The Verona Newly Diagnosed Type 2 Diabetes Study

The Verona Newly Diagnosed Type 2 Diabetes Study (VNDS) is an ongoing study aiming at building a biobank of patients with newly diagnosed type 2 diabetes mellitus. All patients referred to the Division of Endocrinology and Metabolic Diseases of University of Verona School of Medicine, whose diabetes has been diagnosed in the last six months, are asked to participate in this research. The clinical evidence on which the diagnosis of type 2 diabetes has been made is reviewed and the diagnosis confirmed, according to the current criteria of American Diabetes Association. Patients already treated with antidiabetic drugs undergo a treatment washout of at least one week before metabolic tests are performed. Among the exclusion criteria are age > 75 years, non-Italian ancestry, insulin treatment, presence of anti-GAD antibodies, malignancies, and any condition severely impairing liver and/or kidney function.

All subjects consume a weight-maintaining diet containing 200-250 g of carbohydrate/day for at least three days before studies. Body weight must be stable in all subjects for at least 1 month before studies. No subject should participate in any heavy exercise. Each subject gives informed written consent before participating in the research, which was approved by the Human Investigation Committee of the Verona City Hospital. Measurements of standard clinical phenotypes are collected in all patients. Other diabetes related phenotypes may be collected if their determination is available.

Metabolic tests are carried out on two separate days in random order. On both days, patients are admitted to the Metabolic Clinic Research Center at 07:30 after an overnight fast. All studies are carried out in a quiet, temperature controlled (22° C) room. On one day an oral glucose tolerance test (OGTT) (75 g) is performed to assess beta cell function. On a separate day, a euglycemic insulin clamp is performed to assess insulin sensitivity.

When age of onset and distribution of the disease in the pedigree suggest a potentially monogenic disorder, the relatives of the proband are asked to participate in the study by allowing the collection of standard clinical information and of a fasting blood sample for genetic and phenotypic determinations.

-OGTT: For ethical reasons, the OGTT cannot performed in patients presenting with fasting plasma glucose higher than 15 mmol/l. During the entire test patients are sitting in a comfortable cardiac chair. One teflon (21 g) venous catheter is inserted into an antecubital vein for blood sampling and kept patent with heparinized normal saline solution. After a 30' rest to establish baseline and after collecting a 20 cc blood sample for leukocyte DNA extraction, at time = 0' subjects ingest 75 g of glucose in 300 ml of water over 5 min. Blood samples to measure glucose, C-peptide and insulin concentrations are collected at times -10', 0', +15', +30', +45', +60', +90', +120', +150', +180', +210' and +240', +270' and +300'. Urines are collected to measure glycosuria.

-Euglycemic Insulin Clamp: During the entire test patients are lying in bed. One teflon catheter is introduced into an antecubital vein for the infusion of test substances. Another teflon catheter is placed retrogradely into a wrist vein for sampling arterialized venous blood, according to the "hot box" technique. After a 30' rest in bed to establish baseline, indirect calorimetry (at least 40') is performed. At the end of calorimetric measures, baseline blood samples are collected and a standard euglycemic insulin clamp is carried out. After an insulin intravenous prime of 4.8 pmol/min/m^2 BSA and a subsequent continuous infusion of 240 pmol/min/m^2 BSA, plasma glucose is allowed to decline until it reaches 5.5 mmol/l, after which glucose clamping starts with a glucose concentration goal of 5 mmol/l. The duration of the glucose clamp is at least of 120', but it is prolonged, if and as needed, to ensure at least 60' of insulin clamp at euglycemia in each patient. Timed blood samples were collected to measure hormone and substrate levels. In the last 45' of the clamp indirect calorimetry is repeated to assess substrate oxidation and energy production rates. Urine is collected to measure urea excretion rate.

In both metabolic tests, all blood samples are collected in pre-chilled tubes and readily spun at 1,500 g. Plasma and serum specimens are stored at -80° C.

-Analytical procedures: Plasma glucose concentration is measured in duplicate at bedside. Serum C-peptide and insulin concentrations are measured by chemiluminescence. Glycated hemoglobin and serum lipids were measured by standard in-house methods. GAD-antibodies are measured by immunoradiometry (CentAK, Medipan, Germany), according to manufacturer's instructions.

-Genotyping: A leukocyte DNA sample is collected in each subject and the DNA is extracted through standard salting out method. Genotyping is performed by RFLP (Restriction Fragment Length Polymorphism), which consists in a PCR (Polymerase Chain Reaction) followed by proper enzymatic digestion and resolution on agarose gel. Alternatively, it is performed were assessed by the high-throughput genotyping Veracode technique (Illumina Inc, CA), applying the GoldenGate Genotyping Assay according to manufacturer's instructions.