (1978 –1985) and characteristics of the study population Overall, 597 offspring participated in the study (56% of those eligible), but 30 subjects (3%) met the exclusion criteria (previously known T1DM or T2DM, n ⫽ 10; and GADab positive, n ⫽ 20) for analyses of incretin hormones and glucagon, giving a data set of 567 offspring (53%) (O-GDM, 163; O-T1DM, 146; O-NoGDM, 133; O-BP, 125) (Table 1 and Figure 1). Participants and subjects lost to follow-up were comparable in total and within the groups, and the mothers of O-GDM and O-NoGDM were comparable with respect to most risk factors for GDM, as previously described (2). Per definition, women with GDM had higher blood glucose levels than women in the NoGDM group: fasting glucose, 5.2 vs 4.7 mmol/L; and 2-hour glucose, 7.9 vs 5.2 mmol/L (P ⬍ .0001 for both). In women with T1DM, the mean blood glucose was 8.9 (2.8) mmol/L in the first trimester and 6.8 (1.8) mmol/L in the third trimester. Data on maternal characteristics and basic metabolic characteristics in the offspring at follow-up are given in Table 1 and have been published previously (2, 3). Incretin hormones and glucagon during OGTT (Tables 2 and 3) GLP-1 concentration in the fasting state was significantly lowerinthetwogroupsexposedtomaternaldiabetes(O-GDM and O-T1DM) compared to O-BP (P ⫽ .040 and P ⫽ .008, respectively). During the OGTT, the levels of GLP-1 did not differ significantly between the groups. No significant differences in the levels of GIP were found between the four groups, either in the fasting state or during the OGTT.
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1971
Table 1. Baseline Data in Mothers and Offspring (1978 –1985) and Follow-up Characteristics in the Adult Offspring (2003–2005) n (total ⫽ 567) Maternal data BMI, kg/m2 Pregestational overweight (BMI ⱖ 25 kg/m2) Family history of diabetes Nordic Caucasian (ethnicity) Offspring birth data Male sex Birth weight, g Birth weight SDS SGA LGA Offspring follow-up data Age, y Abnormal glucose tolerance (IFG, IGT, or T2DM)c BMI, kg/m2 Overweight (BMI ⱖ 25 kg/m2) Physical activity ⱖ 30 min/d Healthy diet with a high content of vegetables and fruits Family social class, group V or VI
O-GDM
O-T1DM
O-NoGDM
O-BP
P
163
146
133
125
24.93 (5.95)a,b 37% (61/163)a,b
21.62 (2.32) 7% (9/138)
22.67 (4.28)a 23% (26/113)
21.29 (3.10) 11% (14/123)
⬍.0001 ⬍.0001
29% (48/163)a 91% (149/163)
22% (31/143) 99% (144/146)a
36% (44/122)a 93% (123/133)
16% (20/123) 92% (115/125)
.002 .039
55% (90/163) 3414.31 (533.84) 0.1964 (1.3377)b 10% (17/163) 18% (29/163)
45% (65/146) 3282.23 (754.58)a 1.1188 (1.7524)a 6% (8/146)a 42% (61/146)a
45% (60/133) 3488.31 (507.20) ⫺0.1797 (1.1804) 16% (21/133) 12% (17/133)
50% (62/125) 3477.26 (485) ⫺0.1678 (1.1019) 15% (18/124) 11% (14/124)
.21 .012 ⬍.0001 .029 ⬍.0001
21.5 (1.8)a 20% (33/163)a
22.6 (2.2) 12% (17/129)
21.1 (2.1)a 12% (16/132)
22.8 (2.2) 4% (5/128)
⬍.0001 .001
24.86 (4.92)a 39% (63/163)a 56% (91/163) 15% (24/163)
24.82 (5.17)a 40% (59/146)a 47% (68/146) 20% (29/146)
24.41 (4.61)a 28% (37/133) 54% (72/133) 13% (17/116)a
23.29 (3.56) 25% (31/125) 49% (61/125) 26% (33/125)
.020 .01 .34 .02
27% (43/162)a
19% (27/146)a
18% (24/132)
8% (10/125)
.001
Abbreviations: IFG, impaired fasting glucose; IGT, impaired glucose tolerance; SGA, small for gestational age; LGA, large for gestational age. Data are expressed as mean (SD) or percentage (number). Data include offspring with normal glucose tolerance, IFG, IGT, screen detected, treatmentnaive T2DM, and GADab-negative status. Analysis of differences (means or proportions) between the four groups was performed by ANOVA or 2, respectively. P values ⬍.05 are bold. a
Compared with the O-BP group, P ⬍ .05 (post hoc test, independent samples t test or 2). P values are multiplied by 4.
b
Compared with the O-NoGDM group, P ⬍ .05 (post hoc test, independent samples t test, or 2). P values are multiplied by 4.
c
Based on OGTT and evaluated according to World Health Organization criteria of 1999 (25).
No significant differences in the levels of glucagon were found between the four groups in the fasting state or at 120 minutes. At 30 minutes, a significant difference was found (P ⫽ .044) between the four groups, with O-GDM presenting the highest level or, in other terms, the least suppression. However, post hoc testing did not reveal individual significant differences between exposure and control groups (O-GDM vs O-BP, P ⫽ .072; O-GDM vs O-NoGDM, P ⫽ .064; O-T1DM vs O-BP, P ⫽ .616). No differences were found regarding 0 to 30- and 0 to 120-minute increments as well as total AUCs for any of the three hormones (Table 3). Multiple linear regression analyses When adjusted for potential confounders, the level of fasting GLP-1 was still significantly lower in the O-T1DM compared to the O-BP (, ⫺0.085; 95% CI, ⫺0.150 to ⫺0.020; P ⫽ .010), whereas the level in O-GDM was no longer significantly different from O-BP (, ⫺0.057; 95% CI, ⫺0.124 to 0.010; P ⫽ .092). As in the crude analyses, levels of GLP-1 did not differ between the four groups at 30 and 120 minutes during the OGTT, and no significant associations were found between the four groups and the levels of GIP and
glucagon, either in the fasting state or at 30 or 120 minutes during OGTT (Supplemental Table 1). The potential effect of offspring body weight—at birth and follow-up Inclusion of birth weight SDS and offspring BMI in the models did not change the associations between the exposed offspring (O-GDM and O-T1DM) and the outcomes (GLP-1, GIP, and glucagon). Levels of fasting GLP-1 were still significantly lower in O-T1DM compared to O-BP in the analysis including birth weight SDS (, ⫺0.084; 95% CI, ⫺0.153 to ⫺0.016; P ⫽ .016) and in analyses including offspring BMI (, ⫺0.084; 95% CI, ⫺0.149 to ⫺0.019; P ⫽ .012). Maternal blood glucose during pregnancy as exposure variable From the maternal OGTT in pregnancy (only from mothers of O-GDM and O-NoGDM), measures of blood glucose in the fasting state and at 120 minutes were included in the models. Maternal fasting blood glucose was in crude analyses associated with lower levels of fasting GLP-1 in the adult
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1972
Kelstrup et al
Table 2.
Incretin and Glucagon Levels in Adult Offspring
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
Levels of GLP-1, GIP, and Glucagon During OGTT
n (total ⫽ 567) GLP-1, pmol/La 0 min 30 min 120 min GIP, pmol/La 0 min 30 min 120 min Glucagon, pmol/L 0 min 30 min 120 min
O-GDM
O-T1DM
O-NoGDM
O-BP
P
163
146
133
125
13.40 (12.12–14.83)b 23.83 (21.89 –25.96) 16.23 (14.48 –18.19)
13.05 (12.03–14.16)b 23.44 (21.36 –25.72) 17.23 (15.92–18.65)
15.49 (14.06 –17.07) 23.96 (21.92–26.18) 16.83 (15.40 –18.40)
16.45 (14.59 –18.55) 24.67 (21.74 –27.99) 18.37 (16.26 –20.76)
.003 .91 .40
7.46 (6.71– 8.30) 52.55 (46.45–59.46) 17.43 (15.27–19.89)
8.57 (7.71–9.54) 51.30 (46.06 –57.15) 17.16 (15.85–18.58)
8.24 (7.21–9.42) 43.32 (37.52–50.03) 16.82 (15.390 –18.40)
8.09 (6.99 –9.36) 47.44 (41.14 –54.69) 18.76 (16.51–21.32)
.40 .15 .59
7.37 (4.15) 6.10 (3.63) 3.76 (3.15)
6.68 (3.21) 5.39 (3.52) 4.01 (2.70)
7.16 (3.41) 5.18 (3.63) 3.76 (2.17)
6.88 (3.48) 5.19 (2.83) 3.57 (2.47)
.36 .044 .15
Data are expressed as mean (SD) unless otherwise indicated. P values ⬍.05 are bold. The table includes offspring with normal glucose tolerance, impaired fasting glucose, impaired glucose tolerance, screen detected, treatment-naive T2DM, and GADab-negative status. Analyses of differences in means between the four groups were performed by one-way ANOVA. a
Data are given as geometric mean (95% CI) because of log-transformation to obtain normal distribution before entering the one-way ANOVA analyses.
b
Compared with O-BP, P ⬍ .05 (post hoc test, independent samples t test and Bonferroni correction).
offspring (, ⫺0.051; 95% CI, ⫺0.098 to ⫺0.003; P ⫽ .036) and associated with higher levels of GIP after 30 minutes (, 0.063; 95% CI, 0.001 to 0.126; P ⫽ .048). No association was found with GLP-1 and GIP at other time points, and none was found with glucagon at all measure points. When confounders and mediators were included in the models, no significant association was found between maternal fasting glucose and the three hormones during offspring OGTT (Supplemental Table 1). Maternal blood glucose at 120 minutes was in crude analyses associated with higher levels of glucagon after 30 and 120 minutes during the OGTT in the adult offspring (, 0.308; 95% CI, 0.123 to 0.493; P ⫽ .001; and , 0.247; 95% CI, 0.083 to 0.411; P ⫽ .003, respectively). When confounders were included in the model, the significant association to the levels of glucagon at 120 minutes in the offspring perTable 3.
sisted (, 0.184; 95% CI, 0.010 to 0.357; P ⫽ .038) (Supplemental Table 1), which also was the case when adult offspringBMIwasincludedasamediator;asignificantassociation to levels of glucagon at the end of offspring OGTT was found (, 0.098; 95% CI, 0.022 to 0.174; P ⫽ .012). In mothers with T1DM, neither first trimester nor third trimester mean maternal blood glucose level was associated with fasting or stimulated levels of GLP-1, GIP, or glucagon in the adult O-T1DM (Supplemental Table 1). Levels of GLP-1, GIP, and glucagon in subjects being overweight or with abnormal glucose tolerance The levels of GLP-1, GIP, and glucagon were investigated in the total cohort at follow-up according to the presence of overweight or abnormal OGTT, respectively
AUC for GLP-1, GIP, and Glucagon
n (total ⫽ 567) GLP-1a AUC_incremental 0 –30 AUC_incremental 0 –120 AUC_total GIPa AUC_incremental 0 –30 AUC_incremental 0 –120 AUC_total Glucagonb AUC_incremental 0 –30 AUC_incremental 0 –120 AUC_total
O-GDM
O-T1DM
O-NoGDM
O-BP
P
163
146
133
125
170 (143–202) 1096 (932–1288) 2660 (2405–2942)
154 (128 –187) 837 (690 –1014) 2447 (2262–2647)
144 (119 –174) 756 (610 –936) 2551 (2342–2779)
180 (142–228) 917 (698 –1204) 2822 (2486 –3203)
.418 .064 .238
659 (572–761) 4268 (3770 – 4832) 5249 (4683–5884)
621 (543–711) 4004 (3538 – 4532) 5349 (4812–5947)
536 (454 – 633) 3525 (3001– 4140) 4523 (3969 –5153)
565 (479 – 666) 3706 (3220 – 4266) 4980 (4393–5644)
.215 .209 .204
⫺19 (40) ⫺216 (284) 668 (390)
⫺19 (46) ⫺161 (604) 640 (638)
⫺30 (34) ⫺272 (246) 587 (270)
⫺25 (42) ⫺253 (289) 576 (294)
.088 .087 .237
Data include offspring with normal glucose tolerance, impaired fasting glucose, impaired glucose tolerance, and screen detected, treatment-naive T2DM. Analyses between proportions in the four groups were performed by one-way ANOVA. a
Data are given as geometric mean (CI).
b
Data are given as mean (SD).
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1973
Table 4. Levels of GLP-1, GIP, and Glucagon in Subjects With Overweight or Prediabetes/T2DM Compared to Normal Subjects n (total ⫽ 567) GLP-1a 0 min 30 min 120 min GIPa 0 min 30 min 120 min Glucagonb 0 min 30 min 120 min
Overweightc
Normal Weight
Prediabetes/T2DMd
Normal OGTT
190
377
71
495
13.50 (9.50 –20.63) 21.00 (15.00 –31.00) 15.00 (10.00 –21.00)
14.00 (10.50 –20.25) 25.00 (18.00 –35.00) 18.00 (13.00 –25.75)
.154 ⬍.0001 ⬍.0001
13.00 (8.50 –20.00) 20.00 (16.00 –28.00) 15.00 (10.00 –20.00)
14.00 (10.50 –20.50) 24.00 (17.00 –35.00) 17.00 (13.00 –25.00)
.159 .038 .014
7.00 (5.00 –13.00) 56.00 (35.25– 89.25) 37.00 (23.00 –59.50)
6.50 (5.00 –13.00) 50.00 (30.00 –79.00) 40.00 (25.00 – 65.00)
.769 .082 .384
7.50 (5.00 –16.50) 66.00 (40.25–101.50) 42.00 (24.00 –71.00)
6.50 (5.00 –13.00) 51.00 (30.00 –79.00) 39.00 (25.00 – 63.00)
.251 .016 .423
7.87 (3.83) 6.28 (3.55) 4.15 (2.86)
6.62 (3.41) 5.11 (2.99) 4.12 (6.52)
⬍.0001 ⬍.0001 .962
7.67 (3.73) 6.58 (3.82) 3.96 (2.56)
6.95 (3.58) 5.35 (3.12) 4.16 (5.89)
.117 .003 .781
P
P
Data include offspring with screen detected, treatment-naive T2DM and GADab-negative status. P values ⬎.05 are bold. a Analyses of differences were performed by Mann-Whitney U test. Data are not normally distributed and are given as median (25th–75th percentile). b
Analyses of differences in means were performed by independent t test. Data are given as mean (SD).
c
Overweight defined as BMI ⬎ 25 kg/m2.
d
Prediabetes (impaired fasting glucose), impaired glucose tolerance, or T2DM due to World Health Organization criteria (25).
(Table 4). In subjects being overweight, significantly lower levels of GLP-1 were found at 30 and 120 minutes during OGTT, and significantly higher glucagon levels were found in the fasting state and after 30 minutes at OGTT, compared to subjects being normal weight. GIP was comparable in overweight and normal-weight offspring at all measure points. In subjects with abnormal OGTT, levels of GLP-1 were significantly lower at 30 and 120 minutes, and levels of both GIP and glucagon at 30 minutes were higher compared to subjects with normal glucose tolerance.
Discussion Principal findings Adult offspring exposed to intrauterine hyperglycemia due to maternal GDM or T1DM presented significantly lower fasting levels of the incretin hormone GLP-1 compared to unexposed subjects, and the difference remained statistically significant in O-T1DM after adjustment for potential confounding factors and mediators. Furthermore, high maternal blood glucose levels at 120 minutes during the OGTT in pregnancy were associated with reduced postprandial suppression of glucagon in the offspring in adjusted analyses. Other studies One other study has evaluated the postprandial response of gut hormones in offspring from pregnancies complicated by maternal GDM (16). After a liquid meal challenge in 42 children aged 5–10 years, a suppressed
response of GLP-1 (incremental AUC) in O-GDM, independent of greater adiposity, was found. No difference in fasting levels was found. The authors concluded that fetal exposure to maternal GDM may alter the activity of the gut hormones and that this may contribute to the increased risk of obesity. In this context, our finding of lower levels of fasting GLP-1 in subjects exposed to intrauterine hyperglycemia is novel, but direct comparison between the two studies is not possible due to differences in the ages of offspring (children vs adults), the type of test (meal vs OGTT), and sample size. It has been debated whether the loss of the incretin effect is a primary event in the pathogenesis of T2DM or whether the condition is secondary to the development of T2DM, now with a general consensus for the latter (32– 35). Obesity, insulin resistance, and glucose intolerance have all been found associated with decreased secretion of GLP-1 (fasting and stimulated), hyperglucagonemia, and increased GIP secretion (35, 36). Our data are in accordance with this. Fetal programming of metabolism The hypothesis of fetal programming of metabolism is to some extent supported by our findings. We found significantly decreased levels of GLP-1 in the fasting state in O-T1DM compared to O-BP (both groups having low genetic risk of T2DM) in both crude and adjusted analyses. However, when comparing levels of fasting GLP-1 in the two groups with a genetic high risk of T2DM (O-GDM vs O-NoGDM), no significant difference was found. This may be due to a lack of power or because a high genetic
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predisposition may exceed the environmental effect of intrauterine exposure to maternal hyperglycemia. About 60% of the variations in circulating incretin levels are explained by genetic factors (37, 38), but little is known about these variants. Recently, however, a variant in the GIP receptor was reported to associate with BMI, glucose levels at 120 minutes during an OGTT, lower incretin effect, and T2DM (39, 40). Furthermore, numerous other factors may have potential for fetal programming (eg, maternal overweight and altered lipid profile), which in a genetically high-risk population could potentially overrule or potentate the effects of maternal hyperglycemia. We found that higher levels of maternal blood glucose during OGTT in pregnancy were associated with impaired suppression of glucagon after a glucose load in the adult offspring, suggesting that the glucagon function may also be altered/programmed by intrauterine hyperglycemia. This is a novel finding that calls for confirmation in other cohorts. Strengths and weaknesses of the study The present study is the first to evaluate circulating incretin and glucagon levels during the OGTT in adult offspring of women with diabetes during pregnancy and the first study to include offspring of women with T1DM. The study is based on a relatively large sample size and a high participation rate, and detailed objective information about pregnancy and delivery was available from maternal hospital files. Furthermore, information is included on many potential confounding factors at follow-up time. The study design includes four different groups, which enables evaluation of potential environmental effects of exposure to intrauterine hyperglycemia, separate from the effects of genetic predisposition. Our data on maternal blood glucose in pregnancy may today be considered to be rough estimates because selfmonitored glucose measurements (blood glucose profile) and glycated hemoglobin more accurately reflect blood glucose level during the daily lives of women during pregnancy. Unfortunately, these measures were not introduced into clinical practice in the period of 1978 –1985. A limitation of our study is the use of former Danish diagnostic criteria for GDM and the generalizability of our result thereby reduced. Information on potential confounding lifestyle factors in the adult offspring is based on self-estimated information in a questionnaire with the inherent theoretical risk of recall bias. Due to the large size of our cohort, our data are based on venous blood sampling during an OGTT, which gives a more limited insight into the pathophysiology, compared to more intensive and comprehensive studies including isoglycemic iv glucose infusion combined with incretin injections or clamping techniques. By this limita-
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
tion, we were able to evaluate the circulating levels of the incretin hormones during an OGTT, but we are not able to estimate the incretin effect. Conclusion Lower levels of fasting GLP-1 and impaired glucagon suppression in adult offspring exposed to maternal diabetes during pregnancy are diabetogenic traits and may contribute to glucose intolerance. However, further studies of the clinical implications and basal pathogenic mechanisms are needed to investigate the incretin function in offspring exposed to intrauterine hyperglycemia and to evaluate whether the findings can be attributed to an effect of fetal programming.
Acknowledgments We kindly thank J. Døssing, S. Polmann, K. M. Larsen, M. Wahl, and E. Stage for helpful assistance during data collection and all the persons who were participants in the study. Address all correspondence and requests for reprints to: Louise Kelstrup, MD, Center for Pregnant Women with Diabetes, Department of Obstetrics, Research Unit 7821, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: [email protected]. This work was supported by The Lundbeck Foundation, Civilingeniør H. C. Bechgaard’s Foundation, The Danish Diabetes Association, The Danish Medical Research Council, the Gangsted Foundation, A. P. Moeller og Hustru Chastine McKinney Moeller’s Foundation, Aase og Ejner Danielsen’s Foundation, The Augustinus Foundation, and The Research Foundation of Rigshospitalet. Disclosure Summary: The authors have nothing to disclose.
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ARTICLE
Incretin and Glucagon Levels in Adult Offspring Exposed to Maternal Diabetes in Pregnancy Louise Kelstrup, Tine D. Clausen, Elisabeth R. Mathiesen, Torben Hansen, Jens J. Holst, and Peter Damm Center for Pregnant Women with Diabetes (L.K., T.D.C., E.R.M., P.D.) and Departments of Obstetrics (L.K., P.D.) and Endocrinology (E.R.M.), Rigshospitalet, DK-2100 Copenhagen, Denmark; Department of Gynaecology and Obstetrics (T.D.C.), Nordsjaellands Hospital, 3400 Hillerød, Denmark; Institute of Clinical Medicine (E.R.M., P.D.), Faculty of Health Science, University of Copenhagen, 2200 Copenhagen, Denmark; Novo Nordisk Foundation (NNF) Center for Basic Metabolic Research (T.H.), Section of Metabolic Genetics, University of Copenhagen, 2200 Copenhagen, Denmark; Faculty of Health Sciences (T.H.), University of Southern Denmark, 5230 Odense M, Denmark; and NNF Center for Basic Metabolic Research and Department of Biomedical Sciences (J.J.H.), University of Copenhagen, 2200 Copenhagen, Denmark
Context: Fetal exposure to maternal diabetes is associated with increased risk of type 2 diabetes mellitus (T2DM) later in life. The pathogenesis of T2DM involves dysfunction of the incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), as well as hyperglucagonemia. Objective: Our aim was to investigate circulating plasma levels of GLP-1, GIP, and glucagon during the oral glucose tolerance test (OGTT) in adult offspring of women with diabetes in pregnancy. Design and Participants: We conducted a follow-up study of 567 offspring, aged 18 –27 years. We included two groups exposed to maternal diabetes in utero: offspring of women with diet-treated gestational diabetes mellitus (O-GDM; n ⫽ 163) or type 1 diabetes (O-T1DM; n ⫽ 146). Two reference groups were included: offspring of women with risk factors for GDM, but normoglycemia during pregnancy (O-NoGDM; n ⫽ 133) and offspring from the background population (O-BP; n ⫽ 125). The subjects underwent a 75-g OGTT with venous samples at 0, 30, and 120 minutes. Results: Fasting plasma levels of GLP-1 were lower in the two diabetes-exposed groups compared to O-BP (O-GDM, P ⫽ .040; O-T1DM, P ⫽ .008). Increasing maternal blood glucose during OGTT in pregnancy was associated with reduced postprandial suppression of glucagon in the offspring. Lower levels of GLP-1 and higher levels of glucagon during the OGTT were present in offspring characterized by overweight or prediabetes/T2DM at follow-up, irrespective of exposure status. Conclusion: Lower levels of fasting GLP-1 and impaired glucagon suppression in adult offspring exposed to maternal diabetes during pregnancy are diabetogenic traits that may contribute to glucose intolerance in these persons, but further investigations are needed. (J Clin Endocrinol Metab 100: 1967–1975, 2015)
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received October 31, 2014. Accepted March 11, 2015. First Published Online March 17, 2015
doi: 10.1210/jc.2014-3978
Abbreviations: AUC, area under the curve; BMI: body mass index; CI, confidence interval; GDM, gestational diabetes mellitus; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide-1; O-BP, offspring of mothers from the background Danish population without risk factors for diabetes; O-GDM, offspring of mothers with GDM; OGTT, oral glucose tolerance test; O-NoGDM, offspring of mothers with risk factors for GDM, but normoglycemic in pregnancy; O-T1DM, offspring of mothers with pregestational T1DM; SDS, birth weight SD score; T1DM, type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
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Incretin and Glucagon Levels in Adult Offspring
etal exposure to intrauterine hyperglycemia due to maternal diabetes is associated with increased risk of obesity, metabolic syndrome, prediabetes, and type 2 diabetes mellitus (T2DM) in the offspring (1–3), as well as impaired insulin function (4). The underlying pathology behind these associations is not known, but a fuelmediated mechanism of fetal programming is hypothesized (5, 6). The pathology in T2DM involves multiple defects of different organ systems, including the loss of the incretin effect, fasting hyperglucagonemia, and impaired suppression of glucagon in the postprandial state (7– 10). The incretin effect is the steeper rise in insulin release after oral ingestion of glucose, compared to the insulin response when glucose is infused iv to similar concentrations (isoglycemia). The effect is mediated through the insulinotropic action on the -cells by the two incretin hormones: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), both secreted from endocrine cells in the small intestine in response to oral intake of nutrients (8, 11, 12). The action of GLP-1, GIP, and glucagon has been investigated in different groups with high risk of T2DM (13–15), but only one study in children exposed to maternal gestational diabetes mellitus (GDM) has been published. The authors concluded that fetal exposure to maternal GDM may alter the activity of the gut hormones and that this may contribute to the increased risk of obesity (16). The aim of our study was to investigate the circulating levels of GLP-1, GIP, and glucagon in both fasting and oral glucose-stimulated conditions in adult offspring of women with either GDM or type 1 diabetes mellitus (T1DM) in pregnancy.
F
Subjects and Methods Study design A follow-up study was conducted of adult offspring of both sexes from all pregnancies complicated by either GDM or T1DM during 1978 –1985 at the Center for Pregnant Women with Diabetes, Rigshospitalet, Copenhagen, Denmark. An equal number of control subjects, randomly selected from healthy mothers giving birth at the Department of Obstetrics, Rigshospitalet, in the same period were invited to participate. Maternal baseline data, including blood glucose levels during pregnancy, and other relevant data from the period of pregnancy and the time of delivery were accessible from the mothers’ medical records at the archives of the Department of Obstetrics. Coupling between the mothers’ medical record and the adult offspring was possible through the Danish Civil Registrar System. The materials and methods have previously been described in detail (2). The study was in accordance with the Declaration of Helsinki and was approved by the regional
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
ethical committee. All participants gave written consent before inclusion.
Participants’ exposure status and genetic predisposition to diabetes (Figure 1) The offspring exposed to intrauterine hyperglycemia were subdivided according to maternal diabetes type and differences in genetic predisposition to T2DM as follows: 1. O-GDM: Offspring of women with diet-treated GDM. This group was estimated to have a high genetic predisposition to overweight and T2DM (17). 2. O-T1DM: Offspring of women with pregestational T1DM, and with an assumed low genetic predisposition to obesity and T2DM similar to the general Danish population. Likewise, the unexposed offspring were classified, according to maternal risk factors to GDM and predisposition to T2DM as follows: 3. O-NoGDM: Offspring of women without GDM, documented by a normal oral glucose tolerance test (OGTT) in pregnancy. The mothers underwent an OGTT due to the presence of risk factors for developing GDM. Based on the maternal phenotype, subjects in this group were assumed to have a high genetic predisposition to overweight and T2DM, comparable to the OGDM group. 4. O-BP: Offspring of women from the Danish background population, with an assumed low genetic predisposition to obesity and T2DM because the Danish population in general is a low-risk population.
Exclusion criteria Offspring with previously known diabetes (both T2DM and T1DM) and offspring being positive for GAD65 auto-antibodies (GAD65ab) were excluded (Figure 1).
Diabetes in pregnancy in the period 1978 –1985 In Denmark, screening for GDM has traditionally been based on risk factors (18 –20). During the period 1978 –1985, the following risk factors were used: ⱖ20% overweight before pregnancy, family history of diabetes, previous GDM, previous delivery of a child weighing ⬎4500 g, and glucosuria (18, 21). Women with risk indicators and two consecutive fasting capillary blood glucose measurements ⱖ4.1 mmol were offered a 3-hour 50-g OGTT. The OGTT was defined as abnormal if more than two of seven values during the test exceeded the mean ⫹ 3 SD for a reference group of normal-weight nonpregnant women without a family history of diabetes (20, 22). All mothers with GDM in this study were diet-treated only. Offspring of mothers with insulin-treated GDM (15% of all women with GDM) were excluded from participation to minimize the risk of misclassification among the mothers in the GDM group (eg, undiagnosed T2DM, early-stage T1DM, and MODY). Mothers of O-T1DM fulfilled three criteria: onset of diabetes at age ⱕ40 years, a classical history of hyperglycemic symptoms before disease diagnosis, and insulin treatment started ⱕ6 months after diagnosis. Mothers of O-NoGDM had risk factors qualifying for an OGTT, but the glucose values during the OGTT were all below the mean ⫹ 2 SD of the reference group.
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doi: 10.1210/jc.2014-3978
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1969
Potential eligible offspring Born between 1978-85 n=1066
Subjects exposed to intrauterine hyperglycemia in pregnancy (n=556 )
High genetic predisposition to overweight and T2DM (n=295)
N=469
N=30
Low genetic predisposition to overweight and T2DM (n=261)
Subject of mothers without diabetes (n=510)
High genetic predisposition to overweight and T2DM (n=254)
Low genetic predisposition to overweight and T2DM (n=256)
O-GDM
O-T1DM
O-NoGDM.
O-BP
Lost to follow up: n=127 (43%)
Lost to follow up: n=101 (39%)
Lost to follow up: n=113 (45%)
Lost to follow up: n=128 (50%)
O-GDM Exclusion due to: Diabetes: n=1 GADab pos.: n=4 (2%)
O-T1DM Exclusion due to: Diabetes: n=7 GADab pos.: n=7 (5%)
O-NoGDM. Exclusion due to: Diabetes: n=2 GADab pos.: n=6 (3%)
O-BP Exclusion due to Diabetes: n=0 GADab pos.: n=3 (1%)
O-GDM (participants) n=163 (55%)
O-T1DM (participants) n=146 (56%)
O-NoGDM (participants) n=133 (52%)
O-BP (participants) n=125 (49%)
Follow up in 2003-2005 Age 18-25 years Total included n=567 (53%) Figure 1. Study design. Flowchart of subjects participating in the study (n ⫽ 567), subjects lost to follow-up (n ⫽ 469; 44%), subjects excluded due to predefined criteria of previously known diabetes (both T2DM and T1DM), and offspring being positive for GAD65ab (n ⫽ 30; 3%).
Mothers of O-BP were from the local community and were routinely referred for antenatal care and delivery at Rigshospitalet, Copenhagen.
Examination of adult offspring at follow-up Venous blood samples were obtained in the fasting state, and participants without known diabetes underwent a 120-min 75-g OGTT with venous sampling at 30 and 120 minutes. Anthropometric measuresintermsof weight(kilograms)and height (meters) were obtained, andaquestionnairewithinformationonhealth,medication,diet,levels of physical activity, and socioeconomic parameters was completed.
Exposure variables Five different estimates of intrauterine hyperglycemia were used as exposure variables: 1) assignment into the four groups (O-GDM, O-T1DM, O-NoGDM, O-BP) was used as a surrogate measure of different exposures to intrauterine exposure and/or genetic predisposition to T2DM and obesity; 2) and 3) maternal OGTT data at 0 and 120 minutes, obtained from mothers of O-GDM and O-NoGDM only; 4) and 5), mean maternal blood glucose in the first and third trimesters from mothers of O-T1DM only.
Definition of variables Primary outcome variables Our main outcomes were GLP-1, GIP, and glucagon at three time points during OGTT: 0, 30, and 120 minutes. Secondary outcomes were incremental 0 –30 minutes, 0 –120 minutes, and total area under the curve (AUC) calculated by the trapezoidal method (23).
Covariates with references to the mother were: pregestational overweight (body mass index [BMI], kg/m2), family history of diabetes (unspecified diabetes in a first-degree relative on the maternal side [yes vs no]), and ethnicity (defined as Nordic Caucasian if the mother originated from Denmark, Norway, Sweden, or Iceland [yes vs no]). Covariates associ-
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J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
ated with the offspring were: gender (male sex [yes vs no]), age (years), level of physical activity (ⱖ30 vs ⬍30 min/d), quality of diet composition (divided into a diet with a high content of vegetables and fruits [“green diet”] vs a diet rich in meat and animal fat [“high animal fat diet”]), and socioeconomic position (based on the highest occupational status of the parents at the time of follow-up and coded into family social class I–V in accordance with the standards of the Danish National Institute of Social Research, similar to the British Registrar General’s Classification I–V). We added a social class VI representing people on transfer income, including sickness benefits and disability pension, and dichotomized the variable into family social class (I–IV vs V–VI) (24). OGTTs were evaluated according to World Health Organization criteria (25). Two covariates were considered to be mediators: birth weight SD score (SDS), calculated as the deviation from the population mean value in SD units adjusted for sex and gestational age (26); and offspring BMI at follow-up.
test. Correction for multiple comparisons was done by the Bonferroni method: we multiplied P values in the post hoc test by 4, because of the four possible comparisons (O-BP compared with the three other groups, and furthermore O-GDM with O-NoGDM) due to our hypothesis of an influence of intrauterine exposure to maternal hyperglycemia as well as genetic predisposition to obesity and T2DM. To test for associations between maternal blood glucose levels in pregnancy and the levels of the GLP-1, GIP, and glucagon during OGTT and to control for the effect of confounding, multiple linear regression analyses were done. Eight covariates chosen due to previous studies and theoretical considerations were included in the regression analyses. Two other covariates considered to be mediators were included in the basic models one by one to evaluate the effect. All tests were two-tailed, and a significance level of 0.05 was chosen. Data were processed using SPSS version 18 (SPSS Inc).
Biochemical analyses
Results
GLP-1, GIP, and glucagon concentrations in plasma were measured after extraction of plasma with 70% ethanol (vol/ vol, final concentration). The plasma concentrations of GLP-1 were measured (27) against standards of synthetic GLP-1 (7– 36) amide using antiserum code no. 89390, which is specific for the amidated C terminus of GLP-1 and therefore mainly reacts with GLP-1 of intestinal origin. The assay reacts equally with intact GLP-1 and with GLP-1 (3–36) amide, the primary metabolite. For the GIP RIA (28, 29), we used the C-terminally directed antiserum no. 867, which cross-reacts fully with human GIP but not with the so-called GIP 8000, whose chemical nature and relationship to GIP secretion is uncertain. The antiserum, which is similar to the previously employed R65, reacts equally with intact GIP and GIP 3– 42, the primary metabolite. Human GIP and 125-I human GIP (70 MBq/nmol) were used for standards and tracer. Because of the rapid and intravascular conversion of both GLP-1 and GIP to their primary metabolites, it is essential to determine the sum of the intact hormone and the metabolite for estimation of the plasma level of these hormones. The glucagon RIA was directed against the C terminus of the glucagon molecule (antibody code no. 4305) and therefore mainly measures glucagon of pancreatic origin (30, 31). For all three assays, sensitivity was ⬍1 pmol/L, intra-assay coefficient of variation was below 6% at 20 pmol/L, and recovery of standard, added to plasma before extraction, was about 100% when corrected for losses inherent in the plasma extraction procedure. GAD65ab were detected by ELISA (GAD65 Autoantibody Kit; RSR Ltd) and defined as positive when ⱖ5 U/mL.
Statistical analyses Normally distributed continuous data are presented as mean (SD), whereas non-normally distributed data are presented either as median (25th-75th percentiles) or as geometric mean (95% confidence interval [CI]). Results from the linear regression analyses are presented as regression coefficient (), 95% CI, and P value. Overall differences between the groups were analyzed with one-way ANOVA, 2 test, or Mann-Whitney U test when appropriate. As a post hoc test, and according to the hypothesis, specific comparisons between the groups were carried out by independent t test or 2
Data on mothers (1978 –1985) and characteristics of the study population Overall, 597 offspring participated in the study (56% of those eligible), but 30 subjects (3%) met the exclusion criteria (previously known T1DM or T2DM, n ⫽ 10; and GADab positive, n ⫽ 20) for analyses of incretin hormones and glucagon, giving a data set of 567 offspring (53%) (O-GDM, 163; O-T1DM, 146; O-NoGDM, 133; O-BP, 125) (Table 1 and Figure 1). Participants and subjects lost to follow-up were comparable in total and within the groups, and the mothers of O-GDM and O-NoGDM were comparable with respect to most risk factors for GDM, as previously described (2). Per definition, women with GDM had higher blood glucose levels than women in the NoGDM group: fasting glucose, 5.2 vs 4.7 mmol/L; and 2-hour glucose, 7.9 vs 5.2 mmol/L (P ⬍ .0001 for both). In women with T1DM, the mean blood glucose was 8.9 (2.8) mmol/L in the first trimester and 6.8 (1.8) mmol/L in the third trimester. Data on maternal characteristics and basic metabolic characteristics in the offspring at follow-up are given in Table 1 and have been published previously (2, 3). Incretin hormones and glucagon during OGTT (Tables 2 and 3) GLP-1 concentration in the fasting state was significantly lowerinthetwogroupsexposedtomaternaldiabetes(O-GDM and O-T1DM) compared to O-BP (P ⫽ .040 and P ⫽ .008, respectively). During the OGTT, the levels of GLP-1 did not differ significantly between the groups. No significant differences in the levels of GIP were found between the four groups, either in the fasting state or during the OGTT.
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Table 1. Baseline Data in Mothers and Offspring (1978 –1985) and Follow-up Characteristics in the Adult Offspring (2003–2005) n (total ⫽ 567) Maternal data BMI, kg/m2 Pregestational overweight (BMI ⱖ 25 kg/m2) Family history of diabetes Nordic Caucasian (ethnicity) Offspring birth data Male sex Birth weight, g Birth weight SDS SGA LGA Offspring follow-up data Age, y Abnormal glucose tolerance (IFG, IGT, or T2DM)c BMI, kg/m2 Overweight (BMI ⱖ 25 kg/m2) Physical activity ⱖ 30 min/d Healthy diet with a high content of vegetables and fruits Family social class, group V or VI
O-GDM
O-T1DM
O-NoGDM
O-BP
P
163
146
133
125
24.93 (5.95)a,b 37% (61/163)a,b
21.62 (2.32) 7% (9/138)
22.67 (4.28)a 23% (26/113)
21.29 (3.10) 11% (14/123)
⬍.0001 ⬍.0001
29% (48/163)a 91% (149/163)
22% (31/143) 99% (144/146)a
36% (44/122)a 93% (123/133)
16% (20/123) 92% (115/125)
.002 .039
55% (90/163) 3414.31 (533.84) 0.1964 (1.3377)b 10% (17/163) 18% (29/163)
45% (65/146) 3282.23 (754.58)a 1.1188 (1.7524)a 6% (8/146)a 42% (61/146)a
45% (60/133) 3488.31 (507.20) ⫺0.1797 (1.1804) 16% (21/133) 12% (17/133)
50% (62/125) 3477.26 (485) ⫺0.1678 (1.1019) 15% (18/124) 11% (14/124)
.21 .012 ⬍.0001 .029 ⬍.0001
21.5 (1.8)a 20% (33/163)a
22.6 (2.2) 12% (17/129)
21.1 (2.1)a 12% (16/132)
22.8 (2.2) 4% (5/128)
⬍.0001 .001
24.86 (4.92)a 39% (63/163)a 56% (91/163) 15% (24/163)
24.82 (5.17)a 40% (59/146)a 47% (68/146) 20% (29/146)
24.41 (4.61)a 28% (37/133) 54% (72/133) 13% (17/116)a
23.29 (3.56) 25% (31/125) 49% (61/125) 26% (33/125)
.020 .01 .34 .02
27% (43/162)a
19% (27/146)a
18% (24/132)
8% (10/125)
.001
Abbreviations: IFG, impaired fasting glucose; IGT, impaired glucose tolerance; SGA, small for gestational age; LGA, large for gestational age. Data are expressed as mean (SD) or percentage (number). Data include offspring with normal glucose tolerance, IFG, IGT, screen detected, treatmentnaive T2DM, and GADab-negative status. Analysis of differences (means or proportions) between the four groups was performed by ANOVA or 2, respectively. P values ⬍.05 are bold. a
Compared with the O-BP group, P ⬍ .05 (post hoc test, independent samples t test or 2). P values are multiplied by 4.
b
Compared with the O-NoGDM group, P ⬍ .05 (post hoc test, independent samples t test, or 2). P values are multiplied by 4.
c
Based on OGTT and evaluated according to World Health Organization criteria of 1999 (25).
No significant differences in the levels of glucagon were found between the four groups in the fasting state or at 120 minutes. At 30 minutes, a significant difference was found (P ⫽ .044) between the four groups, with O-GDM presenting the highest level or, in other terms, the least suppression. However, post hoc testing did not reveal individual significant differences between exposure and control groups (O-GDM vs O-BP, P ⫽ .072; O-GDM vs O-NoGDM, P ⫽ .064; O-T1DM vs O-BP, P ⫽ .616). No differences were found regarding 0 to 30- and 0 to 120-minute increments as well as total AUCs for any of the three hormones (Table 3). Multiple linear regression analyses When adjusted for potential confounders, the level of fasting GLP-1 was still significantly lower in the O-T1DM compared to the O-BP (, ⫺0.085; 95% CI, ⫺0.150 to ⫺0.020; P ⫽ .010), whereas the level in O-GDM was no longer significantly different from O-BP (, ⫺0.057; 95% CI, ⫺0.124 to 0.010; P ⫽ .092). As in the crude analyses, levels of GLP-1 did not differ between the four groups at 30 and 120 minutes during the OGTT, and no significant associations were found between the four groups and the levels of GIP and
glucagon, either in the fasting state or at 30 or 120 minutes during OGTT (Supplemental Table 1). The potential effect of offspring body weight—at birth and follow-up Inclusion of birth weight SDS and offspring BMI in the models did not change the associations between the exposed offspring (O-GDM and O-T1DM) and the outcomes (GLP-1, GIP, and glucagon). Levels of fasting GLP-1 were still significantly lower in O-T1DM compared to O-BP in the analysis including birth weight SDS (, ⫺0.084; 95% CI, ⫺0.153 to ⫺0.016; P ⫽ .016) and in analyses including offspring BMI (, ⫺0.084; 95% CI, ⫺0.149 to ⫺0.019; P ⫽ .012). Maternal blood glucose during pregnancy as exposure variable From the maternal OGTT in pregnancy (only from mothers of O-GDM and O-NoGDM), measures of blood glucose in the fasting state and at 120 minutes were included in the models. Maternal fasting blood glucose was in crude analyses associated with lower levels of fasting GLP-1 in the adult
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Table 2.
Incretin and Glucagon Levels in Adult Offspring
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
Levels of GLP-1, GIP, and Glucagon During OGTT
n (total ⫽ 567) GLP-1, pmol/La 0 min 30 min 120 min GIP, pmol/La 0 min 30 min 120 min Glucagon, pmol/L 0 min 30 min 120 min
O-GDM
O-T1DM
O-NoGDM
O-BP
P
163
146
133
125
13.40 (12.12–14.83)b 23.83 (21.89 –25.96) 16.23 (14.48 –18.19)
13.05 (12.03–14.16)b 23.44 (21.36 –25.72) 17.23 (15.92–18.65)
15.49 (14.06 –17.07) 23.96 (21.92–26.18) 16.83 (15.40 –18.40)
16.45 (14.59 –18.55) 24.67 (21.74 –27.99) 18.37 (16.26 –20.76)
.003 .91 .40
7.46 (6.71– 8.30) 52.55 (46.45–59.46) 17.43 (15.27–19.89)
8.57 (7.71–9.54) 51.30 (46.06 –57.15) 17.16 (15.85–18.58)
8.24 (7.21–9.42) 43.32 (37.52–50.03) 16.82 (15.390 –18.40)
8.09 (6.99 –9.36) 47.44 (41.14 –54.69) 18.76 (16.51–21.32)
.40 .15 .59
7.37 (4.15) 6.10 (3.63) 3.76 (3.15)
6.68 (3.21) 5.39 (3.52) 4.01 (2.70)
7.16 (3.41) 5.18 (3.63) 3.76 (2.17)
6.88 (3.48) 5.19 (2.83) 3.57 (2.47)
.36 .044 .15
Data are expressed as mean (SD) unless otherwise indicated. P values ⬍.05 are bold. The table includes offspring with normal glucose tolerance, impaired fasting glucose, impaired glucose tolerance, screen detected, treatment-naive T2DM, and GADab-negative status. Analyses of differences in means between the four groups were performed by one-way ANOVA. a
Data are given as geometric mean (95% CI) because of log-transformation to obtain normal distribution before entering the one-way ANOVA analyses.
b
Compared with O-BP, P ⬍ .05 (post hoc test, independent samples t test and Bonferroni correction).
offspring (, ⫺0.051; 95% CI, ⫺0.098 to ⫺0.003; P ⫽ .036) and associated with higher levels of GIP after 30 minutes (, 0.063; 95% CI, 0.001 to 0.126; P ⫽ .048). No association was found with GLP-1 and GIP at other time points, and none was found with glucagon at all measure points. When confounders and mediators were included in the models, no significant association was found between maternal fasting glucose and the three hormones during offspring OGTT (Supplemental Table 1). Maternal blood glucose at 120 minutes was in crude analyses associated with higher levels of glucagon after 30 and 120 minutes during the OGTT in the adult offspring (, 0.308; 95% CI, 0.123 to 0.493; P ⫽ .001; and , 0.247; 95% CI, 0.083 to 0.411; P ⫽ .003, respectively). When confounders were included in the model, the significant association to the levels of glucagon at 120 minutes in the offspring perTable 3.
sisted (, 0.184; 95% CI, 0.010 to 0.357; P ⫽ .038) (Supplemental Table 1), which also was the case when adult offspringBMIwasincludedasamediator;asignificantassociation to levels of glucagon at the end of offspring OGTT was found (, 0.098; 95% CI, 0.022 to 0.174; P ⫽ .012). In mothers with T1DM, neither first trimester nor third trimester mean maternal blood glucose level was associated with fasting or stimulated levels of GLP-1, GIP, or glucagon in the adult O-T1DM (Supplemental Table 1). Levels of GLP-1, GIP, and glucagon in subjects being overweight or with abnormal glucose tolerance The levels of GLP-1, GIP, and glucagon were investigated in the total cohort at follow-up according to the presence of overweight or abnormal OGTT, respectively
AUC for GLP-1, GIP, and Glucagon
n (total ⫽ 567) GLP-1a AUC_incremental 0 –30 AUC_incremental 0 –120 AUC_total GIPa AUC_incremental 0 –30 AUC_incremental 0 –120 AUC_total Glucagonb AUC_incremental 0 –30 AUC_incremental 0 –120 AUC_total
O-GDM
O-T1DM
O-NoGDM
O-BP
P
163
146
133
125
170 (143–202) 1096 (932–1288) 2660 (2405–2942)
154 (128 –187) 837 (690 –1014) 2447 (2262–2647)
144 (119 –174) 756 (610 –936) 2551 (2342–2779)
180 (142–228) 917 (698 –1204) 2822 (2486 –3203)
.418 .064 .238
659 (572–761) 4268 (3770 – 4832) 5249 (4683–5884)
621 (543–711) 4004 (3538 – 4532) 5349 (4812–5947)
536 (454 – 633) 3525 (3001– 4140) 4523 (3969 –5153)
565 (479 – 666) 3706 (3220 – 4266) 4980 (4393–5644)
.215 .209 .204
⫺19 (40) ⫺216 (284) 668 (390)
⫺19 (46) ⫺161 (604) 640 (638)
⫺30 (34) ⫺272 (246) 587 (270)
⫺25 (42) ⫺253 (289) 576 (294)
.088 .087 .237
Data include offspring with normal glucose tolerance, impaired fasting glucose, impaired glucose tolerance, and screen detected, treatment-naive T2DM. Analyses between proportions in the four groups were performed by one-way ANOVA. a
Data are given as geometric mean (CI).
b
Data are given as mean (SD).
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Table 4. Levels of GLP-1, GIP, and Glucagon in Subjects With Overweight or Prediabetes/T2DM Compared to Normal Subjects n (total ⫽ 567) GLP-1a 0 min 30 min 120 min GIPa 0 min 30 min 120 min Glucagonb 0 min 30 min 120 min
Overweightc
Normal Weight
Prediabetes/T2DMd
Normal OGTT
190
377
71
495
13.50 (9.50 –20.63) 21.00 (15.00 –31.00) 15.00 (10.00 –21.00)
14.00 (10.50 –20.25) 25.00 (18.00 –35.00) 18.00 (13.00 –25.75)
.154 ⬍.0001 ⬍.0001
13.00 (8.50 –20.00) 20.00 (16.00 –28.00) 15.00 (10.00 –20.00)
14.00 (10.50 –20.50) 24.00 (17.00 –35.00) 17.00 (13.00 –25.00)
.159 .038 .014
7.00 (5.00 –13.00) 56.00 (35.25– 89.25) 37.00 (23.00 –59.50)
6.50 (5.00 –13.00) 50.00 (30.00 –79.00) 40.00 (25.00 – 65.00)
.769 .082 .384
7.50 (5.00 –16.50) 66.00 (40.25–101.50) 42.00 (24.00 –71.00)
6.50 (5.00 –13.00) 51.00 (30.00 –79.00) 39.00 (25.00 – 63.00)
.251 .016 .423
7.87 (3.83) 6.28 (3.55) 4.15 (2.86)
6.62 (3.41) 5.11 (2.99) 4.12 (6.52)
⬍.0001 ⬍.0001 .962
7.67 (3.73) 6.58 (3.82) 3.96 (2.56)
6.95 (3.58) 5.35 (3.12) 4.16 (5.89)
.117 .003 .781
P
P
Data include offspring with screen detected, treatment-naive T2DM and GADab-negative status. P values ⬎.05 are bold. a Analyses of differences were performed by Mann-Whitney U test. Data are not normally distributed and are given as median (25th–75th percentile). b
Analyses of differences in means were performed by independent t test. Data are given as mean (SD).
c
Overweight defined as BMI ⬎ 25 kg/m2.
d
Prediabetes (impaired fasting glucose), impaired glucose tolerance, or T2DM due to World Health Organization criteria (25).
(Table 4). In subjects being overweight, significantly lower levels of GLP-1 were found at 30 and 120 minutes during OGTT, and significantly higher glucagon levels were found in the fasting state and after 30 minutes at OGTT, compared to subjects being normal weight. GIP was comparable in overweight and normal-weight offspring at all measure points. In subjects with abnormal OGTT, levels of GLP-1 were significantly lower at 30 and 120 minutes, and levels of both GIP and glucagon at 30 minutes were higher compared to subjects with normal glucose tolerance.
Discussion Principal findings Adult offspring exposed to intrauterine hyperglycemia due to maternal GDM or T1DM presented significantly lower fasting levels of the incretin hormone GLP-1 compared to unexposed subjects, and the difference remained statistically significant in O-T1DM after adjustment for potential confounding factors and mediators. Furthermore, high maternal blood glucose levels at 120 minutes during the OGTT in pregnancy were associated with reduced postprandial suppression of glucagon in the offspring in adjusted analyses. Other studies One other study has evaluated the postprandial response of gut hormones in offspring from pregnancies complicated by maternal GDM (16). After a liquid meal challenge in 42 children aged 5–10 years, a suppressed
response of GLP-1 (incremental AUC) in O-GDM, independent of greater adiposity, was found. No difference in fasting levels was found. The authors concluded that fetal exposure to maternal GDM may alter the activity of the gut hormones and that this may contribute to the increased risk of obesity. In this context, our finding of lower levels of fasting GLP-1 in subjects exposed to intrauterine hyperglycemia is novel, but direct comparison between the two studies is not possible due to differences in the ages of offspring (children vs adults), the type of test (meal vs OGTT), and sample size. It has been debated whether the loss of the incretin effect is a primary event in the pathogenesis of T2DM or whether the condition is secondary to the development of T2DM, now with a general consensus for the latter (32– 35). Obesity, insulin resistance, and glucose intolerance have all been found associated with decreased secretion of GLP-1 (fasting and stimulated), hyperglucagonemia, and increased GIP secretion (35, 36). Our data are in accordance with this. Fetal programming of metabolism The hypothesis of fetal programming of metabolism is to some extent supported by our findings. We found significantly decreased levels of GLP-1 in the fasting state in O-T1DM compared to O-BP (both groups having low genetic risk of T2DM) in both crude and adjusted analyses. However, when comparing levels of fasting GLP-1 in the two groups with a genetic high risk of T2DM (O-GDM vs O-NoGDM), no significant difference was found. This may be due to a lack of power or because a high genetic
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predisposition may exceed the environmental effect of intrauterine exposure to maternal hyperglycemia. About 60% of the variations in circulating incretin levels are explained by genetic factors (37, 38), but little is known about these variants. Recently, however, a variant in the GIP receptor was reported to associate with BMI, glucose levels at 120 minutes during an OGTT, lower incretin effect, and T2DM (39, 40). Furthermore, numerous other factors may have potential for fetal programming (eg, maternal overweight and altered lipid profile), which in a genetically high-risk population could potentially overrule or potentate the effects of maternal hyperglycemia. We found that higher levels of maternal blood glucose during OGTT in pregnancy were associated with impaired suppression of glucagon after a glucose load in the adult offspring, suggesting that the glucagon function may also be altered/programmed by intrauterine hyperglycemia. This is a novel finding that calls for confirmation in other cohorts. Strengths and weaknesses of the study The present study is the first to evaluate circulating incretin and glucagon levels during the OGTT in adult offspring of women with diabetes during pregnancy and the first study to include offspring of women with T1DM. The study is based on a relatively large sample size and a high participation rate, and detailed objective information about pregnancy and delivery was available from maternal hospital files. Furthermore, information is included on many potential confounding factors at follow-up time. The study design includes four different groups, which enables evaluation of potential environmental effects of exposure to intrauterine hyperglycemia, separate from the effects of genetic predisposition. Our data on maternal blood glucose in pregnancy may today be considered to be rough estimates because selfmonitored glucose measurements (blood glucose profile) and glycated hemoglobin more accurately reflect blood glucose level during the daily lives of women during pregnancy. Unfortunately, these measures were not introduced into clinical practice in the period of 1978 –1985. A limitation of our study is the use of former Danish diagnostic criteria for GDM and the generalizability of our result thereby reduced. Information on potential confounding lifestyle factors in the adult offspring is based on self-estimated information in a questionnaire with the inherent theoretical risk of recall bias. Due to the large size of our cohort, our data are based on venous blood sampling during an OGTT, which gives a more limited insight into the pathophysiology, compared to more intensive and comprehensive studies including isoglycemic iv glucose infusion combined with incretin injections or clamping techniques. By this limita-
J Clin Endocrinol Metab, May 2015, 100(5):1967–1975
tion, we were able to evaluate the circulating levels of the incretin hormones during an OGTT, but we are not able to estimate the incretin effect. Conclusion Lower levels of fasting GLP-1 and impaired glucagon suppression in adult offspring exposed to maternal diabetes during pregnancy are diabetogenic traits and may contribute to glucose intolerance. However, further studies of the clinical implications and basal pathogenic mechanisms are needed to investigate the incretin function in offspring exposed to intrauterine hyperglycemia and to evaluate whether the findings can be attributed to an effect of fetal programming.
Acknowledgments We kindly thank J. Døssing, S. Polmann, K. M. Larsen, M. Wahl, and E. Stage for helpful assistance during data collection and all the persons who were participants in the study. Address all correspondence and requests for reprints to: Louise Kelstrup, MD, Center for Pregnant Women with Diabetes, Department of Obstetrics, Research Unit 7821, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. E-mail: [email protected]. This work was supported by The Lundbeck Foundation, Civilingeniør H. C. Bechgaard’s Foundation, The Danish Diabetes Association, The Danish Medical Research Council, the Gangsted Foundation, A. P. Moeller og Hustru Chastine McKinney Moeller’s Foundation, Aase og Ejner Danielsen’s Foundation, The Augustinus Foundation, and The Research Foundation of Rigshospitalet. Disclosure Summary: The authors have nothing to disclose.
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