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Endocrinology and Metabolism Group (B.K.T., J.C., J.E.D., S.D.K., H.S.R.), Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, and Centre for Reproductive Medicine (C.R.K.), University Hospitals, Coventry CV2 2DX, United Kingdom
Address all correspondence and requests for reprints to: Dr. Harpal S. Randeva, MBCHB, FRCP, Ph.D., Endocrinology and Metabolism Group, Department of Biological Sciences, Warwick Medical School, The University of Warwick, Coventry CV4 7AL, United Kingdom. E-mail: hrandeva{at}bio.warwick.ac.uk.
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Abstract |
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Objectives: We investigated the mRNA expression of visfatin from sc and omental (om) adipose tissue and sc adipocytes in women with PCOS compared with matched normal women, as well as visfatin protein in adipose tissue; plasma visfatin was also assessed.
Design: Real-time RT-PCR and Western blotting were used to assess the relative mRNA and protein expression of visfatin. Biochemical measurements were performed.
Results: There was significant up-regulation of visfatin mRNA in both sc (P < 0.05) and om (P < 0.05) adipose tissue of PCOS women, when compared with normal controls; these findings were also reflected in isolated sc adipocytes (PCOS > controls; P < 0.05). In addition to elevated plasma visfatin levels in women with PCOS (mean ± SD, 30.2 ± 10.4 vs. 11.2 ± 6.2 ng/ml; P < 0.01) when compared with normal controls, visfatin protein levels were significantly greater in both sc and om adipose tissue of PCOS women (P < 0.05 and P < 0.01, respectively).
Conclusions: The precise reason for the up-regulation of visfatin seen in women with PCOS, a proinflammatory state, is unknown. Additional studies are needed to clarify the potential role of visfatin in the pathophysiology of PCOS.
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Introduction |
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The metabolic syndrome is closely associated with excessive accumulation of body fat. Besides its role in energy storage, adipose tissue, an endocrine organ, produces several hormones and cytokines that have wide-ranging effects on carbohydrate and lipid metabolism and therefore appear to play an important role in the pathogenesis of diabetes, IR, and atherosclerosis (3). However, it is apparent that accumulation of visceral adipose tissue poses a greater cardio-metabolic risk than sc adipose tissue, even though the latter is the larger adipose tissue depot of the two (4); removal of visceral rather than sc adipose tissue improves insulin sensitivity (5). Additionally, differences in gene expression of adipocyte-secreted molecules suggest that there are intrinsic adipose tissue depot-specific differences in the endocrine function of adipose tissue.
Visfatin (pre-B-cell colony-enhancing factor, PBEF), is a recently described adipokine reported to be preferentially produced by human visceral adipose tissue as compared with sc adipose tissue (6). It reportedly has insulin-mimetic actions, mediated by activation of the insulin receptor in a manner distinct from that of insulin (6). Also, plasma levels increased with obesity and correlated positively with visceral adiposity (6). Interestingly, others have not noted this association (7). Moreover, plasma concentrations of visfatin are increased in subjects with T2DM (8).
With the aforementioned in mind and the observation that women with PCOS have an increased prevalence of visceral obesity and the metabolic syndrome (1, 2), we studied the mRNA and protein expression of visfatin in both sc and visceral adipose tissue depots in these women and matched controls and their respective plasma visfatin levels.
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Subjects and Methods |
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After an overnight fast, blood samples and abdominal (sc) and omental (om) adipose tissue were obtained (0800–1000 h) from adult female patients undergoing elective surgery for infertility investigation. The sc biopsies were obtained from the same site, i.e. from a 3-cm horizontal midline incision approximately 3 cm above the symphysis pubis. All samples were obtained during the early follicular phase (d 2–4 from the first day of spontaneous bleeding episode); serum/plasma were immediately aliquoted on ice and stored at –80 C. Adipose tissue biopsies were frozen in liquid nitrogen immediately and stored at –80 C. The sc adipose tissues from subjects were placed into a sterile container containing Medium 199 (Sigma-Aldrich, Gillingham, UK) for primary adipocyte isolation. The control group had no discernible cause for infertility (unexplained infertility). Subjects underwent laparoscopy to rule out fallopian tube(s) obstruction. Furthermore, in most centers in the United Kingdom, and most certainly in our department, either a HysteroContrast Sonography or a laparoscopy is employed to assess fallopian tube(s) patency before commencing treatment (for example, clomiphene citrate) to correct anovulation, as the case may be for PCOS women, and thereby to rule out concurrent fallopian tube(s) obstruction. All patients underwent anthropometric measurements, i.e. weight, height, and waist to hip circumference ratio (WHR). Equal numbers of women (n = 8) were recruited in each group, one that consisted of women with PCOS and the other healthy controls (Table 1
). Eight women were overweight, i.e. with body mass index (BMI) between 25 and 30 (four PCOS and four controls); eight were obese, i.e. with BMI between 30 and 35 (four PCOS and four controls). The Local Research Ethics Committee approved the study, and all patients involved gave their informed consent, in accordance with the guidelines in The Declaration of Helsinki 2000.
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Biochemical and hormonal analysis
Assays for glucose, insulin, LH, FSH, 17ß-estradiol (E2), progesterone, testosterone, androstenedione, dehydroepiandrostenedione sulfate (DHEA-S), and SHBG were performed using an automated analyzer (Abbott Architect; Abbott Laboratories, Abbott Park, IL). The estimate of IR by homeostasis model assessment (HOMA) score was calculated as Iox Go/22.5, where Io is the fasting insulin and Go is the fasting glucose, as described by Matthews et al. (10).
Plasma visfatin levels were measured using a commercially available ELISA (EIA kit; Phoenix Pharmaceuticals, Belmont, CA) according to the manufacturer’s protocol, with a coefficient of variation of less than 6%.
Isolation of primary adipocytes
Primary adipocytes were isolated by a method modified from that of Rodbell (11) and Gliemann et al. (12). Fat samples were washed with a large volume of PBS, meticulously cleaned of obvious blood vessels, connective tissue, and skin, and then chopped with sterile scissors. The cleaned samples were washed again with saline, and the floating, clean fat recovered by centrifugation. Adipocytes were isolated by collagenase digest (Hanks’ balanced salt solution containing 3 mg/ml collagenase type II and 1.5% BSA) in a shaking water bath at 37 C for up to 60 min. Mature adipocytes were separated from the stromal vascular cells through an inert oil, bis-3,5,5-trimethylhexyl phthalate (Fluka Chemicals, Gillingham, UK). Total RNA extraction and cDNA synthesis from isolated adipocytes were performed immediately as described below. To exclude potential contamination of the adipocytes, von Willebrand factor was examined, revealing its absence in adipocytes, employing human umbilical vascular endothelial cells as the positive control; moreover, there was negligible CD45 and CD14 gene expression when compared with adipose tissue as the positive control (data not shown).
Total RNA extraction and cDNA synthesis
Total RNA was extracted from whole adipose tissue samples and isolated adipocytes using QIAGEN RNeasy Lipid Tissue Mini Kit according to the manufacturer’s guidelines (QIAGEN, Crawley, UK). First-strand cDNA synthesis was carried out using Moloney Murine Leukemia Virus reverse transcriptase and random hexamers as primers.
RT-PCR
Quantitative PCR of visfatin, CD45, and CD14 were performed on a Roche Light Cycler system (Roche Molecular Biochemicals, Mannheim, Germany). PCR were carried out in a reaction mixture consisting of 5.0 µl reaction buffer and 2.0 mM MgCl2 (Biogene, Kimbolton, UK), 1.0 µl of each primer (10 ng/µl), 2.5 µl cDNA and 0.5 µl Light Cycler DNA Master SYBR Green I (Roche). Protocol conditions consisted of denaturation of 95 C for 15 sec, followed by 40 cycles of 94 C for 1 sec, 58 C for 10 sec, and 72 C for 12 sec, followed by melting-curve analysis. For analysis, quantitative amounts of genes of interest were standardized against the housekeeping gene ß-actin. The RNA levels were expressed as a ratio, using the delta-delta method for comparing relative expression results between treatments in real-time PCR (13). The following primers were used for our study: visfatin (228 bp), 5'-AAGAGACTGCTGGCATAGGA-3' and 5'-ACCACAGATACAG GCACTGA-3'; CD45 (194 bp), 5'-AGGAGAGTGAATGCCTTCAG-3' and 5'-GCC TCTACTTGAACCATCAG-3'; and CD14 (173 bp), 5'-CGCAACACAGGAATGGAGAC-3' and 5'-CCAGCGAACGACAGATTGAG-3'.
Ten microliters of the reaction mixture was subsequently electrophoresed on a 1% agarose gel and visualized by ethidium bromide, using a 1-kb DNA ladder (Life Technologies, Inc.-BRL, Paisley, UK) to estimate the band sizes. As a negative control for all the reactions, preparations lacking RNA or reverse transcriptase were used in place of the cDNA. RNAs were assayed from three independent biological replicates.
Sequence analysis
The PCR products from the adipose tissue and adipocyte samples were purified from the 1% agarose gel using the QIAquick Gel Extraction Kit (QIAGEN). PCR products were then sequenced in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Centre for Biotechnology Information, confirming the identity of our products.
Western blotting
Protein lysates were prepared by homogenizing adipose tissue in radioimmunoprecipitation lysis buffer (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s instructions. Equal amounts of Laemmli buffer (5 M urea, 0.17 M SDS, 0.4 M dithiothreitol, and 50 mM Tris-HCl, pH 8.0) were added, mixed, and placed in a boiling water bath for 5 min. Lysates were allowed to cool at room temperature. The proteins (38.7 µg/lane) were separated by SDS-PAGE (10% resolving gel) and transferred to polyvinylidene difluoride (PVDF) membranes at 100 V for 1 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. PVDF membranes were then blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% BSA for 2 h. After three washes with TBS/0.1% Tween, the PVDF membranes were incubated with primary rabbit-antihuman antibody for visfatin (Bethyl Laboratories Inc., Montgomery, TX) at a 1:10,000 dilution or primary rabbit-antihuman antibody for ß-actin (Cell Signaling Technology Inc., Beverly, MA) at a 1:1000 dilution overnight at 4 C. The membranes were washed thoroughly for 60 min with TBS/0.1% Tween before incubation with the secondary antirabbit horseradish-peroxidase-conjugated Ig (1:2000) for 1 h at room temperature. Antibody complexes were visualized using chemiluminescence (ECL; Amersham Pharmacia, Little Chalfont, UK). Visfatin peptide was used as the positive control and water as the negative control (data not shown).
Statistical analyses
For the real-time PCR measurements, Western immunoblotting, and blood analysis, results were evaluated between groups by using unpaired Student’s t test, with significance determined at the level of P < 0.05. The Wilcoxon matched-pairs test and the Mann-Whitney U test were used for comparisons within and between groups, respectively, and P < 0.05 was considered significant. For Western immunoblotting experiments, the densities were measured using a scanning densitometer coupled to scanning software ImageQuant (Molecular Dynamics, Amersham Pharmacia). Spearman rank correlation was used for calculation of associations between variables, and multiple regression analysis contained insulin, HOMA, testosterone, and SHBG; P < 0.05 was considered significant.
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Results |
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Table 1
shows the anthropometric, biochemical, and hormonal data in both PCOS and control women, and the data are shown as the mean ± SD. As expected, insulin, testosterone, and androstenedione levels, HOMA, and free androgen index were significantly higher in women with PCOS. ELISA analysis of plasma visfatin levels revealed that PCOS patients have significantly elevated levels when compared with controls (30.2 ± 10.4 vs. 11.2 ± 6.2 ng/ml; P < 0.01; Table 1). Serum progesterone levels in all women confirmed follicular phase of the menstrual cycle (1.6 ± 0.3 nmol/liter; follicular phase, <6 nmol/liter; luteal phase, 6–64 nmol/liter).
mRNA expression of visfatin in normal and PCOS women
Using RT-PCR analysis, we detected visfatin mRNA in adipose tissue and in adipocytes, and subsequent sequencing of the PCR products confirmed the gene identity. Furthermore, real-time RT-PCR analysis corrected over ß-actin showed a significant up-regulation of visfatin in both sc and om adipose tissue of PCOS women, when compared with normal controls (Fig. 1, A and B; P < 0.05). Similar findings were noted in isolated sc adipocytes from these women, where PCOS women had significantly higher expression of visfatin compared with controls (Fig. 1C; P < 0.05). Interestingly, visfatin was seen to be significantly up-regulated in om adipose tissue of PCOS patients when compared with corresponding sc adipose tissue (Fig. 1D; P < 0.01), whereas there was no significant difference in the expression of visfatin in the om and sc adipose tissue depots of normal controls (Fig. 1D; P > 0.05).
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Once again, RT-PCR analysis detected CD45 and CD14 mRNA in adipose tissue of both groups of women, and subsequent sequencing of the PCR products confirmed the identity of both genes (Fig. 2A-B). The levels of CD45 and CD14 mRNA, a marker for macrophage index, were assessed in normal and PCOS women, both in sc and om adipose tissues. Real-time RT-PCR analysis revealed that there was no statistical difference in CD45 and CD14 between both groups of women in either adipose tissue depot (P > 0.05).
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The changes noted at the mRNA level were also reflected at the protein level in PCOS women, with significantly greater visfatin expression levels in om vs. sc adipose tissue (Fig. 3A; P < 0.05), but no significant difference in ß-actin protein was noted as per immunoblotting (Fig. 3B; P > 0.05). Interestingly, unlike the mRNA data, visfatin protein was significantly higher in om vs. sc adipose tissue in normal controls (Fig. 3A; P < 0.05). The detected proteins for visfatin and ß-actin have apparent molecular masses of 55 and 45 kDa, respectively (Fig. 3B, inset). Furthermore, when compared with normal controls, visfatin protein levels were significantly greater in both sc and om adipose tissue depots of PCOS women (Fig. 3B; P < 0.05 and P < 0.01, respectively).
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When both groups were analyzed collectively, no significant correlation was noted between plasma visfatin and BMI (P = 0.12) or WHR (P = 0.15). Plasma visfatin was positively associated with insulin (P < 0.01), HOMA (P < 0.01), testosterone (P = 0.03), and E2 (P = 0.046) and negatively with SHBG (P = 0.03). Multiple regression analysis contained insulin, HOMA, testosterone, and SHBG (Table 2). Interestingly, HOMA was the only predictor of plasma visfatin (ß = 1.23; P = 0.02), with insulin failing to reach significance (ß = –0.54; P = 0.09). Similar findings were noted when the groups were analyzed individually. Furthermore, plasma visfatin levels correlated positively with visfatin mRNA from sc (P = 0.01) and om (P = 0.037) and visfatin protein levels from sc (P = 0.002) and om adipose tissue (P = 0.008).
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and 4), in multiple linear regression analysis, only insulin (ß = –1.15l; P = 0.05) and HOMA (ß = 2.35; P = 0.014) were predictors of visfatin protein in om adipose tissue; HOMA just failed to reach significance in sc adipose tissue (P = 0.07).
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Discussion |
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The up-regulation of visfatin in PCOS women, a pro-diabetogenic state, is of interest given that subjects with T2DM have higher plasma and adipose tissue mRNA expression of visfatin levels, compared with controls (8, 14), after accounting for BMI and WHR. In our study, it is unlikely that either BMI or WHR is responsible for higher visfatin levels in PCOS women, given that both groups were matched for these variables. Besides, Hammarstedt et al. (14), like us, did not find any significant correlation between WHR, a surrogate marker for visceral fat, and circulating levels of visfatin. Moreover, like others (7), although we detected a significant positive relationship between visfatin gene expression and BMI and WHR in both adipose tissue depots, we report novel data of no significant correlation between these anthropometric measures and visfatin protein in adipose tissue, in keeping with our plasma visfatin data. Our observations and those of others (7, 14) are in contrast to the original findings by Fukuhara et al. (6) and the more recent findings by Chen et al. (8), where a positive correlation was noted between plasma visfatin and visceral fat mass and also WHR. Moreover, unlike us, they did not study visfatin protein expression in adipose tissue and its relation to BMI/WHR. The precise reason for these differences is not clear at present, but it may be related to ethnic differences, where some studies are from Far Eastern populations (6, 8) and others (7, 14), like ours, from Caucasians.
The insulin-mimetic action of visfatin has been proposed to contribute to the development of the metabolic syndrome (6). Women with PCOS, as in our study, have a higher incidence of IR. We noted a positive correlation between plasma visfatin levels and both insulin and HOMA, as previously reported by some authors (8, 15) but not others (7); however, in multiple linear regression analysis, only HOMA was significantly associated with plasma visfatin levels. We went on further to determine the relationship between visfatin protein in adipose tissue and metabolic parameters and report new findings that insulin and HOMA are significantly correlated to visfatin protein in om but not sc adipose tissue. In addition, we noted a significant correlation between plasma visfatin and protein visfatin levels in both om and sc adipose tissue, a finding not described before.
In our control group, similar to others (7, 14), we found no difference in visfatin mRNA levels between om and sc adipose tissue, but we did observe significantly higher visfatin protein expression in om when compared with sc tissue. In our PCOS group, om visfatin mRNA expression and protein level was significantly higher than sc adipose tissue. The precise reason for higher omental visfatin in both control and PCOS women is unclear. Recently, macrophages in human visceral adipose tissue have been described to be a source of visfatin (16). In our study, in both controls and PCOS women, as expected, there was higher CD14 and CD45 gene expression, hallmarks of macrophages, in om vs. sc adipose tissue depots. More importantly, PCOS women did not have significantly higher CD14 and CD45 levels in either adipose tissue depot compared with controls. Therefore, although they may contribute to the visfatin pool, it is unlikely that macrophages are the key reason for our observations. Furthermore, in isolated sc adipocytes, visfatin expression was significantly higher in PCOS women. Unfortunately, because of technical limitations in om adipose tissue procurement, we were unable to obtain sufficient amounts of sample per patient to perform adipocyte separation in om adipose tissue depots. These limitations notwithstanding, it is clear that both adipose tissue and adipocytes in PCOS women express more visfatin, with a parallel increase in plasma visfatin.
Studies in adipocytes from women with PCOS reveal adipocyte insensitivity to inhibition of lipolysis by insulin as well as a decrease in maximal rates of adipocyte glucose uptake (17). We hypothesize that the elevated visfatin levels in both plasma and adipose tissue in PCOS women in our study may suggest a role of visfatin signaling in target tissues or the dysregulation in biosynthesis and thereby the increased need for insulin receptor signaling in PCOS adipose tissue. In addition, we speculate that the increased visfatin in PCOS women may be a compensatory response to IR in specific tissues or a marker of tissue-specific inflammatory cytokine action and that this may account, in particular, for the higher om visfatin expression. Finally, visfatin may not only have an endocrine role in modulating insulin sensitivity (6), but also have autocrine/paracrine effects, given that it is expressed in pure adipocytes, as shown here and by others (16).
A limitation of our study may relate to the number of subjects studied. However, obtaining BMI/WHR-matched and menstrual cycle-synchronized blood and tissue samples impeded subject recruitment. This may in part explain why in bivariate analysis, androgens (testosterone and DHEA-S) significantly correlated with only insulin and HOMA, and testosterone with visfatin (data not shown); however, in multivariate analysis, insulin and HOMA were the only significant factors correlating with visfatin. It is also important to recognize that potentially several of these correlations may be without causative significance and result from the fact that hyperandrogenism and IR characterizes PCOS. Nonetheless, our observations are consistent and significant and raise interesting questions on the mechanisms regulating visfatin expression. Furthermore, it will be interesting to see whether these findings are also present in matched lean (BMI < 25) PCOS women vs. normal controls.
In conclusion, we present novel data with regard to the increased expression both at the mRNA and protein levels as well as plasma levels of visfatin in women with PCOS. Although the up-regulation of visfatin seen in PCOS women may act in an endocrine manner, we speculate that visfatin may have important autocrine/paracrine effects. The physiological and pathological significance of our findings remain to be elucidated.
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Acknowledgments |
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Footnotes |
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Disclosure statement: The authors have nothing to declare.
First Published Online September 26, 2006
1 B.K.T. and J.C. have contributed equally to the manuscript. 
Abbreviations: BMI, Body mass index; DHEA-S, dehydroepiandrosterone sulfate; E2, 17ß-estradiol; HOMA, homeostasis model assessment; IR, insulin resistance; om, omental; PCOS, polycystic ovary syndrome; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; T2DM, type 2 diabetes mellitus; WHR, waist to hip circumference ratio.
Received May 3, 2006.
Accepted September 20, 2006.
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