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Insulin and Metformin Regulate Circulating and Adipose Tissue Chemerin

Insulin and Metformin Regulate Circulating and Adipose Tissue Chemerin ORIGINAL ARTICLE Insulin and Metformin Regulate Circulating and Adipose Tissue Chemerin 1 1 1 1 1 2 Bee K. Tan, Jing Chen, Syed Farhatullah, Raghu Adya, Jaspreet Kaur, Dennis Heutling, 1,3 1 1,4 1 Krzysztof C. Lewandowski, J. Paul O’Hare, Hendrik Lehnert, and Harpal S. Randeva consequent hyperinsulinemia is more prevalent in lean and OBJECTIVE—To assess chemerin levels and regulation in sera obese women with PCOS when compared with age- and and adipose tissue from women with polycystic ovary syndrome weight-matched normal women (3). (PCOS) and matched control subjects. The metabolic syndrome is associated with excessive RESEARCH DESIGN AND METHODS—Real-time RT-PCR accumulation of central body fat. As well as its role in and Western blotting were used to assess mRNA and protein energy storage, adipose tissue produces several hormones expression of chemerin. Serum chemerin was measured by and cytokines termed ‘adipokines’ that have widespread enzyme-linked immunosorbent assay. We investigated the in vivo effects on carbohydrate and lipid metabolism. They appear effects of insulin on serum chemerin levels via a prolonged to play an important role in the pathogenesis of insulin insulin-glucose infusion. Ex vivo effects of insulin, metformin, and steroid hormones on adipose tissue chemerin protein pro- resistance, diabetes, and atherosclerosis (4). Furthermore, duction and secretion into conditioned media were assessed by it is apparent that accumulation of visceral adipose tissue Western blotting and enzyme-linked immunosorbent assay, poses a greater cardiometabolic risk than subcutaneous respectively. adipose tissue (5) as removal of visceral rather than subcutaneous adipose tissue has been shown to improve RESULTS—Serum chemerin, subcutaneous, and omental adi- insulin sensitivity (6). Moreover, differences in gene ex- pose tissue chemerin were significantly higher in women with PCOS (n  14; P  0.05, P  0.01). Hyperinsulinemic induction pression of adipocyte-secreted molecules (adipokines) in human subjects significantly increased serum chemerin levels suggest that there are inherent adipose tissue depot– (n  6; P  0.05, P  0.01). In adipose tissue explants, insulin specific differences in the endocrine function of adipose significantly increased (n  6; P  0.05, P  0.01) whereas tissue. In relation to this, we have published data on the metformin significantly decreased (n  6; P  0.05, P  0.01) increased levels of vaspin in women with PCOS (7); vaspin chemerin protein production and secretion into conditioned is a recently described adipokine mainly formed in human media, respectively. After 6 months of metformin treatment, visceral adipose tissue that has insulin-sensitizing effects there was a significant decrease in serum chemerin (n  21; P (8). 0.01). Importantly, changes in homeostasis model assessment– Recently, Bozaoglu et al. (9) reported chemerin as a insulin resistance were predictive of changes in serum chemerin (P  0.046). novel adipokine, circulating levels of which significantly correlated with BMI, circulating triglycerides, and blood CONCLUSIONS—Serum and adipose tissue chemerin levels are pressure, features of the metabolic syndrome. In addition, increased in women with PCOS and are upregulated by insulin. chemerin or chemerin receptor knockdown impaired dif- Metformin treatment decreases serum chemerin in these women. ferentiation of 3T3-L1 cells and attenuated the expression Diabetes 58:1971–1977, 2009 of adipocyte genes involved in glucose and lipid homeosta- sis (10). With the aforementioned in mind and the fact that there olycystic ovary syndrome (PCOS), a common is no literature with regards to chemerin in human adipose endocrinopathy affecting 5–10% of women in the tissue and its regulation, in study 1, we assessed circulat- reproductive age, is characterized by menstrual ing chemerin as well as mRNA expression and protein Pdysfunction and hyperandrogenism and is levels of chemerin in subcutaneous and omental adipose associated with insulin resistance and pancreatic -cell tissue depots in women with PCOS against age, BMI, and dysfunction, impaired glucose tolerance (IGT), type 2 waist-to-hip ratio (WHR) in matched control subjects. diabetes, dyslipidemia, and visceral obesity (1,2). The Furthermore, we studied the in vivo (study 2) and ex vivo effects of insulin on circulating chemerin levels via a prolonged insulin-glucose infusion in humans and From the Endocrinology and Metabolism Group, Clinical Sciences Research primary adipose tissue explant cultures, respectively. In Institute, Warwick Medical School, University of Warwick, Coventry, U.K.; study 3 we studied the effects of metformin therapy, the Division of Endocrinology and Metabolism, Magdeburg University Hospital, Magdeburg, Germany; the Department of Endocrinology and widely used in the treatment of PCOS in women, on Metabolic Diseases, The Medical University of Lodz and Polish Mother’s circulating chemerin levels in tandem with associated 4 st Memorial Research Institute, Lodz, Poland; and the 1 Medical Depart- changes to clinical, hormonal, and metabolic parame- ment, University of Lu¨ beck Medical School, Lu¨ beck, Germany. Corresponding author: Harpal S. Randeva, [email protected]. ters in the same cohort of PCOS in women. Additionally, Received 3 November 2008 and accepted 19 May 2009. we studied the ex vivo effects of metformin and steroid Published ahead of print at http://diabetes.diabetesjournals.org on 5 June hormones in human primary adipose tissue explants. 2009. DOI: 10.2337/db08-1528. © 2009 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by RESEARCH DESIGN AND METHODS -nc-nd/3.0/ for details. Study 1. Seventy three subjects were recruited consecutively from the The costs of publication of this article were defrayed in part by the payment of page infertility clinic in accordance with the inclusion/exclusion criteria (PCOS: charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. n  19; control subjects: n  54) as previously described (7); 62 subjects were DIABETES, VOL. 58, SEPTEMBER 2009 1971 INSULIN AND METFORMIN REGULATE CHEMERIN TABLE 1 Clinical, hormonal, and metabolic features of women with PCOS and control subjects PCOS (n  14) Control subjects (n  14) Significance Age (years) 29.5 (28–38) 32.5 (29–35) NS BMI (kg/m ) 30.5 (27.8–30.9) 28.8 (28–30.5) NS WHR 0.89 (0.78–0.99) 0.84 (0.81–0.96) NS Glucose (mmol/l) 5.5 (4.8–6.0) 4.5 (4.3–5.2) P  0.01 Insulin (pmol/l) 78.9 (42.0–91.1) 57.3 (48.5–66.0) NS HOMA-IR 3.0 (2.1–3.6) 2.0 (1.4–2.2) P  0.05 Cholesterol (mmol/l) 5.1 (4.1–5.7) 5.0 (4.8–5.5) NS Triglycerides (mmol/l) 2.0 (1.5–2.3) 0.9 (0.7–1.4) P  0.01 Luteinizing hormone (IU/l) 7.0 (6.0–10.0) 6.0 (5.0–7.0) NS Follicle-stimulating hormone (IU/l) 6.0 (6.0–7.0) 6.5 (5.0–8.0) NS Prolactin (mU/l) 348.0 (305.0–387.0) 304.5 (211.0–322.0) NS E (pmol/l) 353.5 (287.0–471.0) 174.5 (129.0–264.0) P  0.01 Progesterone (nmol/l) 1.6 (1.3–2.1) 2.2 (1.7–2.3) NS 17-OH-P (nmol/l) 2.5 (2.1–2.8) 2.0 (1.2–2.3) NS Testosterone (nmol/l) 1.7 (1.5–2.1) 0.8 (0.6–0.9) P  0.01 Androstenedione (nmol/l) 15.6 (14.2–16.8) 6.3 (5.0–8.2) P  0.01 DHEA-S (mol/l) 5.9 (5.4–6.6) 4.5 (4.0–5.3) P  0.05 SHBG (nmol/l) 31.5 (26.7–35.2) 59.0 (47.7–66.0) P  0.01 FAI 19.5 (14.5–21.2) 3.9 (3.3–6.1) P  0.01 Leptin (ng/ml) 24.9 (19.7–29.0) 24.1 (19.3–28.8) NS Adiponectin (g/ml) 5.67 (4.52–7.5) 6.27 (5.52–9.48) NS Chemerin (ng/ml) 6.02 (5.96–6.04) 2.62 (1.96–3.23) P  0.01 Data are medians (interquartile range). Group comparison by Mann-Whitney U test. FAI  Testosterone (nmol/liter)/SHBG (nmol/liter) 100. NS  not significant. FAI, free androgen index; SHBG, sex hormone–binding globulin. from a previous study (7). Of the 19 PCOS subjects recruited, five withdrew was offered to all women with PCOS independently from the results of before the study could be completed. In the control group, seven subjects did insulin sensitivity testing, as per standard clinical practice. The study not complete the study. From the remaining 47 control subjects, 14 control design was approved by the local research ethics committee of the subjects matched for age, BMI, and WHR were included in the final analysis University of Magdeburg, and written informed consent was obtained from (Table 1). Blood and adipose tissue samples were obtained as previously all participants in accordance with the guidelines in The Declaration of described (7). Subcutaneous adipose tissue was obtained froma3cm Helsinki 2000. horizontal midline incision 3 cm above the symphysis pubis. Omental Biochemical and hormonal analysis. Assays were performed using an adipose tissue was obtained by excisional biopsy from the greater omentum. automated analyzer as previously described (7). The estimate of insulin The local research ethics committee of University Hospitals Coventry and resistance by homeostasis model assessment (HOMA-IR) score was calcu- Warwickshire NHS Trust approved the study, and all patients involved gave lated as previously described (12). Circulating leptin and adiponectin levels their informed consent in accordance with the guidelines in The Declaration were measured with a coated-tube immunoradiometric assay kit (Diagnostic of Helsinki 2000. Systems Laboratories, Sinsheim, Germany) and by a commercially available Study 2. We measured circulating chemerin in six (three men, three women) RIA kit (Millipore, Watford, U.K.), respectively, according to manufacturer’s healthy subjects [age: (mean  SD) 26.5  8 years, BMI: 23.2  2.5 kg/m ]. All protocol. subjects studied were nonsmokers and otherwise healthy. Women volunteers Chemerin levels in sera and conditioned media from human adipose tissue had regular periods, no clinical or biochemical hyperandrogenism, and were explants were measured using a commercially available enzyme-linked immu- not taking any medications. Exclusion criteria for the study, as previously nosorbent assay (ELISA) kit (R & D Systems, Abingdon, U.K.), according to described (7), included age over 40 years, known cardiovascular disease, manufacturer’s protocol, with an intra-assay coefficient of variation of less thyroid disease, neoplasms, current smoking, diabetes, hypertension (blood than 9%. pressure 140/90 mmHg), and renal impairment (serum creatinine 120 Primary explant culture. Adipose tissue organ explants were cultured using mol/l). None of these women were on any medication for at least 6 months a protocol that was a modification of the method described by Fried and before the study, including oral contraceptives, glucocorticoids, ovulation Moustaid-Moussa (13). Adipose tissue explants were cultured for 24 h with or induction agents, antidiabetic and antiobesity drugs, or estrogenic, antiandro- without the addition of insulin, testosterone, 17-estradiol, androstenedione, genic, or antihypertensive medication. The local research ethics committee of dehydroepiandrosterone-sulfate (DHEA-S), or metformin, as previously de- University Hospitals Coventry and Warwickshire NHS Trust approved the scribed (7). study, and all patients involved gave their informed consent in accordance Total RNA extraction and cDNA synthesis. Total RNA was extracted from with the guidelines in The Declaration of Helsinki 2000. To account for the adipose tissue samples and cDNA synthesized as previously described (7). possible diurnal variation in chemerin levels, we obtained a daily control RT-PCR. Quantitative PCR of chemerin was performed on a Roche Light curve by measuring fasting chemerin levels at 30-min intervals from 0800 to Cycler system (Roche Molecular Biochemicals, Mannheim, Germany) as 1000 h. Subsequently, chemerin levels were measured at 2-h intervals until previously described (7). The sequences of the sense and antisense primers 2400 h and then at 0400 h as well as at 30-min intervals from 0800 to 1000 h on used were chemerin (252 bp) 5 -AGACAAGCTGCCGGAAGAGG-3 and 5 - day 2. On the following day, the same subjects were subjected to a prolonged TGGAGAAGGCGAACTGTCCA-3 ; -actin (216 bp) 5 -AAGAGAGGCATCCT- insulin-glucose infusion for 26 h beginning at 0800 h. Insulin (Human Actrapid) CACCCT-3 and 5 -TACATGGCTGGGGTCTTGAA-3 . was administered intravenously as a priming dose of 0.04 units/kg followed by Western blotting. Protein lysates were prepared as previously described (7). 1 1 continuous infusion of 0.5 mU  kg  min . By choosing this rate of insulin Protein samples (30 g/lane) containing SDS sample buffer (5 M urea, 0.17 M infusion we expected to achieve hyperinsulinemia with an approximate four- SDS, 0.4 M dithiothreitol, and 50 mmol/l Tris-HCl, pH 8.0) were subjected to to sixfold elevation of basal insulinemia (11). Fasting blood samples were SDS-PAGE (10% resolving gel) and transferred to polyvinylidene difluoride drawn at 30-min intervals between 0800 and 1000 h on day 1 and day 2 of the (PVDF) membranes (Millipore). PVDF membranes were blocked in Tris- prolonged insulin-glucose infusion (the first and the last2hofthe infusion). buffered saline (TBS) containing 0.1% Tween-20 and 5% BSA for 2 h. The PVDF Intermediate blood samples were taken at 2-h intervals until 2400 h and then membranes were then incubated with polyclonal primary goat anti-human at 0400 h on day 2. Glucose levels were maintained between 4.0 and 6.0 antibody for chemerin (R & D Systems) (1:1,000 dilution) or monoclonal mmol/l. primary rabbit anti-human antibody for -actin (Cell Signaling Technology, Study 3. Subjects were recruited and blood samples were obtained as Beverly, CA) (1:1,000 dilution) overnight at 4°C. The membranes were washed previously described (7). A treatment with metformin in an “off-label use” thoroughly for 60 min with TBS 0.1% Tween before incubation with the 1972 DIABETES, VOL. 58, SEPTEMBER 2009 B. K. TAN AND ASSOCIATES secondary anti-goat horseradish peroxidase–conjugated immunoglobulin A Sc Om (Dako, Ely, U.K.) (1:2,000) or secondary anti-rabbit horseradish peroxidase– conjugated immunoglobulin (Dako) [1:2000], respectively, for1hat room temperature. Antibody complexes were visualized using chemiluminescence ** (ECL ; GE Healthcare, Little Chalfont, U.K.). Human chemerin protein (R & D Systems) was used as positive control and water as negative control (data not shown). Statistics. Data were analyzed by Student’s t test, Mann-Whitney U test, Kruskal-Wallis, or Friedman’s ANOVA (post hoc analysis: Dunn’s test) accord- ing to the number of groups compared; P  0.05 was considered significant. For Western immunoblotting experiments, the densities were measured using a scanning densitometer coupled to scanning software Scion Image (Scion Corporation, Frederick, MD). 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 chemerin and -actin were made only within this linearity range. Spearman Rank correlation Normal PCOS Normal PCOS was used for calculation of associations between variables; P  0.05 was considered significant. Sc Om ** RESULTS Demographic data. Table 1 shows the anthropometric, biochemical, and hormonal data in all subjects. Glucose, HOMA, triglycerides, 17-estradiol (E ), testosterone, an- drostenedione, DHEA-S levels, and free androgen index were significantly higher, whereas sex hormone–binding globulin was significantly lower in women with PCOS. Serum chemerin levels were significantly higher in PCOS subjects than in control subjects (6.02 [5.96–6.04] vs. 2.62 [1.96–3.23] ng/ml; P  0.01; Table 1). Serum Normal PCOS Normal PCOS progesterone levels in all women confirmed follicular phase of the menstrual cycle. FIG. 1. A: Chemerin mRNA expression relative to -actin was mRNA expression and protein levels of chemerin in significantly increased in human subcutaneous (Sc) and omental (Om) adipose tissue depots when comparing PCOS women (n  14) normal and PCOS women. We detected chemerin mRNA with normal control subjects (n  14), using real-time RT-PCR. Data in adipose tissue of all subjects, and subsequent sequenc- are expressed as percent difference of median of human subcutane- ing of the PCR products confirmed gene identity. Real-time ous adipose tissue of normal control subjects. Each experiment was carried out in three replicates. Group comparison by Kruskal-Wallis RT-PCR analysis corrected over -actin showed a signifi- ANOVA and post hoc Dunn’s test; *P < 0.05, **P < 0.01. B: cant increase of chemerin expression in subcutaneous Densitometric analysis of chemerin immune complexes having nor- (*P  0.05) and omental (**P  0.01) adipose tissue of malized to -actin revealed that protein levels of chemerin were significantly increased in human subcutaneous and omental adipose PCOS when compared with normal control subjects tissue depots, respectively, when comparing all women with PCOS (Fig. 1A). However, no significant difference in with all normal control subjects. Data are expressed as percent chemerin mRNA expression was observed when com- difference of median of normal control subjects. Each experiment was carried out in three replicates. Group comparison by Kruskal- paring corresponding omental with subcutaneous adi- Wallis ANOVA (post hoc analysis: Dunn’s test). *P < 0.05, **P < pose tissue in PCOS and normal subjects (Fig. 1A; P 0.01; PSL, phosphostimulated light units. 0.05). Changes noted at mRNA level were also reflected at protein level (Fig. 1B). Study 2: Effects of a prolonged insulin-glucose infu- data of PCOS subjects investigated in study 1 were not sion on serum chemerin levels. Insulin infusion resulted significantly different compared with the PCOS subjects in elevation of fasting insulinemia from 78.1  12.0 pmol/l investigated in study 3. Reasons for subjects not complet- to 294.6  31.0 pmol/l. Insulin levels remained elevated ing study 3 were nausea and gastrointestinal side effects until the end of the prolonged insulin-glucose infusion (n  4), pregnancies (n  5), incompliance (n  2), and (366.0  37.0 pmol/l). Chemerin levels remained unaltered loss of contact (n  2). After 6 months of metformin throughout the control day from 2.06  0.08 ng/ml be- treatment, there were significant decreases in serum tween 0800 and 1000 h to 1.92  0.07 ng/ml between 0800 chemerin, WHR, E , testosterone, glucose, and HOMA-IR and 1000 h the next day (Fig. 2; P  0.05). (Table 2). There was a profound effect of insulin on chemerin Dose-dependent effects of insulin, testosterone, 17- levels over 26 h of insulin infusion: from 2.54  0.32 ng/ml estradiol, androstenedione, DHEA-S, and metformin between 0800 and 1000 h to 3.97  0.37 ng/ml between on chemerin protein production and secretion into 0800 and 1000 h the following day (Fig. 2; *P  0.05). The conditioned media from control human omental adi- increase in chemerin levels was relatively acute approach- pose tissue explants. We found that chemerin protein ing maximal values at 4 h (Fig. 2; 5.08  0.27 ng/ml, **P  production and secretion into conditioned media was 0.01) and persisting throughout the entire period of significantly increased dose dependently by insulin in hyperinsulinemia. control human omental adipose tissue explants (Fig. 3; Study 3: Effects of metformin treatment on serum *P  0.05 and **P  0.01, respectively). Interestingly, chemerin levels. Metformin treatment was started in 34 metformin significantly decreased chemerin protein pro- women with PCOS. Only 21 women completed the study duction and secretion into conditioned media in control and were investigated after 6 months of metformin treat- human omental adipose tissue explants (Fig. 4 and *P ment. The anthropometric, biochemical, and hormonal 0.05 and **P  0.01, respectively). Similar results were DIABETES, VOL. 58, SEPTEMBER 2009 1973 Chemerin/β-actin mRNA Chemerin/β-actin PSL (% difference) expression (% difference) INSULIN AND METFORMIN REGULATE CHEMERIN Control Infusion ** ** 0800- 1200- 1600- 2200- 0400 0800- 1000 1400 1800 2400 day 2 1000 T0 ime 080 0800-1000 1200-1400 1600-1800 2200-2400 0400 day 2 0800-1000 Insulin (pmol/L) 78.1 ± 12.0 276.3 ± 25.1 294.6 ± 31.0 345.4 ± 31.7 365.1 ± 38.1 339.7 ± 32.5 366.0 ± 37.0 10% Glucose Infusion (ml/h) - 116.2 ± 11.9 120.4 ± 8.9 122.5 ± 11.4 125.8 ± 9.3 138.3 ± 11.7 128.3 ± 13.2 Glucose (mmol/L) 4.3 ± 0.5 4.8 ± 0.6 4.3 ± 0.7 5.6 ± 0.9 5.2 ± 0.5 4.4 ± 1.0 4.7 ± 0.8 FIG. 2. Mean concentrations of chemerin in nanogram per milliliter in all subjects, before and after insulin infusion. Insulin infusion resulted in elevation of fasting insulinemia from 78.1  12.0 pmol/l to 294.6  31.0 pmol/l. Insulin levels remained elevated until the end of the prolonged insulin-glucose infusion (366.0  37.0 pmol/l). Data are means  SD. Group comparison by Student’s t test. *P < 0.05, **P < 0.01. observed in control human subcutaneous adipose tissue In study 3, we analyzed the correlation between the explants (data not shown). With respect to gonadal and change in serum chemerin levels before and after met- adrenal steroids, no significant effects on chemerin protein formin therapy ( chemerin) and the changes ( ) in other production and secretion were observed (data not shown). covariates (Table 3). Chemerin was significantly posi- Association of chemerin with covariates. In study 1, tively associated with WHR, glucose, insulin, HOMA- Spearman’s rank analyses demonstrated that serum and IR, and triglycerides. When subjected to multiple subcutaneous and omental adipose tissue chemerin levels regression analysis with WHR, glucose, insulin, HOMA-IR, were significantly positively associated with BMI, WHR, and triglycerides, only HOMA-IR was predictive of glucose, insulin, HOMA-IR, and circulating triglycerides changes in serum chemerin levels (Table 3), whereas other (P  0.05). However, when subjected to multiple regres- models of multiple regression analysis revealed no other sion analysis, none of these variables were predictive of significant predictors of changes in serum chemerin levels serum chemerin levels (P  0.05). (see supplemental data, available in an online appendix at TABLE 2 Clinical, hormonal, and metabolic features of women with PCOS (n  21) before and after metformin treatment Before metformin After metformin Significance Age (years) 28 (26.5–31.5) 28 (27.5–32.5) NS BMI (kg/m ) 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  0.01 Insulin (pmol/l) 70.0 (54.5–98.0) 60.0 (43.5–81.0) NS HOMA-IR 2.1 (1.7–3.1) 1.6 (1.3–2.3) P  0.05 Cholesterol (mmol/l) 4.9 (4.1–5.3) 5.0 (4.0–5.4) NS Triglycerides (mmol/l) 1.0 (0.7–1.9) 1.2 (1.0–1.7) NS E (pmol/l) 329.8 (164.9–494.7) 207.1 (103.6–310.7) P  0.05 Testosterone (nmol/l) 1.8 (1.4–2.2) 1.3 (1.0–1.8) P  0.05 Androstenedione (nmol/l) 10.9 (8.0–14.0) 9.7 (7.6–12.4) NS DHEA-S (mol/l) 4.4 (2.8–5.8) 5.4 (3.6–6.7) NS SHBG (nmol/l) 27.0 (21.0–41.0) 25.0 (20.5–46.5) NS FAI 6.2 (4.6–8.0) 5.2 (3.1–6.6) NS Leptin (ng/ml) 26.5 (20.7–30.9) 25.1 (17.9–30.0) NS Adiponectin (g/ml) 4.85 (4.14–6.88) 3.69 (2.91–5.55) NS Chemerin (ng/ml) 6.36 (5.80–6.83) 2.19 (2.04–4.02) P  0.01 Data are medians (interquartile range). Group comparison by Mann-Whitney U test. FAI  Testosterone (nmol/liter)/SHBG (nmol/liter) 100. NS  not significant. FAI, free androgen index; SHBG, sex hormone–binding globulin. 1974 DIABETES, VOL. 58, SEPTEMBER 2009 Chemerin (ng/ml) B. K. TAN AND ASSOCIATES A Insulin -11 -9 -7 Dose (M) B10 10 10 Chemerin (18kDa) β - actin (45kDa) ** -11 -9 -7 B10 10 10 Insulin (M) ** -11 -9 -7 B10 10 10 Insulin (M) 11 9 7 FIG. 3. A: Dose-dependent effects of insulin (10 M, 10 M, 10 M) in the presence of 5 mmol/l D-glucose on chemerin net protein production in control human omental adipose tissue explants at 24 h were assessed by Western blotting. Western blot analysis of protein extracts from omental adipose tissue demonstrate that the antibody against chemerin and the antibody against -actin recognized bands with apparent molecular weights of 18 kDa and 45 kDa, respectively (Fig. 3A, inserts). Densitometric analysis of chemerin immune complexes having normalized 9 7 to -actin, respectively, revealed that protein levels of chemerin were significantly increased by insulin (10 M, 10 M) in control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. 11 9 7 B: Dose-dependent effects of insulin (10 M, 10 M, 10 M) in the presence of 5 mmol/l D-glucose on chemerin secretion into conditioned media 9 7 from control human omental adipose tissue after 24 h were measured by ELISA. Chemerin secretion was significantly increased (by 10 M, 10 M) from control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. http://diabetes.diabetesjournals.org/cgi/content/full/db08- PCOS have an increased incidence of IGT and type 2 1528/DC1). diabetes (1,2). The higher serum and adipose tissue chemerin levels in women with PCOS is of interest given DISCUSSION that it has recently been reported that IGT and type 2 diabetes Psammomys obesus animals (a unique polygenic We present novel data showing a significant increase of animal model for obesity and type 2 diabetes) had higher serum and subcutaneous and omental adipose tissue adipose tissue chemerin levels than normal glucose–toler- chemerin mRNA expression as well as protein levels in ant Psammomys obesus animals. In the same study, women with PCOS. More importantly, we demonstrate the significant positive associations with BMI and circulating potent and robust regulation of chemerin in vivo and ex vivo triglycerides in normal glucose–tolerant human subjects by insulin as well as its modulation by metformin treatment. were noted (9). However, no data exists on chemerin and Unfortunately, because of technical limitations in adipose tissue procurement, we were unable to obtain sufficient its regulation in human adipose tissue. In our study, we amounts of sample/patient tissue to perform stromal vascu- found significant positive associations between circulating lar separation in adipose tissue depots. We could not account chemerin as well as chemerin levels in subcutaneous and for a potential adipocyte hypertrophy–related side effect. omental adipose tissue with BMI, WHR, glucose, insulin, These limitations not withstanding, adipose tissue from our HOMA-IR, and circulating triglycerides. However, it is PCOS women, compared with control subjects, express unlikely that either BMI or WHR are responsible for these more chemerin. findings, as both groups were matched for these variables. DIABETES, VOL. 58, SEPTEMBER 2009 1975 Chemerin in conditioned Chemerin/β-actin media (% difference) PSL (% difference) INSULIN AND METFORMIN REGULATE CHEMERIN Metformin Dose (µg/ml) B 0.01 0.10 2.00 Chemerin (18kDa) β - actin (45kDa) ** 25 ** B 0.01 0.10 2.00 Metformin (µg/ml) ** B 0.01 0.10 2.00 Metformin (µg/ml) FIG. 4. A: Dose-dependent effects of metformin (0.01, 0.1, and 2.00 g/ml) in the presence of 5 mmol/l D-glucose on chemerin net protein production in control human omental adipose tissue explants at 24 h were assessed by Western blotting. Western blot analysis of protein extracts from omental adipose tissue demonstrate that the antibody against chemerin and the antibody against -actin recognized bands with apparent molecular weights of 18 kDa and 45 kDa, respectively (Fig. 4A, inserts). Densitometric analysis of chemerin immune complexes having normalized to -actin, respectively, revealed that protein levels of chemerin were significantly decreased by metformin (0.01, 0.1, and 2.00 g/ml) in control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. B: Dose-dependent effects of insulin (0.01, 0.1, and 2.00 g/ml) in the presence of 5 mmol/l D-glucose on chemerin secretion into conditioned media from control human omental adipose tissue after 24 h were measured by ELISA. Chemerin secretion was significantly decreased (by 0.01, 0.1, and 2.00 g/ml) from control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. In addition, caution needs to be exercised as these corre- levels of chemerin seen in our insulin-resistant PCOS lations may be spurious, without causative significance, subjects. Of secondary interest, there appears to be no resulting from the simple fact that our PCOS women had circadian variation in chemerin levels as depicted in Fig. 2. higher levels for all these parameters. It should be emphasized that the primary aim of this study Importantly, in study 2 we derive novel observations of was not to investigate the circadian variation of chemerin. a profound increase in chemerin levels by insulin in vivo. In addition, our study utilized relatively small numbers of This effect of insulin appears to be relatively acute, achiev- subjects because of the challenge imposed by the pro- ing a maximal effect 4 h after commencement of insulin longed insulin clamp study; hence, care needs to be and persisting throughout the entire period of hyperinsu- exercised in interpretation of these results. linemia. Furthermore, this is in agreement with our data More importantly, in study 3 we report for the first time on the regulation of chemerin protein production ex vivo. that metformin (6 months treatment; 850-mg twice daily) It is important to bear in mind that the regulation of significantly decreases circulating chemerin levels with a chemerin in adipose tissue is probably multifactorial. concomitant decrease in insulin resistance in PCOS sub- Moreover, it would be of interest to know whether or not jects. Additionally, although the change in serum chemerin the effects of insulin on chemerin production are also levels were significantly positively associated with applicable to other tissues given our in vivo data. Future changes in WHR, glucose, insulin, HOMA-IR, and triglyc- studies are needed to elucidate the role of other factors erides, when subjected to multiple regression analysis that regulate chemerin production. Taken together, the only HOMA-IR was predictive of serum chemerin levels. above findings could tentatively explain the increased Taken together, we hypothesize that elevated chemerin 1976 DIABETES, VOL. 58, SEPTEMBER 2009 Chemerin in conditioned Chemerin/β-action media (% difference) PSL (% difference) B. K. TAN AND ASSOCIATES TABLE 3 it would be of interest to perform this study with lean Linear regression analysis of variables associated with changes women with PCOS. in serum chemerin levels (before and after metformin treatment), In conclusion, we report novel findings of a significant chemerin, in PCOS subjects (n  21) increase of circulating and adipose tissue chemerin, a novel adipokine, in women with PCOS as well as the Simple Multiple potent and robust regulation of chemerin by insulin in vivo Estimate P Estimate P and ex vivo. More importantly, we present novel data that metformin treatment significantly decreases circulating BMI (kg/m ) 0.156 0.500 — — chemerin levels in women with PCOS. The physiologic and WHR 0.486 0.026 0.293 0.329 pathologic significance of our findings remain to be further Glucose (mmol/l) 0.510 0.018 0.338 0.234 elucidated. Insulin (pmol/l) 0.503 0.020 0.387 0.205 HOMA-IR 0.772 0.010 0.628 0.046 Cholesterol (mmol/l) 0.276 0.226 — — ACKNOWLEDGMENTS Triglycerides (mmol/l) 0.490 0.024 0.456 0.107 The General Charities of the City of Coventry funded this E (pmol/l) 0.240 0.327 — — study. Testosterone (nmol/l) 0.130 0.576 — — No potential conflicts of interest relevant to this article Androstenedione (nmol/l) 0.039 0.867 — — were reported. DHEA-S (mol/l) 0.055 0.814 — — H.S.R. would like to acknowledge S. Waheguru, Univer- SHBG (nmol/l) 0.236 0.304 — — sity of Warwick for his continual support. FAI 0.224 0.330 — — Leptin (ng/ml) 0.003 0.989 — — REFERENCES Adiponectin (g/ml) 0.026 0.911 — — 1. Dunaif A. Insulin resistance and the polycystic ovary syndrome: mecha- In multiple linear regression analysis, values included were WHR, nism and implications for pathogenesis. Endocr Rev 1997;18:774–800 glucose, insulin, HOMA-IR, and triglycerides. FAI, free androgen 2. Wild RA, Painter RD, Coulson PB, Carruth KB, Ranney RB. Lipoprotein index; SHBG, sex hormone– binding globulin. lipid concentrations and cardiovascular risk in women with polycystic ovary syndrome. J Clin Endocrinol Metab 1985;61:946–951 3. Diamanti-Kandarakis E. Insulin resistance in PCOS. Endocrine 2006;30: levels may be a compensatory mechanism to insulin 13–17 4. Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin resistance in our cohort of PCOS subjects. Furthermore, of Endocrinol Metab 2004;89:2548–2556 relevance, an elegant study by Bozaoglu et al. (9) describes 5. Wajchenberg BL. Subcutaneous and visceral adipose tissue: their relation important findings that circulating chemerin levels in type to the metabolic syndrome. Endocr Rev 2000;21:697–738 2 diabetes human subjects were not significantly higher 6. Thorne A, Lonnqvist F, Apelman J, Hellers G, Arner P. A pilot study of than those in normal control subjects. Therefore, our long-term effects of a novel obesity treatment: omentectomy in connection observations are timely as they may explain the findings by with adjustable gastric banding. Int J Obes Relat Metab Disord 2002;26: 193–199 Bozaoglu et al. (9), given that quite probably a proportion 7. Tan BK, Heutling D, Chen J, Farhatullah S, Adya R, Keay SD, Kennedy CR, of their type 2 diabetic study subjects may have been Lehnert H, Randeva HS. Metformin decreases the adipokine vaspin in taking metformin (Bozaoglu et al. had not reported the overweight women with polycystic ovary syndrome concomitant with medications taken by their type 2 diabetic subjects, with improvement in insulin sensitivity and a decrease in insulin resistance. metformin arguably being the most common first-line oral Diabetes 2008;57:1501–1507 hypoglycemic therapy to treat type 2 diabetes employed by 8. Hida K, Wada J, Eguchi J, Zhang H, Baba M, Seida A, Hashimoto I, Okada T, Yasuhara A, Nakatsuka A, Shikata K, Hourai S, Futami J, Watanabe E, most physicians in both developing as well as developed Matsuki Y, Hiramatsu R, Akagi S, Makino H, Kanwar YS. Visceral adipose countries. Therefore, our study highlights metformin ther- tissue-derived serine protease inhibitor: a unique insulin-sensitizing adipo- apy as a confounding factor concerning the regulation of cytokine in obesity. Proc Natl Acad SciUSA 2005;102:10610–10615 circulating chemerin levels. This should alert investiga- 9. Bozaoglu K, Bolton K, McMillan J, Zimmet P, Jowett J, Collier G, Walder K, tors who are studying chemerin biology to consider this Segal D. Chemerin is a novel adipokine associated with obesity and in their analyses. In addition, this point may also apply metabolic syndrome. Endocrinology 2007;148:4687–4694 10. Goralski KB, McCarthy TC, Hanniman EA, Zabel BA, Butcher EC, Parlee to other forms of antidiabetic therapy; hence, caution SD, Muruganandan S, Sinal CJ. Chemerin, a novel adipokine that regulates needs to be exercised appropriately. adipogenesis and adipocyte metabolism. J Biol Chem 2007;282:28175– A limitation of our study may relate to the number of subjects studied. However, obtaining BMI/WHR-matched 11. Lewandowski K, Randeva HS, O’Callaghan CJ, Horn R, Medley GF, and menstrual cycle–synchronized blood and adipose tis- Hillhouse EW, Brabant G, O’Hare P. 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Methods Mol Biol 2001;155:197–212 DIABETES, VOL. 58, SEPTEMBER 2009 1977 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Diabetes Pubmed Central

Insulin and Metformin Regulate Circulating and Adipose Tissue Chemerin

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ORIGINAL ARTICLE Insulin and Metformin Regulate Circulating and Adipose Tissue Chemerin 1 1 1 1 1 2 Bee K. Tan, Jing Chen, Syed Farhatullah, Raghu Adya, Jaspreet Kaur, Dennis Heutling, 1,3 1 1,4 1 Krzysztof C. Lewandowski, J. Paul O’Hare, Hendrik Lehnert, and Harpal S. Randeva consequent hyperinsulinemia is more prevalent in lean and OBJECTIVE—To assess chemerin levels and regulation in sera obese women with PCOS when compared with age- and and adipose tissue from women with polycystic ovary syndrome weight-matched normal women (3). (PCOS) and matched control subjects. The metabolic syndrome is associated with excessive RESEARCH DESIGN AND METHODS—Real-time RT-PCR accumulation of central body fat. As well as its role in and Western blotting were used to assess mRNA and protein energy storage, adipose tissue produces several hormones expression of chemerin. Serum chemerin was measured by and cytokines termed ‘adipokines’ that have widespread enzyme-linked immunosorbent assay. We investigated the in vivo effects on carbohydrate and lipid metabolism. They appear effects of insulin on serum chemerin levels via a prolonged to play an important role in the pathogenesis of insulin insulin-glucose infusion. Ex vivo effects of insulin, metformin, and steroid hormones on adipose tissue chemerin protein pro- resistance, diabetes, and atherosclerosis (4). Furthermore, duction and secretion into conditioned media were assessed by it is apparent that accumulation of visceral adipose tissue Western blotting and enzyme-linked immunosorbent assay, poses a greater cardiometabolic risk than subcutaneous respectively. adipose tissue (5) as removal of visceral rather than subcutaneous adipose tissue has been shown to improve RESULTS—Serum chemerin, subcutaneous, and omental adi- insulin sensitivity (6). Moreover, differences in gene ex- pose tissue chemerin were significantly higher in women with PCOS (n  14; P  0.05, P  0.01). Hyperinsulinemic induction pression of adipocyte-secreted molecules (adipokines) in human subjects significantly increased serum chemerin levels suggest that there are inherent adipose tissue depot– (n  6; P  0.05, P  0.01). In adipose tissue explants, insulin specific differences in the endocrine function of adipose significantly increased (n  6; P  0.05, P  0.01) whereas tissue. In relation to this, we have published data on the metformin significantly decreased (n  6; P  0.05, P  0.01) increased levels of vaspin in women with PCOS (7); vaspin chemerin protein production and secretion into conditioned is a recently described adipokine mainly formed in human media, respectively. After 6 months of metformin treatment, visceral adipose tissue that has insulin-sensitizing effects there was a significant decrease in serum chemerin (n  21; P (8). 0.01). Importantly, changes in homeostasis model assessment– Recently, Bozaoglu et al. (9) reported chemerin as a insulin resistance were predictive of changes in serum chemerin (P  0.046). novel adipokine, circulating levels of which significantly correlated with BMI, circulating triglycerides, and blood CONCLUSIONS—Serum and adipose tissue chemerin levels are pressure, features of the metabolic syndrome. In addition, increased in women with PCOS and are upregulated by insulin. chemerin or chemerin receptor knockdown impaired dif- Metformin treatment decreases serum chemerin in these women. ferentiation of 3T3-L1 cells and attenuated the expression Diabetes 58:1971–1977, 2009 of adipocyte genes involved in glucose and lipid homeosta- sis (10). With the aforementioned in mind and the fact that there olycystic ovary syndrome (PCOS), a common is no literature with regards to chemerin in human adipose endocrinopathy affecting 5–10% of women in the tissue and its regulation, in study 1, we assessed circulat- reproductive age, is characterized by menstrual ing chemerin as well as mRNA expression and protein Pdysfunction and hyperandrogenism and is levels of chemerin in subcutaneous and omental adipose associated with insulin resistance and pancreatic -cell tissue depots in women with PCOS against age, BMI, and dysfunction, impaired glucose tolerance (IGT), type 2 waist-to-hip ratio (WHR) in matched control subjects. diabetes, dyslipidemia, and visceral obesity (1,2). The Furthermore, we studied the in vivo (study 2) and ex vivo effects of insulin on circulating chemerin levels via a prolonged insulin-glucose infusion in humans and From the Endocrinology and Metabolism Group, Clinical Sciences Research primary adipose tissue explant cultures, respectively. In Institute, Warwick Medical School, University of Warwick, Coventry, U.K.; study 3 we studied the effects of metformin therapy, the Division of Endocrinology and Metabolism, Magdeburg University Hospital, Magdeburg, Germany; the Department of Endocrinology and widely used in the treatment of PCOS in women, on Metabolic Diseases, The Medical University of Lodz and Polish Mother’s circulating chemerin levels in tandem with associated 4 st Memorial Research Institute, Lodz, Poland; and the 1 Medical Depart- changes to clinical, hormonal, and metabolic parame- ment, University of Lu¨ beck Medical School, Lu¨ beck, Germany. Corresponding author: Harpal S. Randeva, [email protected]. ters in the same cohort of PCOS in women. Additionally, Received 3 November 2008 and accepted 19 May 2009. we studied the ex vivo effects of metformin and steroid Published ahead of print at http://diabetes.diabetesjournals.org on 5 June hormones in human primary adipose tissue explants. 2009. DOI: 10.2337/db08-1528. © 2009 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by RESEARCH DESIGN AND METHODS -nc-nd/3.0/ for details. Study 1. Seventy three subjects were recruited consecutively from the The costs of publication of this article were defrayed in part by the payment of page infertility clinic in accordance with the inclusion/exclusion criteria (PCOS: charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. n  19; control subjects: n  54) as previously described (7); 62 subjects were DIABETES, VOL. 58, SEPTEMBER 2009 1971 INSULIN AND METFORMIN REGULATE CHEMERIN TABLE 1 Clinical, hormonal, and metabolic features of women with PCOS and control subjects PCOS (n  14) Control subjects (n  14) Significance Age (years) 29.5 (28–38) 32.5 (29–35) NS BMI (kg/m ) 30.5 (27.8–30.9) 28.8 (28–30.5) NS WHR 0.89 (0.78–0.99) 0.84 (0.81–0.96) NS Glucose (mmol/l) 5.5 (4.8–6.0) 4.5 (4.3–5.2) P  0.01 Insulin (pmol/l) 78.9 (42.0–91.1) 57.3 (48.5–66.0) NS HOMA-IR 3.0 (2.1–3.6) 2.0 (1.4–2.2) P  0.05 Cholesterol (mmol/l) 5.1 (4.1–5.7) 5.0 (4.8–5.5) NS Triglycerides (mmol/l) 2.0 (1.5–2.3) 0.9 (0.7–1.4) P  0.01 Luteinizing hormone (IU/l) 7.0 (6.0–10.0) 6.0 (5.0–7.0) NS Follicle-stimulating hormone (IU/l) 6.0 (6.0–7.0) 6.5 (5.0–8.0) NS Prolactin (mU/l) 348.0 (305.0–387.0) 304.5 (211.0–322.0) NS E (pmol/l) 353.5 (287.0–471.0) 174.5 (129.0–264.0) P  0.01 Progesterone (nmol/l) 1.6 (1.3–2.1) 2.2 (1.7–2.3) NS 17-OH-P (nmol/l) 2.5 (2.1–2.8) 2.0 (1.2–2.3) NS Testosterone (nmol/l) 1.7 (1.5–2.1) 0.8 (0.6–0.9) P  0.01 Androstenedione (nmol/l) 15.6 (14.2–16.8) 6.3 (5.0–8.2) P  0.01 DHEA-S (mol/l) 5.9 (5.4–6.6) 4.5 (4.0–5.3) P  0.05 SHBG (nmol/l) 31.5 (26.7–35.2) 59.0 (47.7–66.0) P  0.01 FAI 19.5 (14.5–21.2) 3.9 (3.3–6.1) P  0.01 Leptin (ng/ml) 24.9 (19.7–29.0) 24.1 (19.3–28.8) NS Adiponectin (g/ml) 5.67 (4.52–7.5) 6.27 (5.52–9.48) NS Chemerin (ng/ml) 6.02 (5.96–6.04) 2.62 (1.96–3.23) P  0.01 Data are medians (interquartile range). Group comparison by Mann-Whitney U test. FAI  Testosterone (nmol/liter)/SHBG (nmol/liter) 100. NS  not significant. FAI, free androgen index; SHBG, sex hormone–binding globulin. from a previous study (7). Of the 19 PCOS subjects recruited, five withdrew was offered to all women with PCOS independently from the results of before the study could be completed. In the control group, seven subjects did insulin sensitivity testing, as per standard clinical practice. The study not complete the study. From the remaining 47 control subjects, 14 control design was approved by the local research ethics committee of the subjects matched for age, BMI, and WHR were included in the final analysis University of Magdeburg, and written informed consent was obtained from (Table 1). Blood and adipose tissue samples were obtained as previously all participants in accordance with the guidelines in The Declaration of described (7). Subcutaneous adipose tissue was obtained froma3cm Helsinki 2000. horizontal midline incision 3 cm above the symphysis pubis. Omental Biochemical and hormonal analysis. Assays were performed using an adipose tissue was obtained by excisional biopsy from the greater omentum. automated analyzer as previously described (7). The estimate of insulin The local research ethics committee of University Hospitals Coventry and resistance by homeostasis model assessment (HOMA-IR) score was calcu- Warwickshire NHS Trust approved the study, and all patients involved gave lated as previously described (12). Circulating leptin and adiponectin levels their informed consent in accordance with the guidelines in The Declaration were measured with a coated-tube immunoradiometric assay kit (Diagnostic of Helsinki 2000. Systems Laboratories, Sinsheim, Germany) and by a commercially available Study 2. We measured circulating chemerin in six (three men, three women) RIA kit (Millipore, Watford, U.K.), respectively, according to manufacturer’s healthy subjects [age: (mean  SD) 26.5  8 years, BMI: 23.2  2.5 kg/m ]. All protocol. subjects studied were nonsmokers and otherwise healthy. Women volunteers Chemerin levels in sera and conditioned media from human adipose tissue had regular periods, no clinical or biochemical hyperandrogenism, and were explants were measured using a commercially available enzyme-linked immu- not taking any medications. Exclusion criteria for the study, as previously nosorbent assay (ELISA) kit (R & D Systems, Abingdon, U.K.), according to described (7), included age over 40 years, known cardiovascular disease, manufacturer’s protocol, with an intra-assay coefficient of variation of less thyroid disease, neoplasms, current smoking, diabetes, hypertension (blood than 9%. pressure 140/90 mmHg), and renal impairment (serum creatinine 120 Primary explant culture. Adipose tissue organ explants were cultured using mol/l). None of these women were on any medication for at least 6 months a protocol that was a modification of the method described by Fried and before the study, including oral contraceptives, glucocorticoids, ovulation Moustaid-Moussa (13). Adipose tissue explants were cultured for 24 h with or induction agents, antidiabetic and antiobesity drugs, or estrogenic, antiandro- without the addition of insulin, testosterone, 17-estradiol, androstenedione, genic, or antihypertensive medication. The local research ethics committee of dehydroepiandrosterone-sulfate (DHEA-S), or metformin, as previously de- University Hospitals Coventry and Warwickshire NHS Trust approved the scribed (7). study, and all patients involved gave their informed consent in accordance Total RNA extraction and cDNA synthesis. Total RNA was extracted from with the guidelines in The Declaration of Helsinki 2000. To account for the adipose tissue samples and cDNA synthesized as previously described (7). possible diurnal variation in chemerin levels, we obtained a daily control RT-PCR. Quantitative PCR of chemerin was performed on a Roche Light curve by measuring fasting chemerin levels at 30-min intervals from 0800 to Cycler system (Roche Molecular Biochemicals, Mannheim, Germany) as 1000 h. Subsequently, chemerin levels were measured at 2-h intervals until previously described (7). The sequences of the sense and antisense primers 2400 h and then at 0400 h as well as at 30-min intervals from 0800 to 1000 h on used were chemerin (252 bp) 5 -AGACAAGCTGCCGGAAGAGG-3 and 5 - day 2. On the following day, the same subjects were subjected to a prolonged TGGAGAAGGCGAACTGTCCA-3 ; -actin (216 bp) 5 -AAGAGAGGCATCCT- insulin-glucose infusion for 26 h beginning at 0800 h. Insulin (Human Actrapid) CACCCT-3 and 5 -TACATGGCTGGGGTCTTGAA-3 . was administered intravenously as a priming dose of 0.04 units/kg followed by Western blotting. Protein lysates were prepared as previously described (7). 1 1 continuous infusion of 0.5 mU  kg  min . By choosing this rate of insulin Protein samples (30 g/lane) containing SDS sample buffer (5 M urea, 0.17 M infusion we expected to achieve hyperinsulinemia with an approximate four- SDS, 0.4 M dithiothreitol, and 50 mmol/l Tris-HCl, pH 8.0) were subjected to to sixfold elevation of basal insulinemia (11). Fasting blood samples were SDS-PAGE (10% resolving gel) and transferred to polyvinylidene difluoride drawn at 30-min intervals between 0800 and 1000 h on day 1 and day 2 of the (PVDF) membranes (Millipore). PVDF membranes were blocked in Tris- prolonged insulin-glucose infusion (the first and the last2hofthe infusion). buffered saline (TBS) containing 0.1% Tween-20 and 5% BSA for 2 h. The PVDF Intermediate blood samples were taken at 2-h intervals until 2400 h and then membranes were then incubated with polyclonal primary goat anti-human at 0400 h on day 2. Glucose levels were maintained between 4.0 and 6.0 antibody for chemerin (R & D Systems) (1:1,000 dilution) or monoclonal mmol/l. primary rabbit anti-human antibody for -actin (Cell Signaling Technology, Study 3. Subjects were recruited and blood samples were obtained as Beverly, CA) (1:1,000 dilution) overnight at 4°C. The membranes were washed previously described (7). A treatment with metformin in an “off-label use” thoroughly for 60 min with TBS 0.1% Tween before incubation with the 1972 DIABETES, VOL. 58, SEPTEMBER 2009 B. K. TAN AND ASSOCIATES secondary anti-goat horseradish peroxidase–conjugated immunoglobulin A Sc Om (Dako, Ely, U.K.) (1:2,000) or secondary anti-rabbit horseradish peroxidase– conjugated immunoglobulin (Dako) [1:2000], respectively, for1hat room temperature. Antibody complexes were visualized using chemiluminescence ** (ECL ; GE Healthcare, Little Chalfont, U.K.). Human chemerin protein (R & D Systems) was used as positive control and water as negative control (data not shown). Statistics. Data were analyzed by Student’s t test, Mann-Whitney U test, Kruskal-Wallis, or Friedman’s ANOVA (post hoc analysis: Dunn’s test) accord- ing to the number of groups compared; P  0.05 was considered significant. For Western immunoblotting experiments, the densities were measured using a scanning densitometer coupled to scanning software Scion Image (Scion Corporation, Frederick, MD). 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 chemerin and -actin were made only within this linearity range. Spearman Rank correlation Normal PCOS Normal PCOS was used for calculation of associations between variables; P  0.05 was considered significant. Sc Om ** RESULTS Demographic data. Table 1 shows the anthropometric, biochemical, and hormonal data in all subjects. Glucose, HOMA, triglycerides, 17-estradiol (E ), testosterone, an- drostenedione, DHEA-S levels, and free androgen index were significantly higher, whereas sex hormone–binding globulin was significantly lower in women with PCOS. Serum chemerin levels were significantly higher in PCOS subjects than in control subjects (6.02 [5.96–6.04] vs. 2.62 [1.96–3.23] ng/ml; P  0.01; Table 1). Serum Normal PCOS Normal PCOS progesterone levels in all women confirmed follicular phase of the menstrual cycle. FIG. 1. A: Chemerin mRNA expression relative to -actin was mRNA expression and protein levels of chemerin in significantly increased in human subcutaneous (Sc) and omental (Om) adipose tissue depots when comparing PCOS women (n  14) normal and PCOS women. We detected chemerin mRNA with normal control subjects (n  14), using real-time RT-PCR. Data in adipose tissue of all subjects, and subsequent sequenc- are expressed as percent difference of median of human subcutane- ing of the PCR products confirmed gene identity. Real-time ous adipose tissue of normal control subjects. Each experiment was carried out in three replicates. Group comparison by Kruskal-Wallis RT-PCR analysis corrected over -actin showed a signifi- ANOVA and post hoc Dunn’s test; *P < 0.05, **P < 0.01. B: cant increase of chemerin expression in subcutaneous Densitometric analysis of chemerin immune complexes having nor- (*P  0.05) and omental (**P  0.01) adipose tissue of malized to -actin revealed that protein levels of chemerin were significantly increased in human subcutaneous and omental adipose PCOS when compared with normal control subjects tissue depots, respectively, when comparing all women with PCOS (Fig. 1A). However, no significant difference in with all normal control subjects. Data are expressed as percent chemerin mRNA expression was observed when com- difference of median of normal control subjects. Each experiment was carried out in three replicates. Group comparison by Kruskal- paring corresponding omental with subcutaneous adi- Wallis ANOVA (post hoc analysis: Dunn’s test). *P < 0.05, **P < pose tissue in PCOS and normal subjects (Fig. 1A; P 0.01; PSL, phosphostimulated light units. 0.05). Changes noted at mRNA level were also reflected at protein level (Fig. 1B). Study 2: Effects of a prolonged insulin-glucose infu- data of PCOS subjects investigated in study 1 were not sion on serum chemerin levels. Insulin infusion resulted significantly different compared with the PCOS subjects in elevation of fasting insulinemia from 78.1  12.0 pmol/l investigated in study 3. Reasons for subjects not complet- to 294.6  31.0 pmol/l. Insulin levels remained elevated ing study 3 were nausea and gastrointestinal side effects until the end of the prolonged insulin-glucose infusion (n  4), pregnancies (n  5), incompliance (n  2), and (366.0  37.0 pmol/l). Chemerin levels remained unaltered loss of contact (n  2). After 6 months of metformin throughout the control day from 2.06  0.08 ng/ml be- treatment, there were significant decreases in serum tween 0800 and 1000 h to 1.92  0.07 ng/ml between 0800 chemerin, WHR, E , testosterone, glucose, and HOMA-IR and 1000 h the next day (Fig. 2; P  0.05). (Table 2). There was a profound effect of insulin on chemerin Dose-dependent effects of insulin, testosterone, 17- levels over 26 h of insulin infusion: from 2.54  0.32 ng/ml estradiol, androstenedione, DHEA-S, and metformin between 0800 and 1000 h to 3.97  0.37 ng/ml between on chemerin protein production and secretion into 0800 and 1000 h the following day (Fig. 2; *P  0.05). The conditioned media from control human omental adi- increase in chemerin levels was relatively acute approach- pose tissue explants. We found that chemerin protein ing maximal values at 4 h (Fig. 2; 5.08  0.27 ng/ml, **P  production and secretion into conditioned media was 0.01) and persisting throughout the entire period of significantly increased dose dependently by insulin in hyperinsulinemia. control human omental adipose tissue explants (Fig. 3; Study 3: Effects of metformin treatment on serum *P  0.05 and **P  0.01, respectively). Interestingly, chemerin levels. Metformin treatment was started in 34 metformin significantly decreased chemerin protein pro- women with PCOS. Only 21 women completed the study duction and secretion into conditioned media in control and were investigated after 6 months of metformin treat- human omental adipose tissue explants (Fig. 4 and *P ment. The anthropometric, biochemical, and hormonal 0.05 and **P  0.01, respectively). Similar results were DIABETES, VOL. 58, SEPTEMBER 2009 1973 Chemerin/β-actin mRNA Chemerin/β-actin PSL (% difference) expression (% difference) INSULIN AND METFORMIN REGULATE CHEMERIN Control Infusion ** ** 0800- 1200- 1600- 2200- 0400 0800- 1000 1400 1800 2400 day 2 1000 T0 ime 080 0800-1000 1200-1400 1600-1800 2200-2400 0400 day 2 0800-1000 Insulin (pmol/L) 78.1 ± 12.0 276.3 ± 25.1 294.6 ± 31.0 345.4 ± 31.7 365.1 ± 38.1 339.7 ± 32.5 366.0 ± 37.0 10% Glucose Infusion (ml/h) - 116.2 ± 11.9 120.4 ± 8.9 122.5 ± 11.4 125.8 ± 9.3 138.3 ± 11.7 128.3 ± 13.2 Glucose (mmol/L) 4.3 ± 0.5 4.8 ± 0.6 4.3 ± 0.7 5.6 ± 0.9 5.2 ± 0.5 4.4 ± 1.0 4.7 ± 0.8 FIG. 2. Mean concentrations of chemerin in nanogram per milliliter in all subjects, before and after insulin infusion. Insulin infusion resulted in elevation of fasting insulinemia from 78.1  12.0 pmol/l to 294.6  31.0 pmol/l. Insulin levels remained elevated until the end of the prolonged insulin-glucose infusion (366.0  37.0 pmol/l). Data are means  SD. Group comparison by Student’s t test. *P < 0.05, **P < 0.01. observed in control human subcutaneous adipose tissue In study 3, we analyzed the correlation between the explants (data not shown). With respect to gonadal and change in serum chemerin levels before and after met- adrenal steroids, no significant effects on chemerin protein formin therapy ( chemerin) and the changes ( ) in other production and secretion were observed (data not shown). covariates (Table 3). Chemerin was significantly posi- Association of chemerin with covariates. In study 1, tively associated with WHR, glucose, insulin, HOMA- Spearman’s rank analyses demonstrated that serum and IR, and triglycerides. When subjected to multiple subcutaneous and omental adipose tissue chemerin levels regression analysis with WHR, glucose, insulin, HOMA-IR, were significantly positively associated with BMI, WHR, and triglycerides, only HOMA-IR was predictive of glucose, insulin, HOMA-IR, and circulating triglycerides changes in serum chemerin levels (Table 3), whereas other (P  0.05). However, when subjected to multiple regres- models of multiple regression analysis revealed no other sion analysis, none of these variables were predictive of significant predictors of changes in serum chemerin levels serum chemerin levels (P  0.05). (see supplemental data, available in an online appendix at TABLE 2 Clinical, hormonal, and metabolic features of women with PCOS (n  21) before and after metformin treatment Before metformin After metformin Significance Age (years) 28 (26.5–31.5) 28 (27.5–32.5) NS BMI (kg/m ) 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  0.01 Insulin (pmol/l) 70.0 (54.5–98.0) 60.0 (43.5–81.0) NS HOMA-IR 2.1 (1.7–3.1) 1.6 (1.3–2.3) P  0.05 Cholesterol (mmol/l) 4.9 (4.1–5.3) 5.0 (4.0–5.4) NS Triglycerides (mmol/l) 1.0 (0.7–1.9) 1.2 (1.0–1.7) NS E (pmol/l) 329.8 (164.9–494.7) 207.1 (103.6–310.7) P  0.05 Testosterone (nmol/l) 1.8 (1.4–2.2) 1.3 (1.0–1.8) P  0.05 Androstenedione (nmol/l) 10.9 (8.0–14.0) 9.7 (7.6–12.4) NS DHEA-S (mol/l) 4.4 (2.8–5.8) 5.4 (3.6–6.7) NS SHBG (nmol/l) 27.0 (21.0–41.0) 25.0 (20.5–46.5) NS FAI 6.2 (4.6–8.0) 5.2 (3.1–6.6) NS Leptin (ng/ml) 26.5 (20.7–30.9) 25.1 (17.9–30.0) NS Adiponectin (g/ml) 4.85 (4.14–6.88) 3.69 (2.91–5.55) NS Chemerin (ng/ml) 6.36 (5.80–6.83) 2.19 (2.04–4.02) P  0.01 Data are medians (interquartile range). Group comparison by Mann-Whitney U test. FAI  Testosterone (nmol/liter)/SHBG (nmol/liter) 100. NS  not significant. FAI, free androgen index; SHBG, sex hormone–binding globulin. 1974 DIABETES, VOL. 58, SEPTEMBER 2009 Chemerin (ng/ml) B. K. TAN AND ASSOCIATES A Insulin -11 -9 -7 Dose (M) B10 10 10 Chemerin (18kDa) β - actin (45kDa) ** -11 -9 -7 B10 10 10 Insulin (M) ** -11 -9 -7 B10 10 10 Insulin (M) 11 9 7 FIG. 3. A: Dose-dependent effects of insulin (10 M, 10 M, 10 M) in the presence of 5 mmol/l D-glucose on chemerin net protein production in control human omental adipose tissue explants at 24 h were assessed by Western blotting. Western blot analysis of protein extracts from omental adipose tissue demonstrate that the antibody against chemerin and the antibody against -actin recognized bands with apparent molecular weights of 18 kDa and 45 kDa, respectively (Fig. 3A, inserts). Densitometric analysis of chemerin immune complexes having normalized 9 7 to -actin, respectively, revealed that protein levels of chemerin were significantly increased by insulin (10 M, 10 M) in control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. 11 9 7 B: Dose-dependent effects of insulin (10 M, 10 M, 10 M) in the presence of 5 mmol/l D-glucose on chemerin secretion into conditioned media 9 7 from control human omental adipose tissue after 24 h were measured by ELISA. Chemerin secretion was significantly increased (by 10 M, 10 M) from control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. http://diabetes.diabetesjournals.org/cgi/content/full/db08- PCOS have an increased incidence of IGT and type 2 1528/DC1). diabetes (1,2). The higher serum and adipose tissue chemerin levels in women with PCOS is of interest given DISCUSSION that it has recently been reported that IGT and type 2 diabetes Psammomys obesus animals (a unique polygenic We present novel data showing a significant increase of animal model for obesity and type 2 diabetes) had higher serum and subcutaneous and omental adipose tissue adipose tissue chemerin levels than normal glucose–toler- chemerin mRNA expression as well as protein levels in ant Psammomys obesus animals. In the same study, women with PCOS. More importantly, we demonstrate the significant positive associations with BMI and circulating potent and robust regulation of chemerin in vivo and ex vivo triglycerides in normal glucose–tolerant human subjects by insulin as well as its modulation by metformin treatment. were noted (9). However, no data exists on chemerin and Unfortunately, because of technical limitations in adipose tissue procurement, we were unable to obtain sufficient its regulation in human adipose tissue. In our study, we amounts of sample/patient tissue to perform stromal vascu- found significant positive associations between circulating lar separation in adipose tissue depots. We could not account chemerin as well as chemerin levels in subcutaneous and for a potential adipocyte hypertrophy–related side effect. omental adipose tissue with BMI, WHR, glucose, insulin, These limitations not withstanding, adipose tissue from our HOMA-IR, and circulating triglycerides. However, it is PCOS women, compared with control subjects, express unlikely that either BMI or WHR are responsible for these more chemerin. findings, as both groups were matched for these variables. DIABETES, VOL. 58, SEPTEMBER 2009 1975 Chemerin in conditioned Chemerin/β-actin media (% difference) PSL (% difference) INSULIN AND METFORMIN REGULATE CHEMERIN Metformin Dose (µg/ml) B 0.01 0.10 2.00 Chemerin (18kDa) β - actin (45kDa) ** 25 ** B 0.01 0.10 2.00 Metformin (µg/ml) ** B 0.01 0.10 2.00 Metformin (µg/ml) FIG. 4. A: Dose-dependent effects of metformin (0.01, 0.1, and 2.00 g/ml) in the presence of 5 mmol/l D-glucose on chemerin net protein production in control human omental adipose tissue explants at 24 h were assessed by Western blotting. Western blot analysis of protein extracts from omental adipose tissue demonstrate that the antibody against chemerin and the antibody against -actin recognized bands with apparent molecular weights of 18 kDa and 45 kDa, respectively (Fig. 4A, inserts). Densitometric analysis of chemerin immune complexes having normalized to -actin, respectively, revealed that protein levels of chemerin were significantly decreased by metformin (0.01, 0.1, and 2.00 g/ml) in control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. B: Dose-dependent effects of insulin (0.01, 0.1, and 2.00 g/ml) in the presence of 5 mmol/l D-glucose on chemerin secretion into conditioned media from control human omental adipose tissue after 24 h were measured by ELISA. Chemerin secretion was significantly decreased (by 0.01, 0.1, and 2.00 g/ml) from control human omental adipose tissue explants. Data are expressed as percent difference of median of basal. Each experiment was carried out with six different samples from six different subjects in three replicates. Group comparison by Friedman’s ANOVA and post hoc Dunn’s test. *P < 0.05, **P < 0.01. In addition, caution needs to be exercised as these corre- levels of chemerin seen in our insulin-resistant PCOS lations may be spurious, without causative significance, subjects. Of secondary interest, there appears to be no resulting from the simple fact that our PCOS women had circadian variation in chemerin levels as depicted in Fig. 2. higher levels for all these parameters. It should be emphasized that the primary aim of this study Importantly, in study 2 we derive novel observations of was not to investigate the circadian variation of chemerin. a profound increase in chemerin levels by insulin in vivo. In addition, our study utilized relatively small numbers of This effect of insulin appears to be relatively acute, achiev- subjects because of the challenge imposed by the pro- ing a maximal effect 4 h after commencement of insulin longed insulin clamp study; hence, care needs to be and persisting throughout the entire period of hyperinsu- exercised in interpretation of these results. linemia. Furthermore, this is in agreement with our data More importantly, in study 3 we report for the first time on the regulation of chemerin protein production ex vivo. that metformin (6 months treatment; 850-mg twice daily) It is important to bear in mind that the regulation of significantly decreases circulating chemerin levels with a chemerin in adipose tissue is probably multifactorial. concomitant decrease in insulin resistance in PCOS sub- Moreover, it would be of interest to know whether or not jects. Additionally, although the change in serum chemerin the effects of insulin on chemerin production are also levels were significantly positively associated with applicable to other tissues given our in vivo data. Future changes in WHR, glucose, insulin, HOMA-IR, and triglyc- studies are needed to elucidate the role of other factors erides, when subjected to multiple regression analysis that regulate chemerin production. Taken together, the only HOMA-IR was predictive of serum chemerin levels. above findings could tentatively explain the increased Taken together, we hypothesize that elevated chemerin 1976 DIABETES, VOL. 58, SEPTEMBER 2009 Chemerin in conditioned Chemerin/β-action media (% difference) PSL (% difference) B. K. TAN AND ASSOCIATES TABLE 3 it would be of interest to perform this study with lean Linear regression analysis of variables associated with changes women with PCOS. in serum chemerin levels (before and after metformin treatment), In conclusion, we report novel findings of a significant chemerin, in PCOS subjects (n  21) increase of circulating and adipose tissue chemerin, a novel adipokine, in women with PCOS as well as the Simple Multiple potent and robust regulation of chemerin by insulin in vivo Estimate P Estimate P and ex vivo. More importantly, we present novel data that metformin treatment significantly decreases circulating BMI (kg/m ) 0.156 0.500 — — chemerin levels in women with PCOS. The physiologic and WHR 0.486 0.026 0.293 0.329 pathologic significance of our findings remain to be further Glucose (mmol/l) 0.510 0.018 0.338 0.234 elucidated. Insulin (pmol/l) 0.503 0.020 0.387 0.205 HOMA-IR 0.772 0.010 0.628 0.046 Cholesterol (mmol/l) 0.276 0.226 — — ACKNOWLEDGMENTS Triglycerides (mmol/l) 0.490 0.024 0.456 0.107 The General Charities of the City of Coventry funded this E (pmol/l) 0.240 0.327 — — study. Testosterone (nmol/l) 0.130 0.576 — — No potential conflicts of interest relevant to this article Androstenedione (nmol/l) 0.039 0.867 — — were reported. DHEA-S (mol/l) 0.055 0.814 — — H.S.R. would like to acknowledge S. Waheguru, Univer- SHBG (nmol/l) 0.236 0.304 — — sity of Warwick for his continual support. 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Diabetes – Pubmed Central

Published: Jun 5, 2009

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