Abstract
OBJECTIVE
Polycystic ovary syndrome (PCOS) is associated with the metabolic syndrome. Decreased omentin-1 levels are associated with obesity and diabetes. To study the effects of metformin treatment on omentin-1 levels in PCOS subjects and effects of omentin-1 on in vitro migration and angiogenesis.
RESEARCH DESIGN AND METHODS
Serum omentin-1 was measured by ELISA. Angiogenesis was assessed by studying capillary tube formation in human microvascular endothelial cells (HMEC-1) on growth factor reduced Matrigel. Endothelial cell migration assay was performed in a modified Boyden chamber. Nuclear factor-κB (NF-κB) was studied by stably transfecting HMEC-1 cells with a cis-reporter plasmid containing luciferase reporter gene linked to five repeats of NF-κB binding sites. Akt phosphorylation was assessed by Western blotting.
RESULTS
Serum omentin-1 was significantly lower in PCOS women (P < 0.05). After 6 months of metformin treatment, there was a significant increase in serum omentin-1 (P < 0.01). Importantly, changes in hs-CRP were significantly negatively correlated with changes in serum omentin-1 (P = 0.036). In vitro migration and angiogenesis were significantly increased in serum from PCOS women (P < 0.01) compared with matched control subjects; these effects were significantly attenuated by metformin treatment (P < 0.01) plausibly through the regulation of omentin-1 levels via NF-κB and Akt pathways. CRP and VEGF induced in vitro migration, and angiogenesis was significantly decreased by omentin-1.
CONCLUSIONS
Increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin-treated PCOS women.
Polycystic ovary syndrome (PCOS) is a proinflammatory state associated with type 2 diabetes, visceral obesity, and cardiovascular complications, features of the metabolic syndrome (1–5). The metabolic syndrome is associated with accumulation of visceral adipose tissue. Visceral adipose tissue produces cytokines termed “adipokines” implicated in the pathogenesis of diabetes and atherosclerosis (6–9).
Omentin-1 has been described as a novel adipokine preferentially produced by visceral adipose tissue. In vitro experiments revealed that treatment with recombinant omentin-1 enhances insulin-stimulated glucose uptake in human adipocytes. Also, omentin-1 was shown to trigger Akt signaling in both the absence and presence of insulin (10,11). Additionally, omentin plasma levels and omentin gene expression in visceral adipose tissue are decreased in obesity (12). Recently, we have reported novel data showing a significant decrease of adipose tissue and circulating omentin-1 levels in overweight PCOS women (13). Thus, given the above functions of omentin-1, increasing omentin-1 levels in PCOS women may alleviate cardiometabolic dysfunction in these women.
We studied the effects of metformin treatment on omentin-1 levels in PCOS subjects and effects of omentin-1 and serum on in vitro migration and angiogenesis. Researchers have used serum to perform functional experiments in endothelial, cardiac, and neural cells (14–16). Finally, given the link between inflammation and angiogenesis (17), we explored nuclear factor-kappaB (NF-κB), Erk1/2, and Akt pathways, important regulators of inflammation and angiogenesis (18,19).
RESEARCH DESIGN AND METHODS
All PCOS patients met all three criteria of the revised 2003 Rotterdam European Society of Human Reproduction and Embryology (ESHRE)/American Society of Reproductive Medicine (ASRM) PCOS Consensus Workshop Group diagnostic criteria. The three criteria are 1) oligo- and/or anovulation, 2) clinical and/or biochemical signs of hyperandrogenism, and 3) polycystic ovaries (20). Furthermore, all subjects in the control arm had normal findings on pelvic ultrasound scan, regular periods, and no hirsutism/acne. The control group had no discernible cause for infertility (unexplained infertility). No women were amenorrheic. All subjects that were studied did not have endometriosis. Exclusion criteria for the study included age > 40 years, known cardiovascular disease, thyroid disease, neoplasms, current smoking, diabetes, hypertension (blood pressure > 140/90 mmHg), and renal impairment (serum creatinine > 120 μmol/l). None of these women were on any medications for at least 6 months prior to the study, including oral contraceptives, glucocorticoids, ovulation induction agents, antidiabetic and antiobesity drugs, and estrogenic, antiandrogenic, or antihypertensive medication. All patients underwent anthropometric measurements. Blood pressure was measured in a sitting position within a quiet and calm environment after a rest of at least 5 min. The average of three measurements was obtained. Subjects were outpatients of the Department of Reproductive Medicine and Gynaecological Endocrinology of Magdeburg University and the Department of Obstetrics and Gynaecology of Martin-Luther-University Halle. The metabolic study was performed in the Outpatient Department of Endocrinology and Metabolism of Magdeburg University. Blood samples were collected between 0800 and 0900 h, after a 3-day normal carbohydrate diet and an overnight fast. Blood samples were drawn into serum separator tubes that contain no additives or anticoagulants, allowed to clot, centrifuged (3,000 rpm for 10 min) to separate sera, and stored at −80°C.
A treatment with metformin in an “off-label use” was offered to all PCOS women independently from the results of insulin sensitivity testing. In those PCOS women that agreed, therapy was initiated after basal assessment, and the dose of metformin was increased to a maintenance dose of 850 mg twice daily. All patients underwent anthropometric measurements before and after metformin treatment. Carotid intima media thickness (IMT) was measured in PCOS subjects, before and after metformin (see below). A total of 83 women of Caucasian origin with PCOS were recruited, 49 of which did not participate in the metformin arm of the study. The baseline characteristics of the 49 PCOS women were comparable to the 34 PCOS women who participated in the metformin arm of the study. Furthermore, the 21 women who completed the metformin arm of the study were comparable to the 13 PCOS women who did not complete the metformin arm of the study. For the purposes of elucidating the effects of metformin in PCOS women, the 21 PCOS subjects were studied. The study design was approved by the Local Research Ethics Committee of the University of Magdeburg, and written informed consent was obtained from all participants, in accordance with the guidelines in The Declaration of Helsinki 2000.
Intima media thickness.
Measurement of carotid IMT is a widely accepted tool to provide information about preclinical vascular disease. B-mode sonography of the proximal part of the carotid bulb was conducted on both sides, and the segments of the common carotid arteries 1.0 cm proximal were scanned longitudinally with Hitachi EUB-5000 plus-G (Hitachi Ltd., Tokyo, Japan) using a 10-MHz linear-array transducer. All women were investigated in a supine position with the head slightly hyperextended and turned away from the side being scanned. The image was focused on the posterior wall, and the resolution function was used to magnify the arterial far wall. When an optimal image was obtained, it was frozen, and the distance from the junction of the lumen and intima and the junction of the media and adventitia was measured by electronic calipers in end-diastolic phase, to minimize variability during the cardiac cycle. All images were photographed. Five measurements were conducted on each side, and the mean IMT was calculated as the average of these measurements. All measurements were performed by an experienced ultrasound sonographer (intra-observer coefficient of variation was 6.8%) who was blinded to the diagnosis of subjects.
Biochemical and hormonal analysis.
Assays for glucose, insulin, cholesterol, triglycerides, luteinizing hormone, follicular stimulating hormone, testosterone, and rostenedione, dehydroepiandrosterone-sulfate, and sex hormone–binding globulin were performed using an automated analyzer (Abbott Architect, Abbott Laboratories, Abbott Park, USA). The estimate of insulin resistance by homeostasis model assessment (HOMA-IR) score was calculated as previously described (21). High-sensitivity C-reactive protein (hs-CRP) was determined immunoturbidimetrically using a modular system random-access analyzer (Roche Diagnostics). Omentin-1 levels in sera were measured using a commercially available ELISA kit (AXXORA, Nottingham, U.K.), according to manufacturer's protocol, with an intra-assay coefficient of variation of <6%.
Immunodepletion of serum omentin-1 and CRP.
Immunodepletion of serum omentin-1 and CRP were performed using the immunoprecipitation protocol provided by Dr. Kazuyuki Itoh and Montecucco et al., respectively (14,22). One ml of human serum was incubated with mouse-anti-human omentin-1 IgG-protein G-Sepharose conjugate (1–2 μg/100–500 μg of total protein) or control mouse IgG-protein G-Sepharose conjugate; goat-anti-human CRP IgA-protein A-Sepharose conjugate (10 μg/100–500 μg of total protein) or control goat IgA-protein A-Sepharose conjugate, mixed overnight at 4°C and centrifuged to sediment the conjugated Sepharose beads. The supernatants were collected, and similar procedures were repeated eight times. Supernatants and sera were subjected to immunoblotting for omentin-1 as previously described (13). Immunoblotting for CRP is described below. Immunoblotting confirmed that these protocols decreased serum omentin-1 and CRP by ∼80% (data not shown), respectively.
Endothelial cell culture.
Human microvascular endothelial cells (HMEC-1) were obtained from the Center for Disease Control, Atlanta, USA HMEC-1 cells were cultured in MCDB medium (Sigma-Aldrich, Gillingham, U.K.) supplemented with 10% fetal calf serum (Sigma-Aldrich, Gillingham, U.K.), 100 IU/ml penicillin (Sigma-Aldrich, Gillingham, U.K.), 100 μg/ml streptomycin (Sigma-Aldrich), 5 ml of 200 mmol/l l-glutamine/500 ml of media, hydrocortisone 2 μmol/l, and epidermal growth factor 2 ng/ml (Invitrogen, Paisley, U.K.) at 37°C in 5% CO2/95% air. Prior to each experiment, cells were fed with MCDB with addition of 1% fetal calf serum for 16 h.
For endothelial cell signaling experiments, HMECs were preincubated with or without omentin-1 (Kamiya Biomedical Company, Seattle, USA) for 30 min followed by treatment with or without 1% of human serum from normal (n = 39; individually tested) and PCOS women [before (n = 21; individually tested) and after (n = 21; individually tested) 6 months of metformin treatment] at different time points (0, 2, 5, 10, 20, 30, and 60 min). Dose- and time-dependent experiments were performed to determine the optimum concentration and time point.
In vitro angiogenesis assay.
Angiogenesis was assessed by studying the formation of capillary tubelike structures by culturing HMEC-1 cells on growth factor reduced Matrigel (BD Biosciences, San Jose, USA). Matrigel was coated onto the culture plates as per manufacturer's instructions. HMEC-1 cells were preincubated with or without omentin-1 (Kamiya Biomedical Company, Seattle, USA) and/or with or without PI3K/Akt inhibitor (LY294002) (Calbiochem, San Diego, USA) for 30 min and 1 h, respectively, followed by treatment with or without 1% of human serum from normal (n = 39; individually tested) and PCOS women [before (n = 21; individually tested) and after (n = 21; individually tested) 6 months of metformin treatment] or CRP or omentin-1 [PCOS after metformin (n = 21; individually tested)] and CRP [PCOS before metformin (n = 21; individually tested)] immunodepleted sera. Vascular endothelial growth factor (VEGF) served as positive control. Trypsinized HMEC-1 cells were then seeded onto the Matrigel-coated plates at a density of 4–5 × 103/well in fresh media and incubated at 37°C. Dose- and time-dependent experiments were performed to determine the optimum concentration and time point. Capillary tube formation images were captured with a digital microscope camera system (Olympus, Tokyo, Japan). Image Pro Plus software was used to quantify tube length formation; the lengths of tubes in 3–4 randomly selected fields in each of the wells were measured (23–26).
In vitro migration assay.
Endothelial cell migration assay was performed in a modified Boyden chamber using a protocol obtained from BD BioCoat Angiogenesis System (BD Biosciences, San Jose, USA). Endothelial cells were trypsinized; a cell suspension of 4 × 105 cells/ml was prepared, 250 μl of which was added to each of the trans-well inserts. Starvation media (750 μl) was added to the lower chamber and incubated overnight. After this, the cells were labeled by incubating with Hank's balanced salt solution medium (Sigma-Aldrich, Gillingham, U.K.) containing 4 μg/ml Calcein-AM (BD Biosciences, San Jose, USA) for 90 min. The cells were preincubated with or without omentin-1 (Kamiya Biomedical Company, Seattle, USA) and/or with or without PI3K/Akt inhibitor (LY294002) (Calbiochem, San Diego, USA) for 30 min and 1 h, respectively, followed by treatment with or without 1% of human serum from normal (n = 39; individually tested) and PCOS women [before (n = 21; individually tested) and after (n = 21; individually tested) 6 months of metformin treatment] or CRP or omentin-1 [PCOS after metformin (n = 21; individually tested)] and CRP [PCOS before metformin (n = 21; individually tested)] immunodepleted sera. VEGF served as positive control. Dose- and time-dependent experiments were performed to determine the optimum concentration and time point. Cells were fixed with 2% formaldehyde. Fluorescence of migrated cells was read in a fluorescence plate reader with bottom reading capabilities at excitation/emission wavelengths of 494/517 nm. Only those labeled cells that have migrated through the pores of the membrane will be detected (23–26).
NF-κB activation.
We studied NF-κB activation by stably transfecting HMEC-1 cells with a cis-reporter plasmid containing luciferase reporter gene linked to five repeats of NF-κB binding sites (pNF-κB-Luc; Stratagene, LA Jolla, USA). Multiple clones were selected for the analysis of NF-κB activation. HMEC-1 cells were preincubated with or without omentin-1 (Kamiya Biomedical Company, Seattle, USA) for 30 min followed by treatment with or without 1% of human serum from normal (n = 39; individually tested) and PCOS women [before (n = 21; individually tested) and after (n = 21; individually tested) 6 months of metformin treatment] or CRP or omentin-1 [PCOS after metformin (n = 21; individually tested)] and CRP [PCOS before metformin (n = 21; individually tested)] immunodepleted sera for 2 h. Cells were lysed, and luciferase activities were measured. Experiments were also performed with TK plasmid (Promega, Southampton, U.K.) as negative control and tumor necrosis factor α (TNF-α) as positive control. Dose- and time-dependent experiments were performed to determine the optimum concentration and time point.
Western blotting.
Endothelial cells were lysed with SDS sample buffer (5 mol/l urea, 0.17 mol/l SDS, 0.4 mol/l dithiothreitol, and 50 mmol/l Tris-HCl, pH 8.0), mixed, sonicated, boiled, centrifuged (5,000 rpm for 2 min), and then stored at −80°C until use. Eighty μg of each sample, supernatants, and sera were subjected to SDS-PAGE (8% resolving gel) and transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% BSA for 2 h. The PVDF membranes were then incubated with polyclonal primary rabbit-anti-human antibody for phospho-Erk1/2 (Cell Signaling Technology Inc., Beverly, USA) (1:1,000 dilution) or polyclonal primary rabbit-anti-human antibody for phospho-Akt (Ser473) (Cell Signaling Technology Inc., Beverly, USA) (1:1,000 dilution) or monoclonal primary goat-anti-human antibody for CRP (Sigma-Aldrich, Gillingham, U.K.) (1:1,000 dilution) overnight at 4°C. The membranes were washed thoroughly for 60 min with Tris-buffered saline–0.1% Tween before incubation with the secondary anti-rabbit horseradish peroxidase-conjugated immunoglobulin (Dako, Ely, U.K.) (1:2,000) or secondary anti-goat horseradish peroxidase-conjugated immunoglobulin (Dako, Ely, U.K.) (1:2,000) for 1 h at room temperature. Antibody complexes were visualized using chemiluminescence (ECL+; GE Healthcare, Little Chalfont, U.K.). For standardization, the same membranes were stripped and reprobed using polyclonal primary rabbit-anti-human antibodies for total Erk1/2 (Cell Signaling Technology Inc., Beverly, USA) [1:1,000 dilution] or total Akt (Cell Signaling Technology Inc., Beverly, USA) [1:1,000 dilution].
The densities were measured using a scanning densitometer coupled to scanning software Scion Image (Scion Corporation, Frederick, USA). Standard curves were generated to ensure linearity of signal intensity over the range of protein amounts loaded into gel lanes. Comparisons of densitometric signal intensities for phospho-Erk1/2, phospho-Akt, total Erk1/2, and total Akt were made only within this linearity range.
Statistics.
Data were analyzed by Mann–Whitney U test or Kruskal–Wallis ANOVA (post hoc analysis: Dunn's test) according to the number of groups compared. Data are medians (interquartile range). Spearman rank correlation was used for calculation of associations between variables. If individual bivariate correlations achieved statistical significance, multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1. P < 0.05 was considered significant.
RESULTS
Table 1 shows the anthropometric, biochemical, and hormonal data in all subjects. Insulin, HOMA-IR, cholesterol, triglycerides, luteinizing hormone, testosterone, free androgen index, systolic blood pressure, diastolic blood pressure, IMT, and hs-CRP were significantly higher whereas dehydroepiandrosterone-sulfate and sex hormone–binding globulin were significantly lower in PCOS women.
TABLE 1.
Clinical, hormonal, and metabolic features of women with PCOS and control subjects
| Variable | PCOS (n = 21) | Control subjects (n = 39) | Significance |
|---|---|---|---|
| Age (years) | 28 (26.5–31.5) | 28 (23–32) | NS |
| BMI (kg/m2) | 32.8 (29.8–36.5) | 29.1 (27.3–34.1) | NS |
| WHR | 0.82 (0.76–0.88) | 0.81 (0.78–0.86) | NS |
| Glucose (mmol/l) | 5.1 (4.7–5.5) | 4.8 (4.4–5.1) | NS |
| Insulin (pmol/l) | 70.0 (54.5–94.5) | 41.0 (32.0–55.0) | P < 0.01 |
| HOMA-IR | 2.2 (2.0–3.0) | 1.2 (1.0–1.7) | P < 0.01 |
| Cholesterol (mmol/l) | 4.9 (4.1–5.3) | 4.3 (3.8–4.8) | P < 0.05 |
| Triglycerides (mmol/l) | 1.0 (0.8–1.7) | 0.8 (0.6–1.1) | P < 0.05 |
| LH (IU/l) | 7.8 (4.7–11.0) | 3.8 (2.7–5.4) | P < 0.01 |
| FSH (IU/l) | 5.2 (4.0–6.2) | 5.7 (4.3–7.5) | NS |
| Testosterone (nmol/l) | 1.8 (1.5–2.5) | 1.2 (1.0–1.6) | P < 0.01 |
| Androstenedione (nmol/l) | 3.0 (2.3–3.8) | 2.6 (1.9–3.6) | NS |
| DHEA-S (μmol/l) | 4.3 (2.7–5.6) | 6.1 (3.8–7.5) | P < 0.01 |
| SHBG (nmol/l) | 27.0 (20.5–41.0) | 45.0 (34.0–59.0) | P < 0.01 |
| FAI | 6.6 (4.6–9.7) | 3.0 (1.9–4.4) | P < 0.01 |
| SBP (mmHg) | 125.0 (120.0–130.0) | 120.0 (110.0–120.0) | P < 0.01 |
| DBP (mmHg) | 80.0 (75.0–80.0) | 75.0 (70.0–80.0) | P < 0.01 |
| IMT (mm) | 0.53 (0.48–0.58) | 0.42 (0.40–0.44) | P < 0.01 |
| hs-CRP (mg/l) | 3.3 (2.4–5.5) | 1.3 (0.5–2.5) | P < 0.01 |
| Omentin-1 (ng/ml) | 23.7 (20.0–27.9) | 27.6 (25.6–29.4) | P < 0.05 |
Data are median (interquartile range). Group comparison by Mann–Whitney U test. Free androgen index (FAI) = T (nmol/l)/SHBG (nmol/l) × 100. NS = not significant; LH, luteinizing hormone; FSH, follicular stimulating hormone; DHEA-S, dehydroepiandrosterone-sulfate; SHBG, sex hormone–binding globulin; SBP, systolic blood pressure; DBP, diastolic blood pressure.
Serum omentin-1 levels were significantly lower in PCOS subjects compared with control subjects [23.7 (20.0–27.9) versus 27.6 (25.6–29.4) ng/ml; P < 0.05: Table 1]. Serum progesterone levels in all women confirmed follicular phase of the menstrual cycle.
Table 2 shows the effects of metformin treatment on serum omentin-1 levels. Metformin treatment was started in 34 women with PCOS. Only 21 women completed the study and were investigated after 6 months of metformin treatment. Reasons for subjects not completing study 2 were nausea and gastrointestinal side effects (n = 4), pregnancies (n = 5), incompliance (n = 2), and loss of contact (n = 2).
TABLE 2.
Clinical, hormonal, and metabolic features of women with PCOS (n = 21) before and after metformin treatment
| Variable | Before metformin | After metformin | Significance |
|---|---|---|---|
| Age (years) | 28 (26.5–31.5) | 28 (27.5–32.5) | NS |
| BMI (kg/m2) | 32.8 (29.8–36.5) | 31.4 (28.2–35.1) | NS |
| WHR | 0.82 (0.76–0.88) | 0.80 (0.74–0.87) | P < 0.05 |
| Glucose (mmol/l) | 5.1 (4.7–5.5) | 4.8 (4.4–4.9) | P < 0.05 |
| Insulin (pmol/l) | 70.0 (54.5–94.5) | 61.0 (43.5–83.0) | NS |
| HOMA-IR | 2.2 (2.0–3.0) | 1.6 (1.3–2.2) | P < 0.05 |
| Cholesterol (mmol/l) | 4.9 (4.1–5.3) | 5.0 (4.1–5.4) | NS |
| Triglycerides (mmol/l) | 1.0 (0.8–1.7) | 1.2 (1.0–1.9) | NS |
| Testosterone (nmol/l) | 1.8 (1.5–2.5) | 1.3 (1.1–1.8) | P < 0.05 |
| Androstenedione (nmol/l) | 3.0 (2.3–3.8) | 2.8 (2.2–3.6) | NS |
| DHEA-S (μmol/l) | 4.3 (2.7–5.6) | 5.4 (3.6–6.7) | NS |
| SHBG (nmol/l) | 27.0 (20.5–41.0) | 29.0 (21.5–46.5) | NS |
| FAI | 6.6 (4.6–9.7) | 5.3 (3.1–6.6) | NS |
| SBP (mmHg) | 125.0 (120.0–130.0) | 120.0 (120.0–127.5) | NS |
| DBP (mmHg) | 80.0 (75.0–80.0) | 75.0 (75.0–80.0) | NS |
| IMT (mm) | 0.53 (0.48–0.58) | 0.47 (0.41–0.53) | P < 0.05 |
| hs-CRP (mg/l) | 3.3 (2.4–5.5) | 3.1 (2.0–6.2) | NS |
| Omentin-1 (ng/ml) | 23.7 (20.0–27.9) | 59.2 (52.1–60.6) | P < 0.01 |
Data are median (interquartile range). Group comparison by Mann–Whitney U test. Free androgen index (FAI) = T (nmol/l)/SHBG (nmol/l) × 100. NS = not significant; DHEA-S, dehydroepiandrosterone-sulfate; SHBG, sex hormone–binding globulin; SBP, systolic blood pressure; DBP, diastolic blood pressure.
After 6 months of metformin treatment, there was a significant increase in serum omentin-1 [23.7 (20.0–27.9) versus 59.2 (52.1–60.6) ng/ml; P < 0.01: Table 2]. Also, there were significant decreases in waist-to-hip ratio (WHR), testosterone, glucose, HOMA-IR, and IMT.
Correlation of omentin-1 with covariates.
Spearman rank analyses demonstrated that serum omentin-1 levels were significantly negatively correlated with BMI, WHR, glucose, HOMA-IR, and hs-CRP (P < 0.05). However, when subjected to multiple regression analysis, none of these variables were significantly negatively correlated with serum omentin-1 levels (P > 0.05).
Furthermore, we analyzed the correlation between the change in serum omentin-1 levels before and after metformin therapy (Δomentin-1) and the changes (Δ) in other covariates (Table 3). We found that Δomentin-1 was significantly negatively associated with ΔWHR, Δglucose, ΔHOMA-IR, and Δhs-CRP. When subjected to multiple regression analysis, only Δhs-CRP was significantly negatively correlated with Δomentin-1 (β = −0.526; P = 0.036).
TABLE 3.
Linear regression analysis of variables associated with changes in serum omentin-1 levels (before and after metformin treatment), Δomentin-1, in PCOS subjects (n = 21)
| Variable | Simple |
Multiple |
||
|---|---|---|---|---|
| r | P | β | P | |
| BMI (kg/m2) | −0.400 | 0.072 | ||
| WHR | −0.445 | 0.043 | −0.189 | 0.338 |
| Glucose (mmol/l) | −0.471 | 0.031 | −0.038 | 0.906 |
| Insulin (pmol/l) | −0.014 | 0.953 | ||
| HOMA-IR | −0.461 | 0.035 | −0.149 | 0.611 |
| Cholesterol (mmol/l) | 0.100 | 0.667 | ||
| Triglycerides (mmol/l) | 0.390 | 0.080 | ||
| Testosterone (nmol/l) | −0.126 | 0.586 | ||
| Androstenedione (nmol/l) | 0.139 | 0.548 | ||
| DHEA-S (μmol/l) | −0.232 | 0.311 | ||
| SHBG (nmol/l) | 0.131 | 0.570 | ||
| FAI | −0.200 | 0.385 | ||
| SBP (mmHg) | −0.222 | 0.333 | ||
| DBP (mmHg) | 0.032 | 0.889 | ||
| IMT (mm) | −0.032 | 0.892 | ||
| hs-CRP (mg/l) | −0.665 | <0.010 | −0.526 | 0.036 |
Spearman rank correlation was used for calculation of associations between variables. If individual bivariate correlations achieved statistical significance, multiple regression analysis with omentin-1 as dependent variable was performed to test the joint effect of these parameters on omentin-1. Multiple regression analysis contained WHR, glucose, HOMA-IR, and hs-CRP. DHEA-S, dehydroepiandrosterone-sulfate; SHBG, sex hormone–binding globulin; FAI, free androgen index; SBP, systolic blood pressure; DBP, diastolic blood pressure.
Effects of omentin-1 on in vitro migration and angiogenesis.
Given the above, we studied the effects of omentin-1 on in vitro migration and angiogenesis. Capillary tube formation was optimal at 24 h, after which capillary tubes begin to disintegrate. In vitro migration and angiogenesis at 24 h was significantly increased when comparing sera of PCOS women to normal control subjects (Figs. 1 and 2; ##P < 0.01). Also, in vitro migration and angiogenesis were significantly decreased in PCOS women after 6 months of metformin treatment (Figs. 1 and 2; **P < 0.01) and when omentin-1 (200 ng/ml) or the PI3K/Akt inhibitor (LY294002; 10 μmol/l) were added to sera (Figs. 1 and 2; *P < 0.05, **P < 0.01). Furthermore, CRP (1ug/ml) and VEGF (10 ng/ml) induced in vitro migration, and angiogenesis was significantly decreased by omentin-1 (Figs. 1 and 2; *P < 0.05).
FIG. 1.
In vitro tube formation by human serum at 24 h from normal control subjects (n = 39) with or without omentin-1 (200 ng/ml), PCOS women (n = 21) with or without omentin-1 (200 ng/ml) or PI3K/Akt inhibitor (LY294002; 10 μmol/l) or CRP immunodepletion (CRP_IP), PCOS women after 6 months of metformin treatment (n = 21) with or without omentin-1 immunodepletion (Omentin-1_IP), CRP (1ug/ml) with or without addition of omentin-1 (200 ng/ml) (n = 6), and VEGF (10 ng/ml) with or without addition of omentin-1 (200 ng/ml) (n = 6), respectively. Data are expressed as % difference of median of normal control subjects [medians (interquartile range)]. Each experiment was performed in three replicates. Group comparison by Kruskal–Wallis ANOVA (post hoc analysis: Dunn's test) and Mann–Whitney U test, respectively. #P < 0.05, ##P < 0.01; *P < 0.05, **P < 0.01.
FIG. 2.
Endothelial cell migration by human serum at 24 h from normal control subjects (n = 39) with or without omentin-1 (200 ng/ml), PCOS women (n = 21) with or without omentin-1 (200 ng/ml) or PI3K/Akt inhibitor (LY294002; 10 μmol/l) or CRP immunodepletion (CRP_IP), PCOS women after 6 months of metformin treatment (n = 21) with or without omentin-1 immunodepletion (Omentin-1_IP), CRP (1ug/ml) with or without addition of omentin-1 (200 ng/ml) (n = 6), and VEGF (10 ng/ml) with or without addition of omentin-1 (200 ng/ml) (n = 6), respectively. Data are expressed as % difference of median of normal control subjects [medians (interquartile range)]. Each experiment was performed in three replicates. Group comparison by Kruskal–Wallis ANOVA (post hoc analysis: Dunn's test) and Mann-Whitney U test, respectively. #P < 0.05, ##P < 0.01; *P < 0.05, **P < 0.01.
Moreover, in vitro migration and angiogenesis were significantly decreased in CRP (PCOS before metformin) but not significantly altered by omentin-1 (PCOS after metformin) immunodepleted sera (Figs. 1 and 2; P > 0.05, *P < 0.05).
Effects of omentin-1 on NF-κB activation in human microvascular endothelial cells.
In HMEC-1 cells, NF-κB was significantly activated by sera from normal and PCOS women compared with control subjects (Fig. 3; #P < 0.05, ##P < 0.01). NF-κB was significantly activated by sera from PCOS women compared with normal women (Fig. 3; **P < 0.01). Also, NF-κB activation was significantly decreased in PCOS women after 6 months of metformin treatment (Fig. 3; **P < 0.01) and when omentin-1 (200 ng/ml) was added to sera (Fig. 3; *P < 0.05). Furthermore, CRP (1 ug/ml) and TNF-α (10 ng/ml) induced NF-κB activation was significantly decreased by omentin-1 (Fig. 3; *P < 0.05).
FIG. 3.
Effects of serum from normal control subjects (n = 39) with or without omentin-1 (200 ng/ml), PCOS women (n = 21) with or without omentin-1 (200 ng/ml) or CRP immunodepletion (CRP_IP), PCOS women after 6 months of metformin treatment (n = 21) with or without omentin-1 immunodepletion (Omentin-1_IP), CRP (1ug/ml) with or without addition of omentin-1 (200 ng/ml) (n = 6), and TNF-α (10 ng/ml) with or without addition of omentin-1 (200 ng/ml) (n = 6), respectively, on NF-κB activation in HMEC-1 cells stably transfected with pNF-κB-luciferase at 2 h. Cells were lysed, and luciferase activities (RLU) were measured. Data are expressed as % difference of median of basal [medians (interquartile range)]. Each experiment was performed in three replicates. Group comparison by Kruskal–Wallis ANOVA (post hoc analysis: Dunn's test) and Mann–Whitney U test, respectively. #P < 0.05, ##P < 0.01; *P < 0.05, **P < 0.01.
Moreover, NF-κB activation was significantly decreased in CRP (PCOS before metformin) but not significantly altered by omentin-1 (PCOS after metformin) immunodepleted sera (Fig. 3; P > 0.05, *P < 0.05).
Effects of omentin-1 on Erk1/2 and Akt signaling pathways in human microvascular endothelial cells.
In HMEC-1 cells, Erk1/2 and Akt pathways were significantly activated by sera from normal and PCOS women compared with control subjects (Fig. 4A and B; #P < 0.05, ##P < 0.01). Erk1/2 and Akt phosphorylation were significantly increased by sera from PCOS women compared with normal women (Fig. 4A and B; **P < 0.01). Also, Erk1/2 and Akt activation were significantly decreased in PCOS women after 6 months of metformin treatment (Fig. 4A and B; *P < 0.05, **P < 0.01). Furthermore, Akt activation was significantly decreased when omentin-1 (200 ng/ml) was added to sera from PCOS women before metformin treatment (Fig. 4B; *P < 0.05). However, there was no significant difference in Erk1/2 activation (Fig. 4A). The detected proteins for phosphorylated Erk1/2, total Erk1/2, phosphorylated Akt, and total Akthad apparent molecular weights of 44/42, 44/42, 60, and 60 kDa, respectively (Fig. 4A and B, inserts).
FIG. 4.
Effects of serum from normal control subjects (n = 39), PCOS women (n = 21), and PCOS women after 6 months of metformin treatment with or without addition of omentin-1 (200 ng/ml) (n = 21) on Erk1/2 and Akt phosphorylation in HMEC-1 cells at 2 and 5 min, respectively. Data are expressed as % difference of median of basal [medians (interquartile range)]. Each experiment was performed in three replicates. Group comparison by Kruskal–Wallis ANOVA (post hoc analysis: Dunn's test) and Mann–Whitney U test, respectively. #P < 0.05, ##P < 0.01; *P < 0.05, **P < 0.01.
DISCUSSION
We confirm our previous data of decreased circulating omentin-1 in PCOS women. Also, serum omentin-1 levels were significantly negatively correlated with BMI, WHR, glucose, HOMA-IR, and hs-CRP. We report that metformin (6 months treatment; 850 mg twice daily) significantly increases serum omentin-1 levels with concomitant decreases in insulin resistance and carotid IMT in PCOS subjects. Although the change in serum omentin-1 levels were significantly negatively associated with changes in WHR, glucose, HOMA-IR, and hs-CRP, only the change of hs-CRP was significantly negatively correlated with the change of serum omentin-1 levels when subjected to multiple regression analysis. The possible implication is that changes in omentin-1 levels associated with metformin treatment observed in our PCOS subjects are more robustly linked to changes in inflammatory rather than metabolic parameters. Of relevance, there is evidence that inflammatory markers other than CRP are elevated in PCOS subjects (3–5); also, studies have indicated metformin's potential anti-inflammatory effects via modulation of TNF-α, IL-6, IL-18, and IL-1-β levels (27–29).
In PCOS women, metformin corrects the associated endocrine and metabolic abnormalities (30); metformin counters IR by inhibiting hepatic glucose production (31–33). Therefore, glucose may regulate circulating omentin-1 levels; however, it is possible that the effect of metformin on omentin-1 levels could be via modulation of other circulating factors. Further studies are needed to clarify this point.
PCOS is a state of altered steroid milieu. We, like others (34), observed a significant decrease in testosterone levels after metformin treatment. However, treatments of omental adipose tissue explants with gonadal and adrenal steroids revealed no meaningful effects on omentin-1 levels (13). It is therefore unlikely that the effects of metformin on serum omentin-1 levels observed in this study are attributable to altered gonadal and adrenal steroids.
Furthermore, functional assays revealed that in vitro migration and angiogenesis were significantly increased by sera from both normal and PCOS women; in PCOS women, in vitro migration and angiogenesis was attenuated after 6 months of metformin treatment. Addition of omentin-1 to sera from PCOS women before metformin treatment significantly decreased in vitro migration and angiogenesis. Omentin-1 also decreased the effects of CRP and VEGF induced in vitro migration and angiogenesis. However, in vitro migration and angiogenesis were significantly decreased in CRP but not significantly altered by omentin-1 immunodepleted sera. It is important to consider that omentin-1 levels in tissues are unknown; the levels are likely to be significantly higher than in sera as sequestration of omentin-1 in vascular tissue, in particular, within stromal-vascular cells, has been shown (10,35). All of these effects appear to be associated with NF-κB and Akt signaling pathways, both well-established mediators of angiogenesis (36–38). Taken together, increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin-treated PCOS women. It is important to bear in mind that metformin may also modulate angiogenesis via the VEGF system. Of relevance, metformin decreases VEGF levels in type 2 diabetic subjects (39).
The link between angiogenesis and the pathogenesis of atherosclerosis has been well described (17). Given our data of an increase in circulating omentin-1 levels with concomitant decrease in IMT, omentin-1 may have a potential role in the pathogenesis of atherosclerosis.
Omentin-1, by promoting activation of the Akt signaling pathway and in turn modulating the endothelial nitric oxide synthase in endothelial cells, may impact upon endothelial dysfunction, a step that is crucial in the pathogenesis of atherosclerosis (40,41). Our novel data of omentin-1 on angiogenesis in relation to the Akt signaling pathway thus clarifies omentin-1's potentially important role in the pathogenesis of cardiovascular disorders.
A limitation of our study may relate to the lack of a lean PCOS comparator group. Only 3 of the 21 PCOS subjects and 6 of the 39 control subjects had BMI < 25 kg/m2; thus, no meaningful result could be obtained. Future studies should address this point. Furthermore, given ethical constraints, that is, blood samples could and were only obtained at baseline and after 6 months of metformin treatment, it is therefore difficult to be certain as to whether changes in serum omentin-1 levels precede or follow changes in clinical and hormonal indexes as a consequence of metformin treatment. Also, as diet and lifestyle modifications were only subjectively assessed, the changes in serum omentin-1 levels may be partially attributable to diet and lifestyle modifications. Further studies are needed to elucidate this point. Additionally, like others (42), we used WHR, a universally accepted, simple, and cost-effective measure of adiposity as a surrogate marker for central fat mass. However, Cascella et al. has reported that WHR does not distinguish between visceral and subcutaneous fat mass in PCOS women (43). Therefore, more accurate and equally cost-effective methods of assessing central obesity are needed. Finally, given that PCOS is a proinflammatory state associated with clustering of cardiovascular risk factors, it would be of importance to study the effect of metformin therapy in the context of omentin-1 biology on reactive oxygen species in PCOS women, as reactive oxygen species not only induces tissue damage and inflammation but also is increased in dysmetabolic states including PCOS (44).
In conclusion, we provide evidence that increases in omentin-1 levels may play a role but are not sufficient to explain the decreased inflammatory and angiogenic effects of sera from metformin-treated PCOS women. Our findings provide novel insights into the relationship between obesity, insulin resistance, and metformin therapy with dysregulated angiogenesis in PCOS women in the context of omentin-1 biology.
ACKNOWLEDGMENTS
Dr. Dennis Heutling, Division of Endocrinology and Metabolism, Magdeburg University Hospital, Magdeburg, Germany, provided the clinical samples for this study. Dr. Kazuyuki Itoh, Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan, provided the immunoprecipitation protocol for immunodepletion of omentin-1 from human serum. The General Charities of the City of Coventry funded this study. No potential conflicts of interest relevant to this article were reported.
B.K.T. researched data, contributed to discussion, wrote the manuscript, and reviewed/edited the manuscript. R.A. researched data, contributed to discussion, and reviewed/edited the manuscript. S.F. and J.C. researched data. H.L. and H.S.R. contributed to discussion and reviewed/edited the manuscript.
H.S.R. acknowledges S. Waheguru, University of Warwick, for his continual support.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
- 1.Dunaif A: Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev 1997;18:774–800 [DOI] [PubMed] [Google Scholar]
- 2.Wild RA, Painter PC, Coulson PB, Carruth KB, Ranney GB: Lipoprotein lipid concentrations and cardiovascular risk in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1985;61:946–951 [DOI] [PubMed] [Google Scholar]
- 3.Diamanti-Kandarakis E, Paterakis T, Kandarakis HA: Indices of low-grade inflammation in polycystic ovary syndrome. Ann N Y Acad Sci 2006;1092:175–186 [DOI] [PubMed] [Google Scholar]
- 4.Glintborg D, Andersen M: An update on the pathogenesis, inflammation, and metabolism in hirsutism and polycystic ovary syndrome. Gynecol Endocrinol 2010;26:281–296 [DOI] [PubMed] [Google Scholar]
- 5.Sathyapalan T, Atkin SL: Mediators of inflammation in polycystic ovary syndrome in relation to adiposity. Mediators Inflamm 2010; 2010:758656. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kershaw EE, Flier JS: Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 2004;89:2548–2556 [DOI] [PubMed] [Google Scholar]
- 7.Wajchenberg BL: Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr Rev 2000;21:697–738 [DOI] [PubMed] [Google Scholar]
- 8.Thörne A, Lönnqvist F, Apelman J, Hellers G, Arner P: A pilot study of long-term effects of a novel obesity treatment: omentectomy in connection with adjustable gastric banding. Int J Obes Relat Metab Disord 2002;26:193–199 [DOI] [PubMed] [Google Scholar]
- 9.Tan BK, Chen J, Digby JE, Keay SD, Kennedy CR, Randeva HS: Increased visfatin messenger ribonucleic acid and protein levels in adipose tissue and adipocytes in women with polycystic ovary syndrome: parallel increase in plasma visfatin. J Clin Endocrinol Metab 2006;91:5022–5028 [DOI] [PubMed] [Google Scholar]
- 10.Yang RZ, Lee MJ, Hu H, Pray J, Wu HB, Hansen BC, Shuldiner AR, Fried SK, McLenithan JC, Gong DW: Identification of omentin as a novel depot-specific adipokine in human adipose tissue: possible role in modulating insulin action. Am J Physiol Endocrinol Metab 2006;290:E1253–E1261 [DOI] [PubMed] [Google Scholar]
- 11.Schäffler A, Neumeier M, Herfarth H, Fürst A, Schölmerich J, Büchler C: Genomic structure of human omentin, a new adipocytokine expressed in omental adipose tissue. Biochim Biophys Acta 2005;1732:96–102 [DOI] [PubMed] [Google Scholar]
- 12.de Souza Batista CM, Yang RZ, Lee MJ, Glynn NM, Yu DZ, Pray J, Ndubuizu K, Patil S, Schwartz A, Kligman M, Fried SK, Gong DW, Shuldiner AR, Pollin TI, McLenithan JC: Omentin plasma levels and gene expression are decreased in obesity. Diabetes 2007;56:1655–1661 [DOI] [PubMed] [Google Scholar]
- 13.Tan BK, Adya R, Farhatullah S, Lewandowski KC, O'Hare P, Lehnert H, Randeva HS: Omentin-1, a novel adipokine, is decreased in overweight insulin-resistant women with polycystic ovary syndrome: ex vivo and in vivo regulation of omentin-1 by insulin and glucose. Diabetes 2008;57:801–808 [DOI] [PubMed] [Google Scholar]
- 14.Fukushima Y, Tamura M, Nakagawa H, Itoh K: Induction of glioma cell migration by vitronectin in human serum and cerebrospinal fluid. J Neurosurg 2007;107:578–585 [DOI] [PubMed] [Google Scholar]
- 15.Zammit VC, Whitworth JA, Brown MA: Preeclampsia: the effects of serum on endothelial cell prostacyclin, endothelin, and cell membrane integrity. Am J Obstet Gynecol 1996;174:737–743 [DOI] [PubMed] [Google Scholar]
- 16.Kumar A, Kumar A, Michael P, Brabant D, Parissenti AM, Ramana CV, Xu X, Parrillo JE: Human serum from patients with septic shock activates transcription factors STAT1, IRF1, and NF-kappaB and induces apoptosis in human cardiac myocytes. J Biol Chem 2005;280:42619–42626 [DOI] [PubMed] [Google Scholar]
- 17.Krupinski J, Font A, Luque A, Turu M, Slevin M: Angiogenesis and inflammation in carotid atherosclerosis. Front Biosci 2008;13:6472–6482 [DOI] [PubMed] [Google Scholar]
- 18.Brown M, Cohen J, Arun P, Chen Z, Van Waes C: NF-kappaB in carcinoma therapy and prevention. Expert Opin Ther Targets 2008;12:1109–1122 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Lee SJ, Namkoong S, Kim YM, Kim CK, Lee H, Ha KS, Chung HT, Kwon YG, Kim YM: Fractalkine stimulates angiogenesis by activating the Raf-1/MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways. Am J Physiol Heart Circ Physiol 2006;291:H2836–H2846 [DOI] [PubMed] [Google Scholar]
- 20.Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS) (Position Statement). Human Reprod 2004;19:41–47 [DOI] [PubMed] [Google Scholar]
- 21.Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC: Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412–419 [DOI] [PubMed] [Google Scholar]
- 22.Montecucco F, Steffens S, Burger F, Pelli G, Monaco C, Mach F: C-reactive protein (CRP) induces chemokine secretion via CD11b/ICAM-1 interaction in human adherent monocytes. J Leukoc Biol 2008;84:1109–1119 [DOI] [PubMed] [Google Scholar]
- 23.Ahmad S, Hewett PW, Wang P, Al-Ani B, Cudmore M, Fujisawa T, Haigh JJ, le Noble F, Wang L, Mukhopadhyay D, Ahmed A: Direct evidence for endothelial vascular endothelial growth factor receptor-1 function in nitric oxide-mediated angiogenesis. Circ Res 2006;99:715–722 [DOI] [PubMed] [Google Scholar]
- 24.Bussolati B, Dunk C, Grohman M, Kontos CD, Mason J, Ahmed A: Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. Am J Pathol 2001;159:993–1008 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Kuzuya M, Kinsella JL: Reorganization of endothelial cord-like structures on basement membrane complex (Matrigel): involvement of transforming growth factor beta 1. J Cell Physiol 1994;161:267–276 [DOI] [PubMed] [Google Scholar]
- 26.Aranda E, Owen GI: A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA.hy926 endothelial cell line. Biol Res 2009;42:377–389 [PubMed] [Google Scholar]
- 27.Cress A, Banaszewska B, Pawelczyk L, Spaczynski RZ, Duleba AJ: A Combination of Simvastatin and Metformin Reduces Elevated Levels of Cytokines in Women with PCOS (Position Statement). In Proceedings of the 92nd Annual Meeting of the American Endocrine Society, San Diego, CA, June 19–22, 2010, P2–405, The Endocrine Society, Chevy Chase, MD [Google Scholar]
- 28.ELMekkawi SF, ELHosseiny AS, Mansour GM, Abbas AA, Asaad AM, Ali KS: Effect of Metformin Therapy on Serum Interleukin-6 and Interleukin-18 Levels in Patients with Polycystic Ovary Syndrome. New York Sci J 2010;3:83–86 [Google Scholar]
- 29.Takemura Y, Osuga Y, Yoshino O, Hasegawa A, Hirata T, Hirota Y, Nose E, Morimoto C, Harada M, Koga K, Tajima T, Yano T, Taketani Y: Metformin suppresses interleukin (IL)-1beta-induced IL-8 production, aromatase activation, and proliferation of endometriotic stromal cells. J Clin Endocrinol Metab 2007;92:3213–3218 [DOI] [PubMed] [Google Scholar]
- 30.Nardo LG, Rai R: Metformin therapy in the management of polycystic ovary syndrome: endocrine, metabolic and reproductive effects. Gynecol Endocrinol 2001;15:373–380 [DOI] [PubMed] [Google Scholar]
- 31.Johnson AB, Webster JM, Sum CF, Heseltine L, Argyraki M, Cooper BG, Taylor R: The impact of metformin therapy on hepatic glucose production and skeletal muscle glycogen synthase activity in overweight type II diabetic patients. Metabolism 1993;42:1217–1222 [DOI] [PubMed] [Google Scholar]
- 32.Perriello G, Misericordia P, Volpi E, Santucci A, Santucci C, Ferrannini E, Ventura MM, Santeusanio F, Brunetti P, Bolli GB: Acute antihyperglycemic mechanisms of metformin in NIDDM. Evidence for suppression of lipid oxidation and hepatic glucose production. Diabetes 1994;43:920–928 [DOI] [PubMed] [Google Scholar]
- 33.Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, Shulman GI: Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. N Engl J Med 1998;338:867–872 [DOI] [PubMed] [Google Scholar]
- 34.Sahin Y, Unluhizarci K, Yilmazsoy A, Yikilmaz A, Aygen E, Kelestimur F: The effects of metformin on metabolic and cardiovascular risk factors in nonobese women with polycystic ovary syndrome. Clin Endocrinol (Oxf) 2007;67:904–908 [DOI] [PubMed] [Google Scholar]
- 35.Lee JK, Schnee J, Pang M, Wolfert M, Baum LG, Moremen KW, Pierce M: Human homologs of the Xenopus oocyte cortical granule lectin XL35. Glycobiology 2001;11:65–73 [DOI] [PubMed] [Google Scholar]
- 36.Stoltz RA, Abraham NG, Laniado-Schwartzman M: The role of NF-kappaB in the angiogenic response of coronary microvessel endothelial cells. Proc Natl Acad Sci U S A 1996;93:2832–2837 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Shono T, Ono M, Izumi H, Jimi SI, Matsushima K, Okamoto T, Kohno K, Kuwano M: Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress. Mol Cell Biol 1996;16:4231–4239 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Dimmeler S, Zeiher AM: Akt takes center stage in angiogenesis signaling. Circ Res 2000;86:4–5 [DOI] [PubMed] [Google Scholar]
- 39.Shiojima I, Walsh K: Role of Akt signaling in vascular homeostasis and angiogenesis. Circ Res 2002;90:1243–1250 [DOI] [PubMed] [Google Scholar]
- 40.Ersoy C, Kiyici S, Budak F, Oral B, Guclu M, Duran C, Selimoglu H, Erturk E, Tuncel E, Imamoglu S: The effect of metformin treatment on VEGF and PAI-1 levels in obese type 2 diabetic patients. Diabetes Res Clin Pract 2008;81:56–60 [DOI] [PubMed] [Google Scholar]
- 41.Montagnani M, Ravichandran LV, Chen H, Esposito DL, Quon MJ: Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells. Mol Endocrinol 2002;16:1931–1942 [DOI] [PubMed] [Google Scholar]
- 42.Orio F, Jr, Palomba S, Cascella T, Milan G, Mioni R, Pagano C, Zullo F, Colao A, Lombardi G, Vettor R: Adiponectin levels in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2003;88:2619–2623 [DOI] [PubMed] [Google Scholar]
- 43.Cascella T, Palomba S, De Sio I, Manguso F, Giallauria F, De Simone B, Tafuri D, Lombardi G, Colao A, Orio F: Visceral fat is associated with cardiovascular risk in women with polycystic ovary syndrome. Hum Reprod 2008;23:153–159 [DOI] [PubMed] [Google Scholar]
- 44.González F, Rote NS, Minium J, Kirwan JP: Reactive oxygen species-induced oxidative stress in the development of insulin resistance and hyperandrogenism in polycystic ovary syndrome. J Clin Endocrinol Metab 2006;91:336–340 [DOI] [PubMed] [Google Scholar]