Online-Only Appendix

Subjects, biochemical assays and anthropometrical measurements

Details of the study populations are given in Online-OnlyAppendix Table 1. Patients with type 2 diabetes were recruited from the out-patient clinic at StenoDiabetesCenter (n=2,111), from the population-based Inter99 cohort established at Research Centre for Prevention and Health (1,2) (n=352, of these 252 were screen-detected and untreated), and from the ADDITION study (3) (n=1,626; all screen-detected and untreated). Glucose-tolerant subjects were recruited from StenoDiabetesCenter (n=521) or through the population-based Inter99 cohort (n=4,522). The treatment-naïve subjects from the Inter99 cohort included 5,970 subjects with NGT (n=4,522), IFG (n=503), IGT (n=693) and screen-detected and untreated type 2 diabetes (n=252).

The population-based sample of young healthy subjects was established at the Research Centre for Prevention and Health(4) (n=377).In the present study type 2 diabetes patients with diabetes due to known chronic pancreatitis, haemochromatosis, severe insulin resistance, maturity-onset diabetes of the young, maternally inherited diabetes and deafness, patients with a family history of first degree relatives with type 1 diabetes, patients with insulin requirement within the first year after diabetes diagnosis, patients with a fasting serum C-peptide level ≤ 150 pmol/liter at the time of recruitment, or patients tested GAD65 antibody positive were excluded in the present study from the category of clinically defined type 2 diabetes. States of impaired glucose regulation (IFG, IGT, and type 2 diabetes) was diagnosed in accordance with WHO 1999 criteria (5).

For participants from the population-based Inter99 cohort(1,2), the ADDITION study(3), as well as for samples recruited from the outpatient clinic of Steno Diabetes Center blood samples were drawn after a 12-h overnight fast. Plasma-glucose was analyzed by a glucose oxidase method (Granutest, Merck, Darmstadt, Germany). HbA1C was measured by ion-exchange high performance liquid chromatography (normal reference range: 4.1-6.4 %) and serum-insulin (excluding des(31, 32) and intact proinsulin) was measured using the AutoDELFIA insulin kit (Perkin-Elmer, Wallac, Turku, Finland). HOMA-IR (mmol/l×pmol/l) was calculated as fasting plasma-glucose (mmol/l) multiplied by fasting serum-insulin (pmol/l) and divided by 22.5.Insulinogenic indexinsulin was calculated as (s-insulin at 30 minutes [pmol/l] - fasting s-insulin [pmol/l]) / p-glucose at 30 minutes (mmol/l). BIGTT-insulin sensitivity index (BIGTT-SI) and BIGTT-acute insulin response (BIGTT-AIR) usesinformation on sex and BMI combined with analysis of plasma-glucose and serum-insulin levels at the time points 0, 30, and 120 min to provide indexes for Si and AIR that are highly correlated with indexes obtained during an IVGTT, and were calculated as described elsewhere (6).

Each of the 377 young healthy Danish Caucasiansunderwent an intravenous glucose tolerance test (IVGTT) after a 12-h overnight fast, i.e. an intravenous injection of 0.3 g glucose per kg body weight at time zero in combination with intravenous injection of 3 mg tolbutamide per kg body weight after 20 min (4). The insulin sensitivity index was calculated using the Bergman MINMOD computer programme developed specifically for the combined intravenous glucose and tolbutamide tolerance test (7). The plasma-glucose concentration was measured by an automated glucose oxidase method (Granu-test: Merck, Damstadt, Germany) and concentration of insulin (excluding des(31, 32)- and intact proinsulin) in serum was measured by ELISA applying the Dako insulin kit with overnight incubation (code NO. K6219; Dako Diagnostics Ltd., Ely, United Kingdom). Acute serum-insulin response 0-8 minutes (pmol/l×min) was calculated applying the trapezoidal rule as the incremental values. Disposition index was calculated as insulin sensitivity index (10-5×(min×pmol/l)-1) multiplied by acute serum-insulin response 0-8 minutes (pmol/l×min) multiplied by 10-4. Values of Δ p-glucose and s-insulin 2 min. post tolbutamide (pmol/l) were calculated as measured values at 22 minus 19 minuteswith a tolbutamide injection given at 20 minutes.

Height and body weight for all participants were measured in light indoor clothes and without shoes, and BMI was calculated as weight (kg)/(height (m))2.

Online-Only Appendix Table 1Clinical characteristics of 10,705 examined Danish people

Population-based study of young healthy subjects / Inter99 / ADDITION / StenoDiabetesCenter
Treatment-naïve subjects / Glucose tolerant
subjects / Type 2 diabetes
patients / Type 2 diabetes patients / Glucose-tolerant
subjects / Type 2 diabetes patients
n / 377 / 5,970 / 4,522 / 352 / 1,626 / 521 / 2,111
men/women / 186/191 / 2,972/2,998 / 2,102/2,420 / 215/137 / 924/702 / 244/277 / 1,287/824
Age (years) / 25.3 ± 3.5 / 46.1 ± 7.9 / 45.2 ± 7.8 / 51.0 ± 7.1 / 60.2 ± 6.8 / 56.6 ± 10.0 / 62.1 ± 11.3
BMI (kg/m2) / 23.6 ± 3.7 / 26.2 ± 4.5 / 25.5 ± 4.1 / 30.4 ± 5.6 / 31.1 ± 5.4 / 25.8 ± 3.7 / 30.1 ± 5.6
HbA1C (%) / NA / 5.8 ± 0.5 / 5.8 ± 0.4 / 7.0 ± 1.6 / 6.6 ± 1.3 / 5.8 ± 0.5 / 8.1 ± 1.6
Fasting glucose (mmol/l) / 5.0 ± 0.5 / 5.5 ± 0.8 / 5.3 ± 0.4 / 8.3 ± 3.1 / 6.9 ± 2.0* / 5.1 ± 0.4 / 9.7 ± 3.5
Waist (cm) / 78 ± 11 / 86 ± 13 / 84 ± 12 / 98 ± 14 / 104 ± 14 / 87 ± 11 / 104 ± 15
s-Cholesterol (mmol/l) / 4.5 ± 0.8 / 5.5 ± 1.1 / 5.4 ± 1.0 / 5.8 ± 1.1 / 5.7 ± 1.1 / 6.0 ± 1.1 / 5.7 ± 1.2
s-HDL-cholesterol (mmol/l) / 1.2 ± 0.3 / 1.4 ± 0.4 / 1.5 ± 0.4 / 1.3 ± 0.4 / 1.4 ± 0.4 / 1.5 ± 0.4 / 1.1 ± 0.3
s-triglyceride (mmol/l) / 1.1 ± 0.7 / 1.3 ± 1.3 / 1.2 ± 0.9 / 2.1 ± 1.6 / NA / 1.3 ± 0.7 / 2.2 ± 1.9
Systolic BP(mmHg) / 115 ± 12 / 130 ± 17 / 127 ± 16 / 145 ± 21 / 144 ± 19 / 130 ± 17 / 146 ± 23
Diastolic BP (mmHg) / 65 ± 9 / 82 ± 11 / 81 ± 11 / 89 ± 12 / 86 ± 10 / 77 ± 10 / 83 ± 11

Legend to Online-Only Appendix Table 1

Data are means ± standard deviation.* In the ADDITION study fasting glucose is measured on capillary blood. BP, blood pressure; NA, not available; S, serum.

Online-Only Appendix Table 2 Quantitative metabolic traits in the population-based Inter99 cohort including 4,522 middle-aged subjects with normal glucose tolerance stratified according to genotype

rs1111875 (HHEX/KIF11/IDE)
TT / TC / CC / padd / pdom / prec
n / 789 / 2,027 / 1,520
men/women / 374/412 / 937/1090 / 694/826
Age (years) / 45.5±7.7 / 45.3±7.8 / 45.1±7.9
BMI (kg/m2) / 25.4±4.1 / 25.6±4.1 / 25.4±4 / 0.82 / 0.56 / 0.42
Fasting s-insulin (pmol/l) / 32 (23;46) / 31 (22;46) / 37±23 / 0.77 / 0.91 / 0.73
S-insulin at 30 min (pmol/l) / 255 (188;366) / 247 (178;352) / 233 (170;330) / 4×10-8 / 0.00011 / 4×10-7
S-insulin at 120 min (pmol/l) / 141 (88;217) / 137 (88;211) / 135 (85;205) / 0.1 / 0.28 / 0.12
Fasting p-glucose (mmol/l) / 5.3±0.4 / 5.3±0.4 / 5.3±0.4 / 0.61 / 0.28 / 0.91
P-glucose at 30 min (mmol/l) / 8.1±1.5 / 8.2±1.5 / 8.2±1.6 / 0.012 / 0.032 / 0.044
P-glucose at 120 min (mmol/l) / 5.5±1.1 / 5.5±1.1 / 5.5±1.1 / 0.8 / 0.57 / 0.93
HOMA-IR (mmol/l×pmol/l) / 7.3 (5.2;11.0) / 7.5 (5.2;11.2) / 7.5 (5.2;10.7) / 0.82 / 0.97 / 0.72
BIGTT-SI / 10.2 (7.6;13.0) / 10.2 (7.7;12.6) / 10.4 (8.0;13.0) / 0.075 / 0.44 / 0.043
Insulinogenic index insulin / 27.3 (19.5;41.7) / 26.5 (18.2;38.1) / 24.2 (17.1;36.2) / 2×10-10 / 6×10-6 / 6×10-9
BIGTT-AIR / 1,710 (1,400;2,150) / 1,700 (1,370;2,120) / 1,630 (1,310;2,080) / 2×10-5 / 0.0021 / 8×10-5
rs10811661 (CDKN2A/B)
CC / CT / TT / padd / pdom / prec
n / 128 / 1,234 / 3,035
men/women / 60/68 / 587/647 / 1,396/1,639
Age (years) / 45.9±8.2 / 45.4±7.8 / 45.2±7.8
BMI (kg/m2) / 25.9±4.1 / 25.5±4.1 / 25.5±4.1 / 0.64 / 0.29 / 0.88
Fasting s-insulin (pmol/l) / 34 (25;44) / 31 (22;47) / 31 (22;45) / 0.69 / 0.57 / 0.8
S-insulin at 30 min (pmol/l) / 239 (163;352) / 249 (178;355) / 240 (176;341) / 0.066 / 0.95 / 0.037
S-insulin at 120 min (pmol/l) / 156 (94;220) / 141 (93;211) / 135 (86;210) / 0.02 / 0.38 / 0.019
Fasting p-glucose (mmol/l) / 5.3±0.4 / 5.3±0.4 / 5.3±0.4 / 0.25 / 0.6 / 0.13
P-glucose at 30 min (mmol/l) / 8.5±1.6 / 8.1±1.5 / 8.2±1.5 / 0.98 / 0.035 / 0.46
P-glucose at 120 min (mmol/l) / 5.7±1.2 / 5.5±1.1 / 5.5±1.1 / 0.54 / 0.14 / 0.86
HOMA-IR (mmol/l×pmol/l) / 8.0 (5.8;10.8) / 7.4 (5.2;11.1) / 7.5 (5.1;10.9) / 0.8 / 0.52 / 0.95
BIGTT-SI / 9.9 (7.2;12.6) / 10.4 (7.7;12.8) / 10.3 (7.8;12.9) / 0.32 / 0.34 / 0.43
Insulinogenic index insulin / 25.0 (17.6;38.2) / 26.2 (18.2;38.8) / 25.7 (17.9;37.2) / 0.097 / 0.58 / 0.034
BIGTT-AIR / 1,710 (1,370;1,980) / 1,720 (1,360;2,170) / 1,660 (1,350;2,090) / 0.12 / 0.66 / 0.049
rs4402960 (IGF2BP2)
GG / GT / TT / padd / pdom / prec
n / 2,127 / 1,819 / 407
men/women / 990/1,137 / 847/972 / 183/224
Age (years) / 45.2±7.8 / 45.2±7.9 / 45.7±7.8
BMI (kg/m2) / 25.5±4.1 / 25.5±4 / 25.6±4.3 / 0.79 / 0.89 / 0.71
Fasting s-insulin (pmol/l) / 31 (22;44) / 32 (23;46) / 32 (22;48) / 0.28 / 0.47 / 0.25
S-insulin at 30 min (pmol/l) / 246 (176;346) / 242 (176;343) / 237 (179;349) / 0.37 / 0.49 / 0.42
S-insulin at 120 min (pmol/l) / 135 (86;211) / 140 (88;209) / 137 (86;210) / 0.25 / 0.34 / 0.34
Fasting p-glucose (mmol/l) / 5.3±0.4 / 5.3±0.4 / 5.3±0.4 / 0.022 / 0.12 / 0.013
P-glucose at 30 min (mmol/l) / 8.2±1.5 / 8.2±1.5 / 8.2±1.5 / 0.97 / 0.96 / 0.85
P-glucose at 120 min (mmol/l) / 5.5±1.1 / 5.5±1.1 / 5.6±1.1 / 0.1 / 0.096 / 0.42
HOMA-IR (mmol/l×pmol/l) / 7.5 (5.1;10.8) / 7.5 (5.2;11.1) / 7.5 (5.2;11.1) / 0.47 / 0.62 / 0.44
BIGTT-SI / 10.4 (7.9;13.0) / 10.3 (7.8;12.7) / 10.3 (7.6;12.9) / 0.41 / 0.37 / 0.77
Insulinogenic index insulin / 26.0 (18.0;37.9) / 25.8 (18.1;37.7) / 25.3 (17.8;37.7) / 0.32 / 0.5 / 0.29
BIGTT-AIR / 1,670 (1,350;2,090) / 1,680 (1,360;2,110) / 1,670 (1,340;2,170) / 0.46 / 0.5 / 0.62
rs9300039
AA / AC / CC / padd / pdom / prec
n / 39 / 704 / 3,605
men/women / 18/21 / 319/385 / 1,680/1,925
Age (years) / 46.7±7.1 / 44.9±7.9 / 45.3±7.9
BMI (kg/m2) / 25.2±4.5 / 25.2±3.9 / 25.6±4.1 / 0.045 / 0.6 / 0.041
Fasting s-insulin (pmol/l) / 29 (20;38) / 32 (22;47) / 31 (22;45) / 0.71 / 0.82 / 0.64
S-insulin at 30 min (pmol/l) / 244 (181;353) / 242 (179;338) / 243 (176;346) / 0.64 / 0.63 / 0.7
S-insulin at 120 min (pmol/l) / 135 (88;204) / 131 (85;195) / 139 (87;213) / 0.31 / 0.54 / 0.22
Fasting p-glucose (mmol/l) / 5.3±0.4 / 5.3±0.4 / 5.3±0.4 / 0.37 / 0.31 / 0.47
P-glucose at 30 min (mmol/l) / 8.3±1.7s / 8.1±1.5 / 8.2±1.5 / 0.16 / 0.95 / 0.13
P-glucose at 120 min (mmol/l) / 5.4±1 / 5.4±1.1 / 5.5±1.1 / 0.018 / 0.71 / 0.014
HOMA-IR (mmol/l×pmol/l) / 6.5 (4.5;8.9) / 7.6 (5.2;11.1) / 7.5 (5.2;11.0) / 0.82 / 0.72 / 0.74
BIGTT-SI / 11.6 (7.6;13.8) / 10.6 (8.1;13.0) / 10.3 (7.8;12.8) / 0.13 / 0.74 / 0.12
Insulinogenic index insulin / 25.1 (17.8;46.2) / 25.9 (18.9;37.2) / 25.9 (17.9;37.9) / 0.44 / 0.52 / 0.5
BIGTT-AIR / 1,730 (1,400;2,230) / 1,670 (1,360;2,080) / 1,680 (1,350;2,120) / 0.79 / 0.66 / 0.69

Legend to Online-Only Appendix Table 2

Data are median (interquantile range) or means ± standard deviation (age, BMI, and values of plasma-glucose). Values of plasma-glucose, serum-insulin, homeostasis model assessment of insulin resistance (HOMA-IR), insulinogenic index insulin, and BIGTT-acute insulin response (BIGTT-AIR) were logarithmically transformed before statistical analysis. Calculated p-values were adjusted for age, sex, and BMI, where appropriate, assuming an additive (padd), dominant (pdom), or a recessive (prec) model. HOMA-IR was calculated as fasting plasma-glucose (mmol/l) multiplied by fasting serum-insulin (pmol/l) and divided by 22.5. Insulinogenic indexinsulin was calculated as (s-insulin at 30 minutes [pmol/l] - fasting s-insulin [pmol/l]) / p-glucose at 30 minutes (mmol/l).BIGTT-insulin sensitivity index (BIGTT-SI) and BIGTT-AIR usesinformation on sex and BMI combined with analysis of plasma-glucose and serum-insulin levels at the time points 0, 30, and 120 min to provide indexes for Si and AIR which highly correlates with indexes obtained during an IVGTT, and were calculated as described elsewhere (6). P, plasma; s, serum.

References

1. Jørgensen T, Borch-Johnsen K, Thomsen TF, Ibsen H, Glumer C, Pisinger C: A randomized non-pharmacological intervention study for prevention of ischaemic heart disease: Baseline results Inter99 (1). Eur J Cardiovasc Prev Rehab 10:377-386, 2003

2. Glümer C, Jørgensen T, Borch-Johnsen K: Prevalences of diabetes and impaired glucose regulation in a Danish population: the Inter99 study. Diabetes Care 26:2335-2340, 2003

3. Lauritzen T, Griffin S, Borch-Johnsen K, WarehamNJ, Wolffenbuttel BH, Rutten G, for the ADDITION Study Group: The ADDITION study: proposed trial of the cost-effectiveness of an intensive multifactorial intervention on morbidity and mortality among people with Type 2 diabetes detected by screening. Int J Obes Relat Metab Disord 24 Suppl 3:S6-11, 2000

4. Clausen JO, Borch-Johnsen K, Ibsen H, Bergman RN, Hougaard P, Winther K, Pedersen O: Insulin sensitivity index, acute insulin response, and glucose effectiveness in a population-based sample of 380 young healthy Caucasians. Analysis of the impact of gender, body fat, physical fitness, and life-style factors. J Clin Invest 98:1195-1209, 1996

5. World Health Organization Study Group: Definition, Diagnosis and Classification of Diabetes Mellitus and its Complications. Part 1: Diagnosis and Classification of Diabetes Mellitus. Geneva, World Health Organization, 1999,

6. Hansen T, Drivsholm T, Urhammer SA, Palacios RT, Vølund A, Borch-Johnsen K, Pedersen O: The BIGTT test: a novel test for simultaneous measurement of pancreatic -cell function, insulin sensitivity, and glucose tolerance. Diabetes Care 30:257-262, 2007

7. Bergman RN, Prager R, Volund A, Olefsky JM: Equivalence of the insulin sensitivity index in man derived by the minimal model method and the euglycemic glucose clamp. J Clin Invest 79:790-800, 1987

1