PED/PEA-15 Protein, PCOS, Obesity, Insulin Sensitivity Indexes, Metformin, Oral Contraceptives
PED/PEA-15 Protein, PCOS, Obesity, Insulin Sensitivity Indexes, Metformin, Oral Contraceptives
Insulin-resistance plays an important role in polycystic ovary syndrome (PCOS)
 physiopathology. The phosphoprotein enriched in the diabetes (PED/PEA-15), a 15 kDa protein
 related to insulin sensitivity, is over-expressed in type 2 diabetic patients and in PCOS
 women, independently of obesity. The effectiveness of oral contraceptives pills (OCP) or
 metformin (MET) in PCOS management is still uncertain. Aim of this pilot clinical study was
 to compare the effects of OCPs or MET on the expression of PED/PEA-15 in association with
 insulin sensitivity in obese PCOS women. Outcome measures: PED/PEA-15, BMI, plasma glucose
 and insulin, 1/HOMA-IR, homeostasis model assessment of insulin resistance; QUICKI,
 quantitative insulin sensitivity check index; ISI: whole-body insulin sensitivity index.
 Study design: twenty obese PCOS women (age: 24.7±18 yr; BMI: 30±2.4 kg/m2) were randomized
 according to insulin sensitivity to receive 30 µg ethinylestradiol plus 30 mg drospirenone 21
 day/month or MET 1250 mg three times daily for 6 months. Results: At baseline, age and BMI
 were not different in the two groups; PED/PEA-15 protein expression was higher in MET than in
 OCP group (p=0.011), along with higher 1/HOMA-IR (p=0.004), and lower QUICKI and ISI (p=0.003
 and p<0.001, respectively). After treatment, independently of body weight, only in MET group
 PED/PEA-15 decreased (p=0.004), along with insulin and 1/HOMA-IR (p<0.001), and QUICKI and
 ISI increased (p<0.001). Insulin sensitivity indexes improvement correlated significantly
 with PED/PEA-15 protein expression, but not with BMI. Conclusions: PED/PEA-15 protein
 over-expression in obese PCOS women with IR reduced after a six month treatment with MET,
 while remained unchanged in the OCP group. The reduction was independent of body weight, and
 correlated with insulin sensitivity indexes. This effect further supported MET as a more
 effective therapy than OCPs for obese PCOS women with IR, also when fertility is not
 required.
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).
Inclusion Criteria:

 -  female

 -  premenopausal

 -  obesity

 -  PCOS.

 Exclusion Criteria:

 -  pregnancy

 -  type 2 diabetes or impaired glucose tolerance

 -  hypothyroidism

 -  hyperprolactinaemia

 -  Cushing's syndrome

 -  nonclassical congenital adrenal hyperplasia

 -  previous (within the last 6 months) use of oral contraceptives

 -  glucocorticoids

 -  antiandrogens

 -  ovulation induction agents

 -  antidiabetic and antiobesity drugs, or other hormonal drugs.

 None of the subjects was affected by any neoplastic, metabolic, hepatic, and cardiovascular
 disorder or other concurrent medical illness (i.e. diabetes, renal disease, and
 malabsorptive disorders),acute and chronic inflammations based on medical history, physical
 examination, and routine laboratory tests, including measurement of oral temperature, white
 blood cell count and urinalysis.