The study included subjects recruited from the Diabetic and Endocrine Outpatient Clinic of the Laiko University Hospital in Athens. The study was approved by the Scientific Committee of “Laiko” University Hospital (47/14.01.2013).
We recruited patients with pre-diabetes defined by the presence of one of the following criteria: i) IGT defined by serum glucose level at 120minutes ≥ 140 mg/dl but < 200 mg/dl, following a formal 75 g OGTT, ii) IFG defined by fasting serum glucose levels ≥ 100 but < 126 mg/dl) according to ADA criteria (17). Autoimmune thyroid disease was defined by the presence of TgAb and/or TPOAb antibodies. The population was divided into two groups, patients with AITD and patients without AITD.
Αll subjects under any medication known to affect glucose metabolism, those with abnormal thyroid function (thyroid stimulating hormone (TSH) ≥ 5 µIU/ml defined as subclinical/ clinical hypothyroidism or TSH < 0.5 µIU/ml as subclinical/ clinical hyperthyroidism) and individuals with thyroidectomy were excluded. Pregnant women and individuals with a history of hospitalization during the last 6 months, and hemoglobin levels less than 12 g/dl were also excluded from the study.
Oral glucose tolerance test was performed after a 10-h overnight fast. Serum glucose (mg/dl) and insulin levels (µIU/ml), were measured at baseline and at 30-min intervals (30’, 60’, 90’, 120’). Glycosylated haemoglobulin (HbA1c,%) and high-sensitive C-reactive protein (hs-CRP) (mg/L) were also measured. Plasma glucose, total cholesterol, High Density Lipoprotein (HDL)-Cholesterol, Low Density Lipoprotein (LDL)-cholesterol and triglycerides were measured as previously described (18). Percent concentration of HbA1c was performed in vitro in whole blood with an immunological method (tholosimetric suppression immunoanalysis, ΤΙΝΙα) in an automatic analyzer of clinical chemistry (Hitachi 912, Roche, France). Serum insulin was measured with the immunoradiometric assay IRMA (DIAsource ImmunoAssays, Louvain-la-Neuve, Belgium). High-sensitivity CRP (hsCRP, mg/L, high sensitivity CRP enzyme immunoassay test kit, LI7500, Linear Chemicals, S.L., 08390 Montgat, Barcelona, Spain) serum levels were determined by enzyme-linked immunosorbent assay. The intra- and interassay coefficients of variance for hsCRP were 7.5 and 4.1% for low levels and 2.3 and 2.5% for high levels, respectively.
Arterial hypertension was diagnosed according to each individual’s medical and drug history or in the presence of systolic blood pressure (SBP) ≥ 135 mmHg and/or diastolic blood pressure (DBP) ≥ 85 mmHg) [mean value of latest European guidelines (SBP/DBP > 140/90 mm Hg) and the American guidelines (SBP/DBP < 130/80 mm Hg) (18–21). Dyslipidaemia was diagnosed on the basis of LDL levels ≥ 130 mg/dl and the administration of hypolidaemic drugs (22).
Body weight was measured using analogue scales in light clothing; height was measured bare-foot using a stadiometer. Body mass index (BMI, kg/m2) was calculated to assess obesity and waist and waist-to-height ratio (WHR) to assess body fat distribution.
Antibodies to glutamic acid decarboxylase (anti-GAD) were randomly measured in the first 25 persons that were enrolled into the study to test for the case of persons with Latent Autoimmune Diabetes of Adults.
Indices Of Carbohydrate Metabolism
Insulin resistance indices (IRI)
1/fasting insulin, fasting glucose-to-insulin ratio (GIR), the quantitative insulin sensitivity check index (QUICKI), and insulin resistance Homeostasis model assessment (HOMA) (HOMA-IR) were used to assess insulin action (23) in the fasting state, using the following formulas:
QUICKI = 1/ (log(fasting insulin µU/mL) + log(fasting glucose mg/dL) (24)
HOMA-IR = fasting insulin (μΙU/ml) x fasting glucose (mmol/ml)/22.5 (25).
The Matsuda index was used to assess dynamic insulin action using the following formula:
Matsuda index= (10,000/square root of [fasting glucose x fasting insulin] x [mean glucose x mean insulin during OGTT]) (26)
β-cell secretion indices (ISI)
Static ISI (SISI)
HOMA index of β-cell function (HOMA-B) was calculated using the following formula:
HOMA-B = 20 × fasting insulin (μIU/ml)/[fasting glucose (mmol/ml) − 3.5] (27).
First phase and second phase insulin secretion and the incremental area under the insulin to glucose curve (incAUCins/glu) were used using the following formulas (28):
Predicted index of first phase of insulin secretion (1st PHIS) = 1283 + [1.289 x insulin at 30 minutes (pmol/L)] - [138.7 x glucose at 30 minutes (mmol/L)] +[3.772 x insulin at baseline (pmol/L)] (29)
Predicted index of second phase of insulin secretion (2nd PHIS) = 287 + [0.4164 x insulin at 30 minutes (pmol/L)] - [26.07 x glucose at 30 minutes (mmol/L)] + [0.9226 x insulin at baseline (pmol/L)] (29)
IncAUCins/glu by the trapezoidal method from 0’ to 120’ min (23)
The combined index of insulin action and β-cell secretion is expressed by the disposition index (DI) and the following formula is used:
Oral disposition index (DI) = ΔI0–30/ΔG0–30× 1/fasting insulin (27).
The presence of insulin resistance was defined as previously described (28): HOMA-IR > 2.16 and/or QUICKI < 0.34 values.
The serum TSH levels were measured by a sensitive two-site chemiluminescent immunometric assay with analytical sensitivity: 0.004 μIU/mL, and the coefficient of variation (CV) is less than 5.5% for TSH values comprising between 0.3 and 10 μUI/mL. Thyroid antibodies: TgAb < 40 U/mL with analytical sensitivity 20 U/mL with an intra- and inter-assay CV of 3.2% and 4.6%, respectively and TPOAb < 30 U/mL with analytical sensitivity 10 U/mL with an intra- and inter-assay CV of 5.2% and 3.2%, respectively (IMMULITE 2000 SIEMENS Healthcare Diagnostics Products Ltd. Llanberis, Gwynedd LL55 4EL United Kingdom). High-sensitive CRP (reference range <5 mg/L) was determined with a highly sensitive latex-based immunoassay. Anti-GAD antibodies were measured by ELISA (Anachem Ltd, Luton, UK).
A between-group comparison of non-continuous variables was carried out by Chi-squared test corrected by Fisher’s exact test when appropriate, while for. continuous variables a t-test student for parametric and Mann-Whitney U test for non-parametric variables was used. Parametric variables are presented as mean value±standard deviation (SD), and non-parametric variables as median values, interquartile range (IQR), minimum-to-maximum values range). A p value<0.05 was considered as statistically significant. SPSS software (SPSS 16. Inc. Chicago, IL) was used for the statistical analysis.