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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis
Service of Endocrinology, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region
(FISABIO), Valencia, Spain
b
Institute of Health Research INCLIVA, University of Valencia, Valencia, Spain
c
Department of Physiology, University of Valencia, Valencia, Spain
d
Department of Pharmacology and CIBERehd, Faculty of Medicine, University of Valencia, Spain
e
n General de Universidad de Valencia, Valencia, Spain
Fundacio
f
Department of Medicine, University of Valencia, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2015
Received in revised form
17 June 2015
Accepted 7 July 2015
Available online 10 July 2015
Objective: We aim to assess the effect of metformin treatment on metabolic parameters, endothelial
function and inammatory markers in polycystic ovary syndrome (PCOS) subjects.
Methods: The study population consisted of 40 reproductive-age women with PCOS, who underwent
treatment with metformin during a 12-week period, and their corresponding matched controls (n 44).
We evaluated endocrinological parameters, adhesion molecules (vascular cell adhesion molecule 1
(VCAM-1), intercellular cell adhesion molecule 1 (ICAM-1) and E-selectin) and proinammatory cytokines (interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFa)) in serum. In addition, interactions
between human umbilical vein endothelial cells and polymorphonuclear (PMN) cells were assessed by
ow chamber microscopy. In addition, a group of type 2 diabetes patients who underwent treatment
with metformin during a 12-week period was incorporated into the study.
Results: Metformin produced benecial effects on PCOS patients by decreasing polymorphonuclear
(PMN) rolling ux and adhesion. It also decreased levels of ICAM-1, E-selectin, IL-6 and NFa. In addition,
metformin induced an improvement of endocrine and anthropometric parameters in PCOS subjects by
reducing glucose, follicle-stimulating hormone (FSH) and androstendione, and by increasing
dehydroepiandrosterone-sulfate (DHEA-S). Metformin also had benecial effects in type 2 diabetic
subjects by reducing body weight, waist circumference and PMN adhesion, and by increasing PMN
rolling velocity.
Conclusion: Our results highlight the modulating effect of metformin on leukocyte/endothelium interactions. These ndings may explain the potential benecial effect of metformin in reducing the risk of
vascular events in PCOS patients and in insulin resistance conditions.
2015 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Metformin
Mitochondria
Leukocyte
Endothelium
PCOS
Type 2 diabetes
1. Introduction
* Corresponding author. University Hospital Doctor Peset, Avda Gaspar Aguilar
90, 46017, Valencia, Spain.
** Corresponding author. University Hospital Doctor Peset, Avda Gaspar Aguilar
90, 46017, Valencia, Spain.
*** Corresponding author. University Hospital Doctor Peset, Avda Gaspar Aguilar
90, 46017, Valencia, Spain.
E-mail addresses: Victor.Victor@uv.es (V.M. Victor), Milagros.Rocha@uv.es
(M. Rocha), hernandez_antmij@gva.es (A. Hernandez-Mijares).
1
These authors have contributed equally to this work.
http://dx.doi.org/10.1016/j.atherosclerosis.2015.07.017
0021-9150/ 2015 Elsevier Ireland Ltd. All rights reserved.
168
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Table 1
Anthropometric parameters, lipoprotein prole, hydrocarbonated metabolism parameters and circulating androgens of control and PCOS women treated with metformin
during a 12-week period.
Controls (n 44)
Age (years)
Body weight (kg)
BMI (Kg/m2)
Waist circumference (cm)
Systolic BP (mmHg)
Diastolic BP (mmHg)
Total cholesterol (mg/dl)
LDLc (mg/dl)
HDLc (mg/dl)
Triglycerides (mg/dl)
Apolipoprotein A (mg/dl)
Apolipoprotein B (mg/dl)
hsCRP (mg/l)
Glucose (mg/dl)
Insulin (mIU/ml)
HOMA-IR
FSH (mIU/ml)
LH (mIU/ml)
Testosterone (ng/ml)
DHEA-S (mg/dl)
Androstendione (ng/ml)
SHBG (nmol/l)
26.6
77.6
29.3
86.9
109
70
174.2
106.1
51.6
83.7
142.6
80.6
2.87
86.1
9.7
2.14
4.34
5.51
0.43
231.1
2.49
103.8
4.5
17.5
6.6
16.9#
11#
10
31.6
26.8
13.3#
47.2
30.2
21.3
2.49#
10.2
5.3#
1.29#
2.40
5.11
0.20#
108.4#
0.96#
69.4#
7.2
23.1
8.6
17.0
16
12
32.6
27.0
10.0
55.0
18.8
23.3
3.11
10.1
8.8
2.06
1.50
4.88
0.39
170.2
1.68
36.6
23.1
8.6
15.9
16
10
32.1
26.9
7.7
61.4
15.1
26.0
3.52
10.1*
8.5
2.16
1.67*
4.17
0.36
200.2*
1.60*
21.9
Data are expressed as mean SD. # Statistical signicance (p < 0.05) was determined by an unpaired Student's t-test (control vs PCOS).* Statistical signicance (p < 0.05) was
determined by a paired Student's t-test (baseline vs 12 weeks).
from citrated blood samples and were incubated for 45 min with
dextran (3%). The supernatant was centrifuged at 250 g for 25 min
over Fycoll-Hypaque. Lysis buffer was added to the pellet, which
was centrifuged and washed twice at room temperature (100 g,
5 min). PMNs were evaluated in a Scepter device (Millipore, MA,
USA), washed in HBSS medium and stored in complete RPMI media
at 37 C.
2.3. Adhesion assay. Levels of cytokines and adhesion molecules.
Nitric oxide measurements
The parallel plate ow chamber in-vitro model has been
described in detail elsewhere [26,27]. A Luminex 200 ow analyzer
system was used to evaluate ICAM-1, VCAM-1, E-selectin, IL-6 and
TNF-alpha in serum from PCOS subjects (Millipore, Austin, TX,
USA).
Total nitric oxide (NO) in the serum samples was measured with
the total NO/Nitrite/Nitrate Parameter Assay Kit (R & D Systems,
Minneapolis, Minnesota, USA). Concentrations of NO were determined by Griess reaction, in which acidied NO2 produces a
nitrosating agent, which reacts with sulfanilic acid to form the
diazonium ion. This ion is then coupled to N-(1-naphthyl)ethylenediamine to produce the chromophoric molecule that absorbs
light at 540e570 nm.
2.4. Drugs and solutions
Glucose, trypan blue, arginine, TNFa, albumin and bronectin
were obtained from SigmaeAldrich. Dextran was acquired from
Fluka (St. Louis, MO). RPMI 1640 supplemented with 20 mm HEPES,
endothelial cell growth medium culture media, HBSS, DPBS with
(DPBS) or without (DPBS) Ca2 and Mg2, and fetal bovine serum
were obtained from Lonza (Verviers, Belgium). FicollePaque Plus
was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Plastic coverslips with a diameter of 25 mm were
obtained from Nunc (supplied by Thermo Fisher Scientic), sodium
pyruvate and trypsin-EDTA were obtained from Invitrogen (Eugene,
OR). PBS and collagenase, were obtained from Gibco (Life Technologies, Eugene, Oregon).
170
Regarding to the levels of cytokines, PCOS induced an increase of IL6 (Fig. 3A, P < 0.05) and TNFa (Fig. 3B, P < 0.05), while metformin
decreased IL-6 (Fig. 3A, P < 0.05) and TNFa (Fig. 3B, P < 0.05) thus
suggesting an anti-inammatory effect. Regarding levels of NO, no
statistical differences were detected in any of the study groups
(Fig. 4).
4. Discussion
The present study was designed to evaluate the effects of metformin treatment on leukocyte/endothelium interactions, adhesion
molecules and proinammatory cytokines in PCOS patients. We
have observed that metformin can exert benecial effects by
decreasing PMN rolling ux and adhesion, and ICAM-1, E-selectin,
IL-6 and TNFa levels. As expected, we have also seen that it induces
Fig. 1. Effect of metformin treatment on PMN rolling velocity (mm s1) (A), rolling ux
(PMN per minute) (B), and PMN adhesion (PMN per square millimeter) (C). *P < 0.05;
**P < 0.01 and ***P < 0.001 vs. control group. aP < 0.05 vs. before treatment.
Fig. 2. Effect of metformin treatment on VCAM-1 (pg/mL) (A), ICAM-1 (pg/mL) (B), and
E selectin (pg/mL) (C). *P < 0.05 and **P < 0.01 vs. control group. aP < 0.05 vs. before
treatment.
Fig. 3. Effect of metformin treatment on IL-6 levels (A) and TNFa levels (B); both were
evaluated by Luminex 200 ow analyzer system. *P < 0.05 vs. control group. aP < 0.05
vs. before treatment.
Fig. 4. Effect of metformin treatment on NO levels. Total nitric oxide (NO) in the serum
samples was measured with the total NO/Nitrite/Nitrate Parameter Assay Kit (R & D
Systems, Minneapolis, Minnesota, USA).
171
172
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