PED/PEA-15 Protein, PCOS, Obesity, Insulin Sensitivity Indexes, Metformin, Oral Contraceptives

Subjects:

Twenty obese PCOS women (age: 24.7±18 yr; BMI: 30±2.4 kg/m2) were consecutively admitted to the Endocrinology Unit of the Department of Molecular and Clinical Endocrinology and Oncology of the Federico II University of Naples, and were enrolled in this clinical study. The diagnosis of PCOS was made according to the diagnostic criteria for PCOS At study entry the patients were randomized in two groups of treatment, according to insulin sensitivity. OCP group, 10 patients (BMI 29.7± 1.5 kg/m2) received 30 µg ethinylestradiol plus 30 mg drospirenone 21 day/month. MET group, 10 patients (BMI 30.4± 3.1 kg/m2), received metformin 1250 mg three times daily. The duration of follow-up was 6 months. The control group consisted of 10 healthy female volunteers, who were age matched.

Methods:

As all PCOS women were anovulatory, they underwent a progesterone challenge test (100 mg natural progesterone i.m.; Prontogest, Amsa, Rome, Italy), which induced uterine bleeding in all PCOS women. To exclude the presence of type 2 diabetes or abnormal glucose tolerance, the oral glucose tolerance test (OGTT) was performed and the normal glucose response to the OGTT was defined according to the 'Report of the Expert Committee on the diagnosis and classification of diabetes mellitus'.

All anthropometric measurements were taken with subjects wearing only light clothes and without shoes. In each woman, weight and height were measured to calculate the BMI [weight (kg) divided by height squared (m2), kg/m2]. Height was measured to the nearest cm using a wall-mounted stadiometer. Body weight (BW) was determined to the nearest 50 g using a calibrated balance beam scale.

Patients were given a standardized interview to obtain information about the duration of obesity, eating patterns, smok¬ing habits and physical exercise. In particular, subjects were also asked to make a daily record of the amount of physical activity (no exercise; ≤2-3 h/week; ≥2-3 h/week). Preadmission food intake and dietary history were assessed by a skilled dietitian who used a computer-assisted interview (Winfood 1.5, Medimatica srl, Martinsicuro, Italy). All PCOS women received a normo-caloric diet.

Assays:

Blood samples were obtained between 08.00 h and 09.00 h from an antecubital vein after an overnight fast, with the patient in the resting position. The OGTT was performed using 75 g dextrose. Blood samples were obtained at 0, 30, 60, 90, 120, min for plasma glucose and insulin measurements. Fasting plasma glucose (FPG) levels were determined by the glucose oxidase method immediately after the OGTT. Fasting plasma insulin (FPI) samples were promptly centrifuged, plasma was separated and stored at -20°C until assay. FPI was measured by a solid-phase chemiluminescent enzyme immunoassay using commercially available kits (Immunolite Diagnostic Products Co, Los Angeles, CA). 1/HOMA-IR, QUICKI and ISI were calculated.

PED/PEA-15 protein was measured in white-blood cells (WBC) lysates obtained from 10 to 12 ml of freshly collected uncoagulated whole blood, after separation with dextran 6%, using Western blot analysis. For Western blot analysis WBC were solubilized at 4°C in TAT buffer, centrifuged at 500g for 20 min, and supernatant fractions were stored at -20°C until used. The amount of 50 μg of lysate proteins were heated at 100°C in Laemmli buffer. Proteins were separated by 15% SDS-PAGE and then transferred to 0•45-mm Immobilon-P membranes (Millipore, Bedfort, MA). Filters were probed with PED/PEA-15 antiserum at 1:2000 dilution, revealed by enhanced chemiluminescence and autoradiography. The protein bands were quantified by laser densitometry and expressed as percentage of pixels (arbitrary units).