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Metformin Increases the Novel Adipokine Cartonectin/CTRP3 in Women With Polycystic Ovary Syndrome Bee K. Tan, Jing Chen, Jiamiao Hu, Omar Amar, Harman S. Mattu, Raghu Adya, Vanlata Patel, Manjunath Ramanjaneya, Hendrik Lehnert, and Harpal S. Randeva Warwick Medical School (B.K.T., J.C., J.H., O.A., H.S.M., R.A., V.P., H.L., H.S.R.), University of Warwick, Coventry CV4 7AL, United Kingdom; and First Medical Department (M.R., H.L.), University of Lübeck Medical School, 23538 Lübeck, Germany
Context: Recently cartonectin was reported as a novel adipokine, with lower levels in diet-induced obese mice, glucose-lowering effects, and antiinflammatory and cardioprotective properties. Polycystic ovary syndrome (PCOS) is a proinflammatory state associated with obesity, diabetes, dyslipidemia, and cardiovascular complications. Objectives: The objective of the study was to investigate cartonectin levels and regulation in sera and adipose tissue (AT) as well as the effects of metformin of women with PCOS and control subjects. Design: This was a cross-sectional study [PCOS (n ⫽ 83) and control (n ⫽ 39) subjects]. Real-time PCR and Western blotting were used to assess mRNA and protein expression of cartonectin. Serum cartonectin was measured by an ELISA. Results: Serum and omental adipose tissue cartonectin were significantly lower in women with PCOS compared with control subjects (P ⬍ .05 and P ⬍ .01, respectively). Furthermore, cartonectin showed a significant negative association with body mass index, waist to hip ratio, glucose, insulin, total cholesterol, low-density lipoprotein-cholesterol, triglycerides, High sensitivity C-reactive protein (hs-CRP) and intima-media thickness (P ⬍ .05 and P ⬍ .01, respectively); in multiple regression analyses, triglycerides (P ⫽.040) and hs-CRP (P ⫽ .031) were predictive of cartonectin levels (P ⬍ .05). After 6 months of metformin treatment, there was an associated increase in serum cartonectin (P ⬍ .05). Importantly, changes in hs-CRP were significantly negatively correlated with changes in serum cartonectin (P ⫽ .033). Finally, cartonectin protein production and secretion into conditioned media were significantly increased by metformin in control human omental AT explants (P ⬍ .05). Conclusions: Serum and omental AT cartonectin are lower in women with PCOS. Metformin treatment increases serum cartonectin levels in these women and in omental AT explants. (J Clin Endocrinol Metab 98: E1891–E1900, 2013)
olycystic ovary syndrome (PCOS) affects up to 10% of women in the reproductive age (1). PCOS is a proinflammatory state characterized by menstrual dysfunction and hyperandrogenism and is associated with features of the metabolic syndrome, particularly,
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obesity, diabetes, dyslipidemia, and atherosclerosis (1– 4). The metabolic syndrome is associated with excessive accumulation of central body fat. Adipose tissue (AT) produces several cytokines termed adipokines, which have
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received May 13, 2013. Accepted October 9, 2013. First Published Online October 23, 2013
Abbreviations: AT, adipose tissue; BMI, body mass index; CTRP3, C1q complement/TNFrelated protein superfamily 3; DBP, diastolic blood pressure; ⌬, change; DHEA-S, dehydroepiandrosterone sulfate; E2, 17-estradiol; FAI, free androgen index; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment insulin resistance index; hs-CRP, high-sensitivity C-reactive protein; IMT, intima-media thickness; LDL, low-density lipoprotein; PCOS, polycystic ovary syndrome; SBP, systolic blood pressure; WHR, waist to hip ratio.
doi: 10.1210/jc.2013-2227
J Clin Endocrinol Metab, December 2013, 98(12):E1891–E1900
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widespread effects, pivotal in the pathogenesis of insulin resistance, diabetes, and atherosclerosis (5). Furthermore, the accumulation of visceral AT poses a higher cardiometabolic risk than sc AT (6) because elimination of visceral rather than sc AT has been shown to promote insulin sensitivity (7). The link between inflammation and insulin resistance in obesity and diabetes has been well described (8, 9). Importantly, it has been established that inflammation predisposes to the development of atherosclerosis; a major cause of mortality in obese, insulin-resistant, diabetic subjects (10). Recently C1q complement/TNF-related protein superfamily 3 (CTRP3; also known as cartonectin, cartducin, CORS-26) was described as a novel adipokine, the levels of which were lower in diet-induced obese mice, with glucose-lowering effects achieved by suppression of hepatic gluconeogenesis (11). Cartonectin has antiinflammatory properties (12–14) and more recently has been reported as a cardioprotective adipokine (15). We hypothesized that circulating and AT cartonectin levels are altered in women with PCOS (an insulin resistant and proinflammatory state), and if so, metformin, an antidiabetic drug, widely used in the treatment of women with PCOS, could affect circulating and AT cartonectin levels.
Materials and Methods Subjects Study A All women with PCOS met all three criteria of the revised 2003 Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine PCOS Consensus Workshop Group diagnostic criteria. The three criteria are as follows: 1) oligo- and/or anovulation, 2) clinical and/or biochemical signs of hyperandrogenism, and 3) polycystic ovaries (16). Exclusion criteria included age over 40 years; reproductive, cardiovascular, metabolic, renal, and endocrine disorders; neoplasms; and smoking. None of the subjects was on any regular medications for at least 6 months prior to the study. Subjects were initially seen at the infertility clinic and then scheduled for laparoscopy to assess Fallopian tube patency. All subjects underwent anthropometric measurements. After an overnight fast, blood samples, sc and omental AT were obtained (8:00 –10:00 AM) from adult female patients undergoing elective surgery for infertility investigation. Serum was immediately aliquoted on ice and stored at ⫺80°C. Subcutaneous AT was obtained from a 3-cm horizontal midline incision approximately 3 cm above the pubic symphysis. The same fat pad was divided equally into two halves. Each half was either immediately frozen in liquid nitrogen and stored at ⫺80°C or placed into a sterile container containing Medium 199 (Sigma-Aldrich) for primary adipose tissue culture. Samples that were snap frozen were transported on dry ice (⫺80°C) and stored at ⫺80°C in the labora-
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tories of the University of Warwick. A total of 73 subjects were recruited consecutively from the infertility clinic in accordance with the inclusion/exclusion criteria (PCOS: n ⫽ 19; controls: n ⫽ 54). Of the 19 PCOS subjects recruited, five withdrew before the study could be completed. In the control group, seven subjects did not complete the study. From the remaining 47 control subjects, 14 control subjects comparable for age, body mass index (BMI), and waist to hip ratio (WHR) were included in the final analysis. 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.
Study B All women with PCOS met all three criteria of the revised 2003 Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine PCOS Consensus Workshop Group diagnostic criteria. The three criteria are as follows: 1) oligo- and/or anovulation, 2) clinical and/or biochemical signs of hyperandrogenism, and 3) polycystic ovaries (16). Exclusion criteria included age over 40 years; reproductive, cardiovascular, metabolic, renal, and endocrine disorders; neoplasms; and smoking. None of the subjects was on any regular medications for at least 6 months prior to the study. Subjects were outpatients of the Department of Reproductive Medicine and Gynaecological Endocrinology of Magdeburg University and the Department of Obstetrics and Gynaecology of Martin-Luther-University Halle. The metabolic study was performed in the Outpatient Department of Endocrinology and Metabolism of Magdeburg University. A treatment with metformin in an off-label use was offered to all women with PCOS independently from the results of insulin sensitivity testing as per protocol. In those women with PCOS who agreed, therapy was initiated after basal assessment, and the dose of metformin was increased to a maintenance dose of 850 mg twice daily. All subjects underwent anthropometric measurements before and after metformin treatment. Blood pressure was measured in a sitting position within a quiet and calm environment after a rest of at least 5 minutes. The average of three measurements was obtained. Carotid intima-media thickness (IMT) was measured in women with PCOS before and after metformin dose (see Supplemental Data, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Blood samples were collected between 8:00 and 9:00 AM after a 3-day normal carbohydrate diet and an overnight fast. Serum was immediately aliquoted on ice and stored at ⫺80°C. A total of 83 women with PCOS and 39 controls were studied. There were 49 women with PCOS who did not participate in the metformin arm of the study. The baseline characteristics of the 49 women with PCOS were comparable with the 34 women with PCOS who participated in the metformin arm of the study. Furthermore, the 21 subjects who completed the metformin arm of the study were comparable with the 13 women with PCOS who did not complete the metformin arm of the study. For the purposes of elucidating the effects of metformin in women with PCOS, the 21 women with PCOS were studied. Reasons for subjects not completing study B were nausea and gastrointestinal side effects (n ⫽ 4), pregnancies (n ⫽ 5), noncompliance (n ⫽ 2), and loss of contact (n ⫽ 2) [see Supplemental Data and Supplemental Figure 1]. The study design was ap-
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proved by the local Research Ethics Committee of the University of Magdeburg, and written informed consent was obtained from all participants, in accordance with the guidelines in The Declaration of Helsinki 2000.
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Total RNA extraction and cDNA synthesis Total RNA was extracted from AT samples and cDNA synthesized (see Supplemental Data).
Reverse transcription-polymerase chain reaction
Biochemical and hormonal analysis Assays were performed using an automated analyzer (see Supplemental Data). High-sensitivity C-reactive protein (hs-CRP) was determined immunoturbidimetrically using a modular system random-access analyzer (Roche Diagnostics). Circulating leptin was measured with a coated-tube immunoradiometric assay kit (Diagnostic Systems Laboratories), according to the manufacturer’s protocol. Serum TNF-␣ was measured using a commercially available ELISA kit from R&D Systems GmbH, according to the manufacturer’s protocol, with an intraassay coefficient of variation of less than 9%. Circulating leptin and adiponectin were measured with a coated-tube immunoradiometric assay kit (Diagnostic Systems Laboratories) and by a commercially available RIA kit (Millipore), according to the manufacturer’s protocol, with intraassay coefficients of variation of 3.2% and 3.4%, respectively. Serum visfatin was measured using a commercially available enzyme immunoassay kit (Phoenix Pharmaceuticals, Inc, Aviscera), according to the manufacturer’s protocol, with an intraassay coefficient of variation of less than 6%. Cartonectin levels in sera and conditioned media were measured using a commercially available ELISA kit (Aviscera), according to the manufacturer’s protocol, with an intraassay coefficient of variation of less than 10%.
Primary explant culture AT organ explants were cultured using a protocol that was a modification of the method described by Fried and MoustaidMoussa (17) (see Supplemental Data).
Quantitative PCR of cartonectin was performed on a Roche Light Cycler system (Roche Molecular Biochemicals) (see Supplemental Data). The sequences of the sense and antisense primers used were: cartonectin (191 bp) 5⬘-GAGTCTCCACAAACCGGAGG-3⬘ and 5⬘-TCACCTTTGTCGCCCTTCTC-3⬘; and -actin (216 bp) 5⬘-AAGAGAGGCATCCTCACCCT-3⬘ and 5⬘-TACATGGCTGGGGTCTTGAA-3⬘.
Western blotting Protein lysates were prepared and Western blotting performed (see Supplemental Data). We used antibodies for cartonectin (Abcam) and -actin (Cell Signaling Technology Inc).
Statistics Data were analyzed by a Mann-Whitney U test, Wilcoxon matched pairs test, Kruskal-Wallis ANOVA (post hoc analysis: Dunn’s test), and Friedman’s ANOVA (post hoc analysis: Dunn’s test), according to the number of groups compared. Data are medians (interquartile range). The Spearman rank correlation was used for the calculation of associations between variables. Subsequently, if individual bivariate correlations achieved statistical significance, variables were entered into a linear regression model and multiple regression analysis with cartonectin as a dependent variable was performed to test the joint effect of different parameters on cartonectin. All statistical analyses were performed using SPSS version 21.0 (SPSS, Inc). P ⬍ .05 was considered significant.
Table 1. Clinical, Hormonal, and Metabolic Features of Women With PCOS and Control Subjects (Study A) Variable
PCOS (n ⴝ 14)
Controls (n ⴝ 14)
Significance
Age, y BMI, kg/m2 WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L Triglycerides, mmol/L Prolactin, mU/L E2, pmol/L Progesterone, nmol/L 17-OH-P, nmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL Cartonectin, ng/mL
29.5 (28 –38) 30.5 (27.8 –30.9) 0.89 (0.78 – 0.99) 5.5 (4.8 – 6.0) 78.9 (42.0 –91.1) 3.0 (2.1–3.6) 5.1 (4.1–5.7) 2.0 (1.5–2.3) 348.0 (305.0 –387.0) 353.5 (287.0 – 471.0) 1.6 (1.3–2.1) 2.5 (2.1–2.8) 1.7 (1.5–2.1) 15.6 (14.2–16.8) 5.9 (5.4 – 6.6) 31.5 (26.7–35.2) 19.5 (14.5–21.2) 24.9 (19.7–29.0) 5.67 (4.52–7.50) 53.6 (41.0 – 63.1) 5.35 (2.19 – 8.42) 254.2 (193.1–359.6)
32.5 (29 –35) 28.8 (28 –30.5) 0.84 (0.81– 0.96) 4.5 (4.3–5.2) 57.3 (48.5– 66.0) 2.0 (1.4 –2.2) 5.0 (4.8 –5.5) 0.9 (0.7–1.4) 304.5 (211.0 –322.0) 174.5 (129.0 –264.0) 2.2 (1.7–2.3) 2.0 (1.2–2.3) 0.8 (0.6 – 0.9) 6.3 (5.0 – 8.2) 4.5 (4.0 –5.3) 59.0 (47.7– 66.0) 3.9 (3.3– 6.1) 24.1 (19.3–28.8) 6.27 (5.52–9.48) 44.2 (39.0 –50.2) 3.78 (3.19 –5.84) 351.4 (306.1–375.2)
NS NS NS P ⬍ .01 NS P ⬍ .05 NS P ⬍ .01 NS P ⬍ .01 NS NS P ⬍ .01 P ⬍ .01 P ⬍ .05 P ⬍ .01 P ⬍ .01 NS NS NS NS P ⬍ .05
Abbreviations: 17-OH-P, 17-hydroxyprogesterone; NS, not significant. FAI ⫽ T (nanomoles per liter)/SHBG (nanomoles per liter) ⫻ 100. Data are medians (interquartile range). Group comparison was done by a Mann-Whitney U test.
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Results Study A Demographic data Table 1 shows the anthropometric, biochemical, and hormonal data in all subjects. Glucose, homeostasis model assessment insulin resistance index (HOMA-IR), triglycerides, 17-estradiol (E2), T, androstenedione, dehydroepiandrosterone sulfate (DHEA-S) levels and free androgen index (FAI) were significantly higher whereas SHBG was significantly lower in women with PCOS. Serum cartonectin levels were significantly lower in women with
J Clin Endocrinol Metab, December 2013, 98(12):E1891–E1900
PCOS compared with control subjects [254.2 (193.1– 359.6) vs. 351.4 (306.1–375.2) ng/mL; P ⬍ .05: Table 1]. Serum progesterone levels in all subjects confirmed the follicular phase of the menstrual cycle. mRNA expression and protein levels of cartonectin in AT of normal and women with PCOS We detected cartonectin mRNA in AT of all subjects and subsequent sequencing of the PCR products confirmed gene identity. Real-time RT-PCR analysis corrected over -actin showed a significant decrease of cartonectin mRNA expression in omental (*, P ⬍ .05) AT of PCOS compared with control subjects (Figure 1A). However, no significant difference in cartonectin mRNA expression was observed when comparing mRNA expression in sc AT of PCOS with control subjects as well as corresponding omental to sc adipose tissue in PCOS and control subjects (Figure 1A). The changes noted at mRNA level were also reflected at protein level (Figure 1B). Western blot analysis of protein extracts from sc and omental AT demonstrate that the antibody against cartonectin and the antibody against -actin recognized bands with apparent molecular masses of 36 and 42 kDa, respectively.
Figure 1. A, Cartonectin mRNA expression relative to -actin was significantly lower in human omental (om) adipose tissue when comparing women with PCOS (n ⫽ 14) with control subjects (n ⫽ 14), using real-time RT-PCR. Data are the percentage difference of median of human sc adipose tissue of control subjects. Each experiment was carried out in three replicates. Group comparison was done by Kruskal-Wallis ANOVA and post hoc Dunn’s test. *, P ⬍ .05. B, Densitometric analysis of cartonectin immune complexes having normalized to -actin revealed that protein levels of cartonectin were significantly lower in human omental (om) adipose tissue depots when comparing all women with PCOS with all control subjects. Data are the percentage difference of median of human sc adipose tissue of control subjects. Each experiment was carried out in three replicates. Group comparison was done by Kruskal-Wallis ANOVA and post hoc Dunn’s test. *, P ⬍ .05. AU, arbitrary units. C, Dose-dependent effects of metformin (0.01, 0.1, and 2.00 g/mL) in the presence of 5 mmol/L D-glucose on CTRP3 protein production in control human omental AT explants at 24 hours was assessed by Western blotting. Densitometric analysis of CTRP3 immune complexes having normalized to -actin revealed that protein levels of CTRP3 were significantly increased by metformin (2.00 g/mL) in control human omental AT explants. Data are expressed as the percentage difference of median of basal (B). Each experiment was carried out with six different samples from six different control subjects in three replicates. Group comparison was done by Friedman’s ANOVA and post hoc Dunn’s test. *, P ⬍ .05. AU, arbitrary units. D, Dose-dependent effects of metformin (0.01, 0.1, and 2.00 g/mL) in the presence of 5 mmol/L D-glucose on CTRP3 secretion into conditioned media in control human omental AT explants at 24 hours was measured by ELISA. CTRP3 secretion into conditioned media was significantly increased by metformin (2.00 g/mL) in control human omental AT explants. Data are the percentage difference of the median of basal (B) [D-glucose (5 mmol/L)]. Each experiment was carried out with six different samples from six different control subjects in three replicates. Group comparison was done by Friedman’s ANOVA and post hoc Dunn’s test. *, P ⬍ .05.
Study B: effects of metformin treatment on serum cartonectin levels Table 2 shows the anthropometric, biochemical, and hormonal data in all subjects. Insulin, HOMA-IR, total cholesterol, low-density lipoprotein (LDL)-cholesterol, triglycerides, E2, T, androstenedione, FAI, hsCRP, systolic blood pressure (SBP), diastolic blood pressure (DBP), and IMT were significantly higher, whereas high-density lipoprotein (HDL)-cholesterol, DHEA-S, and SHBG were significantly lower in women with PCOS. Serum cartonectin levels were significantly lower in women with PCOS compared with control subjects [200.3 (158.1– 263.6) vs 330.7 (180.3– 436.6) ng/ mL; P ⬍ .01]. After 6 months of met-
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Table 2. Clinical, Hormonal, and Metabolic Features of Women With PCOS and Control Subjects (Study B) Variable
PCOS (n ⴝ 83)
Controls (n ⴝ 39)
Significance
Age, y BMI, kg/m2 WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L HDL-cholesterol, mmol/L LDL-cholesterol, mmol/L Triglycerides, mmol/L E2, pmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI hs-CRP, mg/L SBP, mm Hg DBP, mm Hg IMT, mm Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL Cartonectin (ng/ml)
27 (24.0 –31.0) 31.4 (26.1–34.2) 0.81 (0.77– 0.86) 4.9 (4.5–5.4) 69.0 (44.0 –100.0) 2.0 (1.3–2.9) 5.0 (4.5–5.6) 1.3 (1.1–1.5) 3.1 (2.6 –3.5) 1.1 (0.8 –1.9) 341.9 (200.7–538.4) 2.1 (1.5–2.7) 11.9 (8.4 –16.4) 4.7 (3.1–5.9) 30.0 (21.0 – 42.0) 6.5 (3.3–11.2) 2.8 (1.5– 4.6) 125.0 (120.0 –130.0) 75.0 (75.0 – 80.0) 0.46 (0.44 – 0.52) 21.9 (15.9 –28.5) 5.72 (4.69 –7.21) 46.3 (40.1–57.1) 3.93 (3.41–5.45) 200.3 (158.1–263.6)
28 (23–32) 29.1 (27.3–34.1) 0.81 (0.78 – 0.86) 4.8 (4.4 –5.1) 41.0 (32.0 –55.0) 1.2 (1.0 –1.7) 4.3 (3.8 – 4.8) 1.5 (1.3–1.7) 2.3 (1.9 –2.8) 0.8 (0.6 –1.1) 178.0 (118.9 –292.7) 1.2 (1.0 –1.6) 9.1 (6.6 –12.6) 6.1 (3.8 –7.5) 45.0 (34.0 –59.0) 3.0 (1.9 – 4.4) 1.3 (0.6 –2.3) 120.0 (110.0 –120.0) 75.0 (70.0 –77.5) 0.42 (0.40 – 0.44) 17.3 (10.8 –26.9) 5.84 (4.14 –7.90) 45.8 (40.2– 47.7) 3.73 (2.06 – 6.90) 330.7 (180.3– 436.6)
NS NS NS NS P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .05 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .05 P ⬍ .01 NS NS NS NS P ⬍ .01
Abbreviation: NS, not significant. FAI ⫽ T (nanomoles per liter)/SHBG (nanomoles per liter) ⫻ 100. Data are medians (interquartile range). Group comparison was done by Mann-Whitney U test.
formin treatment (Table 3), there was a significant increase in serum cartonectin [212.9 (105.1–320.6) vs 272.0 (169.8 –386.5) ng/mL; P ⬍ .05]. Also, there were significant decreases in WHR, E2, T, glucose, HOMA-IR, and IMT. Association of cartonectin with covariates In study A, a Spearman rank analysis demonstrated that serum and omental AT cartonectin were significantly negatively associated with WHR, glucose, total cholesterol, and circulating triglycerides (P ⬍ .05). No significant associations were found with respect to sc AT. However, when subjected to multiple regression analysis, none of these variables were predictive of serum or omental AT cartonectin. In study B, a Spearman Rank analysis demonstrated that serum cartonectin was significantly negatively associated with BMI, insulin, LDL-cholesterol, triglycerides, hs-CRP and IMT (Table 4). When subjected to multiple regression analysis, triglycerides and hs-CRP were found to be predictive of serum cartonectin levels. We also analyzed the correlation between the change in serum cartonectin levels before and after metformin therapy (⌬cartonectin) and the changes (⌬) in other covariates (Table 5). We found that ⌬cartonectin was significantly negatively associated with ⌬WHR, ⌬glucose, ⌬LDL-cholesterol, ⌬triglycerides, ⌬hsCRP, and ⌬IMT. When subjected to multiple regression anal-
ysis, only ⌬hs-CRP was predictive of ⌬cartonectin serum levels ( ⫽ ⫺.550; P ⫽ .033). Dose-dependent effects of D-glucose, insulin, metformin, T, E2, androstenedione, and DHEA-S on cartonectin protein production and secretion into conditioned media in control human omental AT explants Given our observations above, we investigated the effects of D-glucose, insulin, metformin, and gonadal and adrenal steroids ex vivo. Cartonectin protein production and secretion into conditioned media were significantly increased by metformin in control human omental AT explants (Figure 1, C and D: *, P ⬍ .05). With respect to D-glucose, insulin, and gonadal and adrenal steroids, no significant effects on cartonectin protein production or secretion into conditioned media was found (data not shown).
Discussion We present novel data showing significantly lower serum and omental AT cartonectin in women with PCOS. Metformin therapy (850 mg twice daily for 6 months) significantly increased serum cartonectin concentrations. Furthermore, cartonectin protein production and secretion
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Table 3. Clinical, Hormonal, and Metabolic Features of Women With PCOS (n ⫽ 21) Before and After Metformin Treatment (Study B) Variable
Before Metformin
After Metformin
Significance
Age, y BMI, kg/m2 WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L HDL-cholesterol, mmol/L LDL-cholesterol, mmol/L Triglycerides, mmol/L E2, pmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI hs-CRP, mg/liter SBP, mm Hg DBP, mm Hg IMT, mm Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL Cartonectin, ng/mL
28 (26.5–31.5) 32.8 (29.8 –36.5) 0.82 (0.76 – 0.88) 5.1 (4.7–5.5) 70.0 (54.5–94.5) 2.2 (2.0 –3.0) 4.9 (4.1–5.3) 1.3 (1.1–1.5) 2.9 (2.2–3.3) 1.0 (0.8 –1.7) 329.8 (164.9 – 494.7) 1.8 (1.5–2.5) 10.5 (8.0 –13.3) 4.3 (2.7–5.6) 27.0 (20.5– 41.0) 6.6 (4.6 –9.7) 4.1 (2.4 –5.9) 125.0 (120.0 –130.0) 80.0 (75.0 – 80.0) 0.53 (0.48 – 0.58) 26.5 (20.7–30.9) 4.85 (4.14 – 6.88) 51.8 (42.3– 60.4) 4.08 (3.52–5.45) 212.9 (105.1–320.6)
28 (27.5–32.5) 31.4 (28.2–35.1) 0.80 (0.74 – 0.87) 4.8 (4.4 – 4.9) 61.0 (43.5– 83.0) 1.6 (1.3–2.2) 5.0 (4.1–5.4) 1.2 (1.1–1.5) 3.0 (2.5–3.3) 1.2 (1.0 –1.9) 207.1 (103.6 –310.7) 1.3 (1.1–1.8) 9.8 (7.7–12.6) 5.4 (3.6 – 6.7) 29.0 (21.5– 46.5) 5.3 (3.1– 6.6) 3.1 (2.1–7.2) 120.0 (120.0 –127.5) 75.0 (75.0 – 80.0) 0.47 (0.41– 0.53) 25.1 (20.0 –30.0) 3.69 (2.91–5.55) 51.0 (40.8 –58.3) 4.15 (3.18 –5.07) 272.0 (169.8 –386.5)
NS NS P ⬍ .05 P ⬍ .05 NS P ⬍ .05 NS NS NS NS P ⬍ .05 P ⬍ .05 NS NS NS NS NS NS NS P ⬍ .05 NS NS NS NS P ⬍ .05
Abbreviation: NS, not significant. FAI ⫽ T (nanomoles per liter)/SHBG (nanomoles per liter) ⫻ 100. Data are medians (interquartile range). Group comparison was done by a Wilcoxon matched-pairs test.
into conditioned media were significantly increased by metformin in omental AT explants. Moreover, a Spearman rank analysis demonstrated that serum cartonectin was significantly negatively associated with BMI, WHR, insulin, LDL-cholesterol, total cholesterol, triglycerides, hs-CRP, and IMT. When subjected to multiple regression analysis, only triglycerides and hs-CRP were found to be predictive of serum cartonectin levels. Finally, changes in cartonectin were significantly negatively associated with changes in WHR, glucose, LDL-cholesterol, triglycerides, hs-CRP, and IMT. When subjected to multiple regression analysis, only changes in hs-CRP were predictive of changes in serum cartonectin. The lower serum and omental AT cartonectin levels in women with PCOS is perplexing, given that it has recently been reported that plasma cartonectin was significantly higher in patients with type 2 diabetes and significant positive associations were found between plasma cartonectin and cardiometabolic risk factors ie, WHR, plasma glucose, lipids, and hs-CRP (18). Interestingly, the same researchers subsequently reported that serum cartonectin was not significantly lower in subjects with the metabolic syndrome compared with controls and that serum cartonectin was significantly negatively associated with waist circumference, DBP, serum glucose, cholesterol, and triglycerides (19). Additionally, in the present study, treat-
ment of human omental AT explants with D-glucose or insulin did not significantly alter cartonectin production in AT. Therefore, it would appear that other mechanisms are involved in the regulation of cartonectin levels in AT. Of relevance, Kopp et al (13) reported that cartonectin reduced lipopolysaccharide induced production of macrophage migration inhibitor factor in nondiabetic controls, with no apparent effects in subjects with type 2 diabetes. Another explanation of elevated cartonectin levels in type 2 diabetes observed by Choi et al (18) could be the effect of medications taken by their study subjects as discussed in their following manuscript (19). In addition, the clinical human studies mentioned above involved only East Asian subjects, whereas our study comprised solely Caucasian subjects. There could be differences in cartonectin levels between different ethnic groups. Finally, there may be other yet-undetermined factors that may account for the significantly higher circulating cartonectin levels found in type 2 diabetes patients. In our studies, we found significant negative associations between serum and omental AT cartonectin levels with cardiometabolic risk factors. Notably, in study B, serum cartonectin levels were significantly negatively associated with BMI, insulin, LDL-cholesterol, triglycerides, hs-CRP, and IMT. Importantly, multiple regression analyses revealed that serum triglycerides and hs-CRP were
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Table 4. Linear Regression Analysis of Variables Associated With Serum Cartonectin Levels in All Subjects (n ⫽ 122) (Study B) Simple Variable
Estimate 2
BMI, kg/m WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L HDL-cholesterol, mmol/L LDL-cholesterol, mmol/L Triglycerides, mmol/L E2, pmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI hs-CRP, mg/L SBP, mm Hg DBP, mm Hg IMT, mm Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL
⫺0.302 ⫺0.013 ⫺0.010 ⫺0.286 ⫺0.107 ⫺0.099 0.127 ⫺0.270 ⫺0.417 ⫺0.122 ⫺0.183 ⫺0.132 0.252 0.147 ⫺0.212 ⫺0.389 ⫺0.012 0.009 ⫺0.259 ⫺0.175 0.138 ⫺0.178 ⫺0.088
Multiple P Value a
.019 .922 .937 .027a .415 .454 .333 .037a .002b .353 .161 .313 .052 .263 .104 .001b .928 .948 .046a .182 .292 .279 .593
Estimate
P Value
0.051
.775
0.112
.502
⫺0.171 ⫺0.322
.256 .040a
⫺0.336
.031a
0.134
.431
In multiple linear regression analysis, values included were BMI, insulin, LDL-cholesterol, triglycerides, hs-CRP, and IMT. a
P ⬍ .05.
b
P ⬍ .01.
predictive of serum cartonectin. Thus, our findings highlight an important connection between circulating cartonectin and the proinflammatory dysmetabolic state present in overweight, insulin-resistant women with PCOS (4). Further studies are needed to clarify the underlying mechanisms that regulate cartonectin levels. In the seminal report of Peterson et al (11), which first described cartonectin as a novel adipokine with glucoselowering effects, the investigators had also observed a profound inverse correlation between leptin and cartonectin (11). In our studies, there were no significant differences in leptin and no significant associations between leptin and cartonectin. Thus, it is unlikely that leptin may account for our observations. Recent studies have reported a sexual dimorphism in circulating cartonectin, with significantly higher cartonectin in females (18, 21). To the best of our knowledge, there are no data on the regulation and effect of steroids on cartonectin. In our studies, both gonadal and adrenal steroids were altered in women with PCOS. However, there were no significant associations between gonadal and/or adrenal steroids and cartonectin. In addition, we found that treatment of human omental AT explants with gonadal or adrenal steroids did not significantly alter cartonectin production in AT. It therefore remains to be clar-
ified as to whether the differences in cartonectin levels are attributable to altered gonadal and/or adrenal steroids. Further studies are needed to elucidate the role of the effects of gonadal and adrenal steroids as well as other factors that regulate cartonectin production. In a recent publication, Schmid et al (22) had emphasized the important role of cartonectin in adipocyte biology, ie, lipolysis and adipokine secretion. Of relevance, catecholamine-induced lipolysis is elevated in visceral adipocytes of women with PCOS (23). This may lead to increased portal fatty acid levels culminating in liver dysfunction causing glucose intolerance, hyperinsulinemia, and dyslipidemia, metabolic aberrations that are often found in women with PCOS (1, 2). The factors driving increased catecholamine-induced lipolysis in visceral adipocytes of women with PCOS remain unexplained (23). We hypothesize that cartonectin may play a pivotal role in this context. Future studies are needed to elucidate this point. To the best of our knowledge, the effects of metformin treatment on cartonectin levels have not been investigated in any human/animal studies. We report for the first time that metformin (6 months treatment; 850 mg twice daily) is associated with a significant increase in serum cartonectin and a parallel decrease in insulin resistance in women
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Table 5. Linear Regression Analysis of Variables Associated With Changes in Serum Cartonectin Levels (Before and After Metformin Treatment), ⌬Cartonectin, in Women With PCOS (n ⫽ 21) Simple Variable ⌬BMI, kg/m ⌬WHR ⌬Glucose, mmol/L ⌬Insulin, pmol/L ⌬HOMA-IR ⌬Total cholesterol, mmol/L ⌬HDL-cholesterol, mmol/L ⌬LDL-cholesterol, mmol/L ⌬Triglycerides, mmol/L ⌬E2, pmol/L ⌬T, nmol/L ⌬Androstenedione, nmol/L ⌬DHEA-S, mol/L ⌬SHBG, nmol/L ⌬FAI ⌬hs-CRP, mg/L ⌬SBP, mm Hg ⌬DBP, mm Hg ⌬IMT, mm ⌬Leptin, ng/mL ⌬Adiponectin, g/mL ⌬Visfatin, ng/mL ⌬TNF-␣, pg/mL 2
Multiple
Estimate
P
⫺0.120 ⫺0.476 ⫺0.447 0.131 0.046 ⫺0.074 ⫺0.003 ⫺0.460 ⫺0.498 ⫺0.160 ⫺0.152 ⫺0.107 ⫺0.241 0.322 ⫺0.401 ⫺0.675 0.398 ⫺0.176 ⫺0.450 0.013 0.214 ⫺0.230 ⫺0.248
.605 .029a .042a .570 .845 .749 .989 .036a .022a .489 .511 .645 .294 .155 .072 .001b .074 .447 .041a .955 .101 .077 .057
Estimate
P
⫺0.263 0.266
.536 .503
⫺0.231 ⫺0.388
.539 .207
⫺0.550
.033a
0.216
.624
In multiple linear regression analysis, values included were ⌬WHR, ⌬glucose, ⌬LDL-cholesterol, ⌬triglycerides, ⌬hs-CRP, and ⌬IMT. a
P ⬍ .05.
b
P ⬍ .01.
with PCOS. More importantly, we observed that cartonectin production was significantly increased by metformin in human omental AT explants. The profound effect of metformin on serum cartonectin levels could be due to its suppressive effect on hepatic glucose production (24 –26). Of relevance, cartonectin has been shown to have glucose-lowering effects through suppression of hepatic gluconeogenesis (11). However, as mentioned above, we did not find a significant difference in AT cartonectin production after D-glucose treatment. Lihn et al (27) and Sell et al (28) had reported that stimulating the AMP-activated protein kinase signaling pathway promotes adiponectin (the foremost adipokine of the C1q complement/TNF-related protein superfamily) expression in human adipose tissue. Because metformin mainly acts through the AMP-activated protein kinase pathway, this could be the mechanism wherein metformin increases cartonectin production and secretion in AT. Our results may account for the elevated cartonectin levels in type 2 diabetes observed by Choi et al (18) as discussed above and highlights metformin therapy as a confounding factor with regard to the regulation of circulating cartonectin levels. This should alert investigators who are studying cartonectin biology to consider this in their analysis. This may also apply to other forms of an-
tidiabetic therapy, and hence, caution needs to be exercised appropriately. In addition, we observed a significant decrease in IMT after metformin treatment in keeping with a recent report by Palomba et al (29). Furthermore, although the change in serum cartonectin was significantly negatively associated with changes in WHR, glucose, LDL-cholesterol, triglyceride, hs-CRP, and IMT, only hsCRP was predictive of the change in serum cartonectin associated with metformin therapy. This suggests that changes in serum cartonectin after metformin treatment observed in our PCOS subjects are more robustly linked to changes in inflammatory rather than metabolic parameters. As such, numerous studies have championed the antiinflammatory action of cartonectin (12–14). Therefore, the elevated serum cartonectin associated with metformin treatment may be an important mechanism through which metformin exerts its antiinflammatory effects (20). Further studies are needed to clarify this concept. In study A, a limitation may relate to the number of subjects studied and hence we would advise caution with regard to our findings; further studies with larger number of study subjects are needed to reaffirm our results. However, obtaining BMI/WHR comparable and menstrual cycle-synchronized blood and AT samples from two AT depots impeded subject recruitment. Nevertheless, our
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observations are highly consistent and significant and raise interesting questions on the mechanisms regulating cartonectin production. In study B, given ethical constraints, ie, blood samples, could and were obtained only at baseline and after 6 months of metformin treatment. It is therefore difficult to be certain as to whether changes in serum cartonectin precede or follow changes in clinical and hormonal indices as a consequence of metformin treatment. Moreover, because diet and lifestyle modifications were only subjectively assessed, the changes in serum cartonectin may be partially attributable to diet and lifestyle modifications. Further studies are needed to elucidate this point. Given the paucity of research into cartonectin, it is still not entirely clear as to how this novel adipokine fits into the homeostatic mechanisms that modulate the various functions of the human body in both health and disease, in particular, with respect to the metabolic syndrome and its associated complications such as type 2 diabetes. The current lack of a definitive receptor for cartonectin and an understanding of its molecular and cellular dynamics have only made the task of understanding the physiological actions of cartonectin more difficult and challenging. Given that cartonectin is by and large expressed in AT stromal vascular cells (13), its well-documented antiinflammatory actions (12–14) as well as the novel data presented in this manuscript, we postulate that cartonectin, potentially, may have a predominant role as an antiinflammatory molecule and that its cardiometabolic actions plausibly are intertwined in the most part via inflammatory pathways. In conclusion, circulating and omental AT cartonectin levels are significantly lower in insulin resistant women with PCOS compared with non-insulin-resistant subjects. Metformin treatment significantly increases circulating cartonectin and it appears that it does this (at least in part) by stimulating cartonectin production and secretion from AT. Because circulating cartonectin is significantly correlated with various cardiometabolic factors, cartonectin may be a useful biomarker of cardiometabolic disease in humans.
Acknowledgments Address all correspondence and requests for reprints to: Dr Bee K. Tan and Dr Harpal S. Randeva, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom. E-mail: [email protected]; or [email protected]. This work was supported by the General Charities of the City of Coventry. Disclosure Summary: The authors have nothing to declare.
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References 1. Dunaif A. Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev. 1997; 18:774 – 800. 2. Wild RA, Painter RD, Coulson PB, Carruth KB, Ranney RB. Lipoprotein 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;30:13–17. 4. Diamanti-Kandarakis E, Paterakis T, Kandarakis HA. Indices of low-grade inflammation in polycystic ovary syndrome. Ann NY Acad Sci. 2006;1092:175–186. 5. Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab. 2004;89:2548 –2556. 6. Wajchenberg BL. Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr Rev. 2000;21:697–738. 7. Thorne A, Lonnqvist F, Apelman J, Hellers G, Arner P. A pilot study of long-term effects of a novel obesity treatment: omentectomy in connection with adjustable gastric banding. Int J Obes Relat Metab Disord. 2002;26:193–199. 8. Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumour necrosis factor-␣: direct role in obesity-linked insulin resistance. Science. 1993;259:87–91. 9. Kubaszek A, Pihlajamäki J, Komarovski V, et al. Promoter polymorphisms of the TNF-␣ (G-308A) and IL-6 (C-174G) genes predict the conversion from impaired glucose tolerance to type 2 diabetes: the Finnish Diabetes Prevention Study. Diabetes. 2003;52:1872– 1876. 10. Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med. 1999;340:115–126. 11. Peterson JM, Wei Z, Wong GW. C1q/TNF-related protein-3 (CTRP3), a novel adipokine that regulates hepatic glucose output. J Biol Chem. 2010;285:39691–39701. 12. Kopp A, Bala M, Weigert J, et al. Effects of the new adiponectin paralogous protein CTRP-3 and of LPS on cytokine release from monocytes of patients with type 2 diabetes mellitus. Cytokine. 2010; 49:51–57. 13. Kopp A, Bala M, Buechler C, et al. C1q/TNF-related protein-3 represents a novel and endogenous lipopolysaccharide antagonist of the adipose tissue. Endocrinology. 2010;151:5267–5278. 14. Hofmann C, Chen N, Obermeier F, et al. C1q/TNF-related protein-3 (CTRP-3) is secreted by visceral adipose tissue and exerts antiinflammatory and antifibrotic effects in primary human colonic fibroblasts. Inflamm Bowel Dis. 2011;17:2462–2471. 15. Yi W, Sun Y, Yuan Y, et al. C1q/tumor necrosis factor-related protein-3, a newly identified adipokine, is a novel antiapoptotic, proangiogenic, and cardioprotective molecule in the ischemic mouse heart. Circulation. 2012;125:3159 –3169. 16. Fauser B. Revised 2003 consensus on diagnostic criteria and longterm health risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004;19:41– 47. 17. Fried SK, Moustaid-Moussa N. Culture of adipose tissue and isolated adipocytes. Methods Mol Biol. 2001;155:197–212. 18. Choi KM, Hwang SY, Hong HC, et al. C1q/TNF-related protein-3 (CTRP-3) and pigment epithelium-derived factor (PEDF) concentrations in patients with type 2 diabetes and metabolic syndrome. Diabetes. 2012;61:2932–2936. 19. Yoo HJ, Hwang SY, Hong HC, et al. Implication of progranulin and C1q/TNF-related protein-3 (CTRP3) on inflammation and atherosclerosis in subjects with or without metabolic syndrome. PLoS One. 2013;8:e55744. 20. Molavi B, Rassouli N, Bagwe S, Rasouli N. A review of thiazolidinediones and metformin in the treatment of type 2 diabetes with focus on cardiovascular complications. Vasc Health Risk Manag. 2007;3:967–973. 21. Wong GW, Krawczyk SA, Kitidis-Mitrokostas C, Revett T, Gimeno
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Hot Topics in Translational Endocrinology—Endocrine Research
Metformin Increases the Novel Adipokine Cartonectin/CTRP3 in Women With Polycystic Ovary Syndrome Bee K. Tan, Jing Chen, Jiamiao Hu, Omar Amar, Harman S. Mattu, Raghu Adya, Vanlata Patel, Manjunath Ramanjaneya, Hendrik Lehnert, and Harpal S. Randeva Warwick Medical School (B.K.T., J.C., J.H., O.A., H.S.M., R.A., V.P., H.L., H.S.R.), University of Warwick, Coventry CV4 7AL, United Kingdom; and First Medical Department (M.R., H.L.), University of Lübeck Medical School, 23538 Lübeck, Germany
Context: Recently cartonectin was reported as a novel adipokine, with lower levels in diet-induced obese mice, glucose-lowering effects, and antiinflammatory and cardioprotective properties. Polycystic ovary syndrome (PCOS) is a proinflammatory state associated with obesity, diabetes, dyslipidemia, and cardiovascular complications. Objectives: The objective of the study was to investigate cartonectin levels and regulation in sera and adipose tissue (AT) as well as the effects of metformin of women with PCOS and control subjects. Design: This was a cross-sectional study [PCOS (n ⫽ 83) and control (n ⫽ 39) subjects]. Real-time PCR and Western blotting were used to assess mRNA and protein expression of cartonectin. Serum cartonectin was measured by an ELISA. Results: Serum and omental adipose tissue cartonectin were significantly lower in women with PCOS compared with control subjects (P ⬍ .05 and P ⬍ .01, respectively). Furthermore, cartonectin showed a significant negative association with body mass index, waist to hip ratio, glucose, insulin, total cholesterol, low-density lipoprotein-cholesterol, triglycerides, High sensitivity C-reactive protein (hs-CRP) and intima-media thickness (P ⬍ .05 and P ⬍ .01, respectively); in multiple regression analyses, triglycerides (P ⫽.040) and hs-CRP (P ⫽ .031) were predictive of cartonectin levels (P ⬍ .05). After 6 months of metformin treatment, there was an associated increase in serum cartonectin (P ⬍ .05). Importantly, changes in hs-CRP were significantly negatively correlated with changes in serum cartonectin (P ⫽ .033). Finally, cartonectin protein production and secretion into conditioned media were significantly increased by metformin in control human omental AT explants (P ⬍ .05). Conclusions: Serum and omental AT cartonectin are lower in women with PCOS. Metformin treatment increases serum cartonectin levels in these women and in omental AT explants. (J Clin Endocrinol Metab 98: E1891–E1900, 2013)
olycystic ovary syndrome (PCOS) affects up to 10% of women in the reproductive age (1). PCOS is a proinflammatory state characterized by menstrual dysfunction and hyperandrogenism and is associated with features of the metabolic syndrome, particularly,
P
obesity, diabetes, dyslipidemia, and atherosclerosis (1– 4). The metabolic syndrome is associated with excessive accumulation of central body fat. Adipose tissue (AT) produces several cytokines termed adipokines, which have
ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2013 by The Endocrine Society Received May 13, 2013. Accepted October 9, 2013. First Published Online October 23, 2013
Abbreviations: AT, adipose tissue; BMI, body mass index; CTRP3, C1q complement/TNFrelated protein superfamily 3; DBP, diastolic blood pressure; ⌬, change; DHEA-S, dehydroepiandrosterone sulfate; E2, 17-estradiol; FAI, free androgen index; HDL, high-density lipoprotein; HOMA-IR, homeostasis model assessment insulin resistance index; hs-CRP, high-sensitivity C-reactive protein; IMT, intima-media thickness; LDL, low-density lipoprotein; PCOS, polycystic ovary syndrome; SBP, systolic blood pressure; WHR, waist to hip ratio.
doi: 10.1210/jc.2013-2227
J Clin Endocrinol Metab, December 2013, 98(12):E1891–E1900
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widespread effects, pivotal in the pathogenesis of insulin resistance, diabetes, and atherosclerosis (5). Furthermore, the accumulation of visceral AT poses a higher cardiometabolic risk than sc AT (6) because elimination of visceral rather than sc AT has been shown to promote insulin sensitivity (7). The link between inflammation and insulin resistance in obesity and diabetes has been well described (8, 9). Importantly, it has been established that inflammation predisposes to the development of atherosclerosis; a major cause of mortality in obese, insulin-resistant, diabetic subjects (10). Recently C1q complement/TNF-related protein superfamily 3 (CTRP3; also known as cartonectin, cartducin, CORS-26) was described as a novel adipokine, the levels of which were lower in diet-induced obese mice, with glucose-lowering effects achieved by suppression of hepatic gluconeogenesis (11). Cartonectin has antiinflammatory properties (12–14) and more recently has been reported as a cardioprotective adipokine (15). We hypothesized that circulating and AT cartonectin levels are altered in women with PCOS (an insulin resistant and proinflammatory state), and if so, metformin, an antidiabetic drug, widely used in the treatment of women with PCOS, could affect circulating and AT cartonectin levels.
Materials and Methods Subjects Study A All women with PCOS met all three criteria of the revised 2003 Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine PCOS Consensus Workshop Group diagnostic criteria. The three criteria are as follows: 1) oligo- and/or anovulation, 2) clinical and/or biochemical signs of hyperandrogenism, and 3) polycystic ovaries (16). Exclusion criteria included age over 40 years; reproductive, cardiovascular, metabolic, renal, and endocrine disorders; neoplasms; and smoking. None of the subjects was on any regular medications for at least 6 months prior to the study. Subjects were initially seen at the infertility clinic and then scheduled for laparoscopy to assess Fallopian tube patency. All subjects underwent anthropometric measurements. After an overnight fast, blood samples, sc and omental AT were obtained (8:00 –10:00 AM) from adult female patients undergoing elective surgery for infertility investigation. Serum was immediately aliquoted on ice and stored at ⫺80°C. Subcutaneous AT was obtained from a 3-cm horizontal midline incision approximately 3 cm above the pubic symphysis. The same fat pad was divided equally into two halves. Each half was either immediately frozen in liquid nitrogen and stored at ⫺80°C or placed into a sterile container containing Medium 199 (Sigma-Aldrich) for primary adipose tissue culture. Samples that were snap frozen were transported on dry ice (⫺80°C) and stored at ⫺80°C in the labora-
J Clin Endocrinol Metab, December 2013, 98(12):E1891–E1900
tories of the University of Warwick. A total of 73 subjects were recruited consecutively from the infertility clinic in accordance with the inclusion/exclusion criteria (PCOS: n ⫽ 19; controls: n ⫽ 54). Of the 19 PCOS subjects recruited, five withdrew before the study could be completed. In the control group, seven subjects did not complete the study. From the remaining 47 control subjects, 14 control subjects comparable for age, body mass index (BMI), and waist to hip ratio (WHR) were included in the final analysis. 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.
Study B All women with PCOS met all three criteria of the revised 2003 Rotterdam European Society of Human Reproduction and Embryology/American Society for Reproductive Medicine PCOS Consensus Workshop Group diagnostic criteria. The three criteria are as follows: 1) oligo- and/or anovulation, 2) clinical and/or biochemical signs of hyperandrogenism, and 3) polycystic ovaries (16). Exclusion criteria included age over 40 years; reproductive, cardiovascular, metabolic, renal, and endocrine disorders; neoplasms; and smoking. None of the subjects was on any regular medications for at least 6 months prior to the study. Subjects were outpatients of the Department of Reproductive Medicine and Gynaecological Endocrinology of Magdeburg University and the Department of Obstetrics and Gynaecology of Martin-Luther-University Halle. The metabolic study was performed in the Outpatient Department of Endocrinology and Metabolism of Magdeburg University. A treatment with metformin in an off-label use was offered to all women with PCOS independently from the results of insulin sensitivity testing as per protocol. In those women with PCOS who agreed, therapy was initiated after basal assessment, and the dose of metformin was increased to a maintenance dose of 850 mg twice daily. All subjects underwent anthropometric measurements before and after metformin treatment. Blood pressure was measured in a sitting position within a quiet and calm environment after a rest of at least 5 minutes. The average of three measurements was obtained. Carotid intima-media thickness (IMT) was measured in women with PCOS before and after metformin dose (see Supplemental Data, published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Blood samples were collected between 8:00 and 9:00 AM after a 3-day normal carbohydrate diet and an overnight fast. Serum was immediately aliquoted on ice and stored at ⫺80°C. A total of 83 women with PCOS and 39 controls were studied. There were 49 women with PCOS who did not participate in the metformin arm of the study. The baseline characteristics of the 49 women with PCOS were comparable with the 34 women with PCOS who participated in the metformin arm of the study. Furthermore, the 21 subjects who completed the metformin arm of the study were comparable with the 13 women with PCOS who did not complete the metformin arm of the study. For the purposes of elucidating the effects of metformin in women with PCOS, the 21 women with PCOS were studied. Reasons for subjects not completing study B were nausea and gastrointestinal side effects (n ⫽ 4), pregnancies (n ⫽ 5), noncompliance (n ⫽ 2), and loss of contact (n ⫽ 2) [see Supplemental Data and Supplemental Figure 1]. The study design was ap-
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proved by the local Research Ethics Committee of the University of Magdeburg, and written informed consent was obtained from all participants, in accordance with the guidelines in The Declaration of Helsinki 2000.
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Total RNA extraction and cDNA synthesis Total RNA was extracted from AT samples and cDNA synthesized (see Supplemental Data).
Reverse transcription-polymerase chain reaction
Biochemical and hormonal analysis Assays were performed using an automated analyzer (see Supplemental Data). High-sensitivity C-reactive protein (hs-CRP) was determined immunoturbidimetrically using a modular system random-access analyzer (Roche Diagnostics). Circulating leptin was measured with a coated-tube immunoradiometric assay kit (Diagnostic Systems Laboratories), according to the manufacturer’s protocol. Serum TNF-␣ was measured using a commercially available ELISA kit from R&D Systems GmbH, according to the manufacturer’s protocol, with an intraassay coefficient of variation of less than 9%. Circulating leptin and adiponectin were measured with a coated-tube immunoradiometric assay kit (Diagnostic Systems Laboratories) and by a commercially available RIA kit (Millipore), according to the manufacturer’s protocol, with intraassay coefficients of variation of 3.2% and 3.4%, respectively. Serum visfatin was measured using a commercially available enzyme immunoassay kit (Phoenix Pharmaceuticals, Inc, Aviscera), according to the manufacturer’s protocol, with an intraassay coefficient of variation of less than 6%. Cartonectin levels in sera and conditioned media were measured using a commercially available ELISA kit (Aviscera), according to the manufacturer’s protocol, with an intraassay coefficient of variation of less than 10%.
Primary explant culture AT organ explants were cultured using a protocol that was a modification of the method described by Fried and MoustaidMoussa (17) (see Supplemental Data).
Quantitative PCR of cartonectin was performed on a Roche Light Cycler system (Roche Molecular Biochemicals) (see Supplemental Data). The sequences of the sense and antisense primers used were: cartonectin (191 bp) 5⬘-GAGTCTCCACAAACCGGAGG-3⬘ and 5⬘-TCACCTTTGTCGCCCTTCTC-3⬘; and -actin (216 bp) 5⬘-AAGAGAGGCATCCTCACCCT-3⬘ and 5⬘-TACATGGCTGGGGTCTTGAA-3⬘.
Western blotting Protein lysates were prepared and Western blotting performed (see Supplemental Data). We used antibodies for cartonectin (Abcam) and -actin (Cell Signaling Technology Inc).
Statistics Data were analyzed by a Mann-Whitney U test, Wilcoxon matched pairs test, Kruskal-Wallis ANOVA (post hoc analysis: Dunn’s test), and Friedman’s ANOVA (post hoc analysis: Dunn’s test), according to the number of groups compared. Data are medians (interquartile range). The Spearman rank correlation was used for the calculation of associations between variables. Subsequently, if individual bivariate correlations achieved statistical significance, variables were entered into a linear regression model and multiple regression analysis with cartonectin as a dependent variable was performed to test the joint effect of different parameters on cartonectin. All statistical analyses were performed using SPSS version 21.0 (SPSS, Inc). P ⬍ .05 was considered significant.
Table 1. Clinical, Hormonal, and Metabolic Features of Women With PCOS and Control Subjects (Study A) Variable
PCOS (n ⴝ 14)
Controls (n ⴝ 14)
Significance
Age, y BMI, kg/m2 WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L Triglycerides, mmol/L Prolactin, mU/L E2, pmol/L Progesterone, nmol/L 17-OH-P, nmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL Cartonectin, ng/mL
29.5 (28 –38) 30.5 (27.8 –30.9) 0.89 (0.78 – 0.99) 5.5 (4.8 – 6.0) 78.9 (42.0 –91.1) 3.0 (2.1–3.6) 5.1 (4.1–5.7) 2.0 (1.5–2.3) 348.0 (305.0 –387.0) 353.5 (287.0 – 471.0) 1.6 (1.3–2.1) 2.5 (2.1–2.8) 1.7 (1.5–2.1) 15.6 (14.2–16.8) 5.9 (5.4 – 6.6) 31.5 (26.7–35.2) 19.5 (14.5–21.2) 24.9 (19.7–29.0) 5.67 (4.52–7.50) 53.6 (41.0 – 63.1) 5.35 (2.19 – 8.42) 254.2 (193.1–359.6)
32.5 (29 –35) 28.8 (28 –30.5) 0.84 (0.81– 0.96) 4.5 (4.3–5.2) 57.3 (48.5– 66.0) 2.0 (1.4 –2.2) 5.0 (4.8 –5.5) 0.9 (0.7–1.4) 304.5 (211.0 –322.0) 174.5 (129.0 –264.0) 2.2 (1.7–2.3) 2.0 (1.2–2.3) 0.8 (0.6 – 0.9) 6.3 (5.0 – 8.2) 4.5 (4.0 –5.3) 59.0 (47.7– 66.0) 3.9 (3.3– 6.1) 24.1 (19.3–28.8) 6.27 (5.52–9.48) 44.2 (39.0 –50.2) 3.78 (3.19 –5.84) 351.4 (306.1–375.2)
NS NS NS P ⬍ .01 NS P ⬍ .05 NS P ⬍ .01 NS P ⬍ .01 NS NS P ⬍ .01 P ⬍ .01 P ⬍ .05 P ⬍ .01 P ⬍ .01 NS NS NS NS P ⬍ .05
Abbreviations: 17-OH-P, 17-hydroxyprogesterone; NS, not significant. FAI ⫽ T (nanomoles per liter)/SHBG (nanomoles per liter) ⫻ 100. Data are medians (interquartile range). Group comparison was done by a Mann-Whitney U test.
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Metformin Increases Cartonectin
Results Study A Demographic data Table 1 shows the anthropometric, biochemical, and hormonal data in all subjects. Glucose, homeostasis model assessment insulin resistance index (HOMA-IR), triglycerides, 17-estradiol (E2), T, androstenedione, dehydroepiandrosterone sulfate (DHEA-S) levels and free androgen index (FAI) were significantly higher whereas SHBG was significantly lower in women with PCOS. Serum cartonectin levels were significantly lower in women with
J Clin Endocrinol Metab, December 2013, 98(12):E1891–E1900
PCOS compared with control subjects [254.2 (193.1– 359.6) vs. 351.4 (306.1–375.2) ng/mL; P ⬍ .05: Table 1]. Serum progesterone levels in all subjects confirmed the follicular phase of the menstrual cycle. mRNA expression and protein levels of cartonectin in AT of normal and women with PCOS We detected cartonectin mRNA in AT of all subjects and subsequent sequencing of the PCR products confirmed gene identity. Real-time RT-PCR analysis corrected over -actin showed a significant decrease of cartonectin mRNA expression in omental (*, P ⬍ .05) AT of PCOS compared with control subjects (Figure 1A). However, no significant difference in cartonectin mRNA expression was observed when comparing mRNA expression in sc AT of PCOS with control subjects as well as corresponding omental to sc adipose tissue in PCOS and control subjects (Figure 1A). The changes noted at mRNA level were also reflected at protein level (Figure 1B). Western blot analysis of protein extracts from sc and omental AT demonstrate that the antibody against cartonectin and the antibody against -actin recognized bands with apparent molecular masses of 36 and 42 kDa, respectively.
Figure 1. A, Cartonectin mRNA expression relative to -actin was significantly lower in human omental (om) adipose tissue when comparing women with PCOS (n ⫽ 14) with control subjects (n ⫽ 14), using real-time RT-PCR. Data are the percentage difference of median of human sc adipose tissue of control subjects. Each experiment was carried out in three replicates. Group comparison was done by Kruskal-Wallis ANOVA and post hoc Dunn’s test. *, P ⬍ .05. B, Densitometric analysis of cartonectin immune complexes having normalized to -actin revealed that protein levels of cartonectin were significantly lower in human omental (om) adipose tissue depots when comparing all women with PCOS with all control subjects. Data are the percentage difference of median of human sc adipose tissue of control subjects. Each experiment was carried out in three replicates. Group comparison was done by Kruskal-Wallis ANOVA and post hoc Dunn’s test. *, P ⬍ .05. AU, arbitrary units. C, Dose-dependent effects of metformin (0.01, 0.1, and 2.00 g/mL) in the presence of 5 mmol/L D-glucose on CTRP3 protein production in control human omental AT explants at 24 hours was assessed by Western blotting. Densitometric analysis of CTRP3 immune complexes having normalized to -actin revealed that protein levels of CTRP3 were significantly increased by metformin (2.00 g/mL) in control human omental AT explants. Data are expressed as the percentage difference of median of basal (B). Each experiment was carried out with six different samples from six different control subjects in three replicates. Group comparison was done by Friedman’s ANOVA and post hoc Dunn’s test. *, P ⬍ .05. AU, arbitrary units. D, Dose-dependent effects of metformin (0.01, 0.1, and 2.00 g/mL) in the presence of 5 mmol/L D-glucose on CTRP3 secretion into conditioned media in control human omental AT explants at 24 hours was measured by ELISA. CTRP3 secretion into conditioned media was significantly increased by metformin (2.00 g/mL) in control human omental AT explants. Data are the percentage difference of the median of basal (B) [D-glucose (5 mmol/L)]. Each experiment was carried out with six different samples from six different control subjects in three replicates. Group comparison was done by Friedman’s ANOVA and post hoc Dunn’s test. *, P ⬍ .05.
Study B: effects of metformin treatment on serum cartonectin levels Table 2 shows the anthropometric, biochemical, and hormonal data in all subjects. Insulin, HOMA-IR, total cholesterol, low-density lipoprotein (LDL)-cholesterol, triglycerides, E2, T, androstenedione, FAI, hsCRP, systolic blood pressure (SBP), diastolic blood pressure (DBP), and IMT were significantly higher, whereas high-density lipoprotein (HDL)-cholesterol, DHEA-S, and SHBG were significantly lower in women with PCOS. Serum cartonectin levels were significantly lower in women with PCOS compared with control subjects [200.3 (158.1– 263.6) vs 330.7 (180.3– 436.6) ng/ mL; P ⬍ .01]. After 6 months of met-
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Table 2. Clinical, Hormonal, and Metabolic Features of Women With PCOS and Control Subjects (Study B) Variable
PCOS (n ⴝ 83)
Controls (n ⴝ 39)
Significance
Age, y BMI, kg/m2 WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L HDL-cholesterol, mmol/L LDL-cholesterol, mmol/L Triglycerides, mmol/L E2, pmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI hs-CRP, mg/L SBP, mm Hg DBP, mm Hg IMT, mm Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL Cartonectin (ng/ml)
27 (24.0 –31.0) 31.4 (26.1–34.2) 0.81 (0.77– 0.86) 4.9 (4.5–5.4) 69.0 (44.0 –100.0) 2.0 (1.3–2.9) 5.0 (4.5–5.6) 1.3 (1.1–1.5) 3.1 (2.6 –3.5) 1.1 (0.8 –1.9) 341.9 (200.7–538.4) 2.1 (1.5–2.7) 11.9 (8.4 –16.4) 4.7 (3.1–5.9) 30.0 (21.0 – 42.0) 6.5 (3.3–11.2) 2.8 (1.5– 4.6) 125.0 (120.0 –130.0) 75.0 (75.0 – 80.0) 0.46 (0.44 – 0.52) 21.9 (15.9 –28.5) 5.72 (4.69 –7.21) 46.3 (40.1–57.1) 3.93 (3.41–5.45) 200.3 (158.1–263.6)
28 (23–32) 29.1 (27.3–34.1) 0.81 (0.78 – 0.86) 4.8 (4.4 –5.1) 41.0 (32.0 –55.0) 1.2 (1.0 –1.7) 4.3 (3.8 – 4.8) 1.5 (1.3–1.7) 2.3 (1.9 –2.8) 0.8 (0.6 –1.1) 178.0 (118.9 –292.7) 1.2 (1.0 –1.6) 9.1 (6.6 –12.6) 6.1 (3.8 –7.5) 45.0 (34.0 –59.0) 3.0 (1.9 – 4.4) 1.3 (0.6 –2.3) 120.0 (110.0 –120.0) 75.0 (70.0 –77.5) 0.42 (0.40 – 0.44) 17.3 (10.8 –26.9) 5.84 (4.14 –7.90) 45.8 (40.2– 47.7) 3.73 (2.06 – 6.90) 330.7 (180.3– 436.6)
NS NS NS NS P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .05 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .01 P ⬍ .05 P ⬍ .01 NS NS NS NS P ⬍ .01
Abbreviation: NS, not significant. FAI ⫽ T (nanomoles per liter)/SHBG (nanomoles per liter) ⫻ 100. Data are medians (interquartile range). Group comparison was done by Mann-Whitney U test.
formin treatment (Table 3), there was a significant increase in serum cartonectin [212.9 (105.1–320.6) vs 272.0 (169.8 –386.5) ng/mL; P ⬍ .05]. Also, there were significant decreases in WHR, E2, T, glucose, HOMA-IR, and IMT. Association of cartonectin with covariates In study A, a Spearman rank analysis demonstrated that serum and omental AT cartonectin were significantly negatively associated with WHR, glucose, total cholesterol, and circulating triglycerides (P ⬍ .05). No significant associations were found with respect to sc AT. However, when subjected to multiple regression analysis, none of these variables were predictive of serum or omental AT cartonectin. In study B, a Spearman Rank analysis demonstrated that serum cartonectin was significantly negatively associated with BMI, insulin, LDL-cholesterol, triglycerides, hs-CRP and IMT (Table 4). When subjected to multiple regression analysis, triglycerides and hs-CRP were found to be predictive of serum cartonectin levels. We also analyzed the correlation between the change in serum cartonectin levels before and after metformin therapy (⌬cartonectin) and the changes (⌬) in other covariates (Table 5). We found that ⌬cartonectin was significantly negatively associated with ⌬WHR, ⌬glucose, ⌬LDL-cholesterol, ⌬triglycerides, ⌬hsCRP, and ⌬IMT. When subjected to multiple regression anal-
ysis, only ⌬hs-CRP was predictive of ⌬cartonectin serum levels ( ⫽ ⫺.550; P ⫽ .033). Dose-dependent effects of D-glucose, insulin, metformin, T, E2, androstenedione, and DHEA-S on cartonectin protein production and secretion into conditioned media in control human omental AT explants Given our observations above, we investigated the effects of D-glucose, insulin, metformin, and gonadal and adrenal steroids ex vivo. Cartonectin protein production and secretion into conditioned media were significantly increased by metformin in control human omental AT explants (Figure 1, C and D: *, P ⬍ .05). With respect to D-glucose, insulin, and gonadal and adrenal steroids, no significant effects on cartonectin protein production or secretion into conditioned media was found (data not shown).
Discussion We present novel data showing significantly lower serum and omental AT cartonectin in women with PCOS. Metformin therapy (850 mg twice daily for 6 months) significantly increased serum cartonectin concentrations. Furthermore, cartonectin protein production and secretion
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Metformin Increases Cartonectin
J Clin Endocrinol Metab, December 2013, 98(12):E1891–E1900
Table 3. Clinical, Hormonal, and Metabolic Features of Women With PCOS (n ⫽ 21) Before and After Metformin Treatment (Study B) Variable
Before Metformin
After Metformin
Significance
Age, y BMI, kg/m2 WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L HDL-cholesterol, mmol/L LDL-cholesterol, mmol/L Triglycerides, mmol/L E2, pmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI hs-CRP, mg/liter SBP, mm Hg DBP, mm Hg IMT, mm Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL Cartonectin, ng/mL
28 (26.5–31.5) 32.8 (29.8 –36.5) 0.82 (0.76 – 0.88) 5.1 (4.7–5.5) 70.0 (54.5–94.5) 2.2 (2.0 –3.0) 4.9 (4.1–5.3) 1.3 (1.1–1.5) 2.9 (2.2–3.3) 1.0 (0.8 –1.7) 329.8 (164.9 – 494.7) 1.8 (1.5–2.5) 10.5 (8.0 –13.3) 4.3 (2.7–5.6) 27.0 (20.5– 41.0) 6.6 (4.6 –9.7) 4.1 (2.4 –5.9) 125.0 (120.0 –130.0) 80.0 (75.0 – 80.0) 0.53 (0.48 – 0.58) 26.5 (20.7–30.9) 4.85 (4.14 – 6.88) 51.8 (42.3– 60.4) 4.08 (3.52–5.45) 212.9 (105.1–320.6)
28 (27.5–32.5) 31.4 (28.2–35.1) 0.80 (0.74 – 0.87) 4.8 (4.4 – 4.9) 61.0 (43.5– 83.0) 1.6 (1.3–2.2) 5.0 (4.1–5.4) 1.2 (1.1–1.5) 3.0 (2.5–3.3) 1.2 (1.0 –1.9) 207.1 (103.6 –310.7) 1.3 (1.1–1.8) 9.8 (7.7–12.6) 5.4 (3.6 – 6.7) 29.0 (21.5– 46.5) 5.3 (3.1– 6.6) 3.1 (2.1–7.2) 120.0 (120.0 –127.5) 75.0 (75.0 – 80.0) 0.47 (0.41– 0.53) 25.1 (20.0 –30.0) 3.69 (2.91–5.55) 51.0 (40.8 –58.3) 4.15 (3.18 –5.07) 272.0 (169.8 –386.5)
NS NS P ⬍ .05 P ⬍ .05 NS P ⬍ .05 NS NS NS NS P ⬍ .05 P ⬍ .05 NS NS NS NS NS NS NS P ⬍ .05 NS NS NS NS P ⬍ .05
Abbreviation: NS, not significant. FAI ⫽ T (nanomoles per liter)/SHBG (nanomoles per liter) ⫻ 100. Data are medians (interquartile range). Group comparison was done by a Wilcoxon matched-pairs test.
into conditioned media were significantly increased by metformin in omental AT explants. Moreover, a Spearman rank analysis demonstrated that serum cartonectin was significantly negatively associated with BMI, WHR, insulin, LDL-cholesterol, total cholesterol, triglycerides, hs-CRP, and IMT. When subjected to multiple regression analysis, only triglycerides and hs-CRP were found to be predictive of serum cartonectin levels. Finally, changes in cartonectin were significantly negatively associated with changes in WHR, glucose, LDL-cholesterol, triglycerides, hs-CRP, and IMT. When subjected to multiple regression analysis, only changes in hs-CRP were predictive of changes in serum cartonectin. The lower serum and omental AT cartonectin levels in women with PCOS is perplexing, given that it has recently been reported that plasma cartonectin was significantly higher in patients with type 2 diabetes and significant positive associations were found between plasma cartonectin and cardiometabolic risk factors ie, WHR, plasma glucose, lipids, and hs-CRP (18). Interestingly, the same researchers subsequently reported that serum cartonectin was not significantly lower in subjects with the metabolic syndrome compared with controls and that serum cartonectin was significantly negatively associated with waist circumference, DBP, serum glucose, cholesterol, and triglycerides (19). Additionally, in the present study, treat-
ment of human omental AT explants with D-glucose or insulin did not significantly alter cartonectin production in AT. Therefore, it would appear that other mechanisms are involved in the regulation of cartonectin levels in AT. Of relevance, Kopp et al (13) reported that cartonectin reduced lipopolysaccharide induced production of macrophage migration inhibitor factor in nondiabetic controls, with no apparent effects in subjects with type 2 diabetes. Another explanation of elevated cartonectin levels in type 2 diabetes observed by Choi et al (18) could be the effect of medications taken by their study subjects as discussed in their following manuscript (19). In addition, the clinical human studies mentioned above involved only East Asian subjects, whereas our study comprised solely Caucasian subjects. There could be differences in cartonectin levels between different ethnic groups. Finally, there may be other yet-undetermined factors that may account for the significantly higher circulating cartonectin levels found in type 2 diabetes patients. In our studies, we found significant negative associations between serum and omental AT cartonectin levels with cardiometabolic risk factors. Notably, in study B, serum cartonectin levels were significantly negatively associated with BMI, insulin, LDL-cholesterol, triglycerides, hs-CRP, and IMT. Importantly, multiple regression analyses revealed that serum triglycerides and hs-CRP were
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Table 4. Linear Regression Analysis of Variables Associated With Serum Cartonectin Levels in All Subjects (n ⫽ 122) (Study B) Simple Variable
Estimate 2
BMI, kg/m WHR Glucose, mmol/L Insulin, pmol/L HOMA-IR Total cholesterol, mmol/L HDL-cholesterol, mmol/L LDL-cholesterol, mmol/L Triglycerides, mmol/L E2, pmol/L T, nmol/L Androstenedione, nmol/L DHEA-S, mol/L SHBG, nmol/L FAI hs-CRP, mg/L SBP, mm Hg DBP, mm Hg IMT, mm Leptin, ng/mL Adiponectin, g/mL Visfatin, ng/mL TNF-␣, pg/mL
⫺0.302 ⫺0.013 ⫺0.010 ⫺0.286 ⫺0.107 ⫺0.099 0.127 ⫺0.270 ⫺0.417 ⫺0.122 ⫺0.183 ⫺0.132 0.252 0.147 ⫺0.212 ⫺0.389 ⫺0.012 0.009 ⫺0.259 ⫺0.175 0.138 ⫺0.178 ⫺0.088
Multiple P Value a
.019 .922 .937 .027a .415 .454 .333 .037a .002b .353 .161 .313 .052 .263 .104 .001b .928 .948 .046a .182 .292 .279 .593
Estimate
P Value
0.051
.775
0.112
.502
⫺0.171 ⫺0.322
.256 .040a
⫺0.336
.031a
0.134
.431
In multiple linear regression analysis, values included were BMI, insulin, LDL-cholesterol, triglycerides, hs-CRP, and IMT. a
P ⬍ .05.
b
P ⬍ .01.
predictive of serum cartonectin. Thus, our findings highlight an important connection between circulating cartonectin and the proinflammatory dysmetabolic state present in overweight, insulin-resistant women with PCOS (4). Further studies are needed to clarify the underlying mechanisms that regulate cartonectin levels. In the seminal report of Peterson et al (11), which first described cartonectin as a novel adipokine with glucoselowering effects, the investigators had also observed a profound inverse correlation between leptin and cartonectin (11). In our studies, there were no significant differences in leptin and no significant associations between leptin and cartonectin. Thus, it is unlikely that leptin may account for our observations. Recent studies have reported a sexual dimorphism in circulating cartonectin, with significantly higher cartonectin in females (18, 21). To the best of our knowledge, there are no data on the regulation and effect of steroids on cartonectin. In our studies, both gonadal and adrenal steroids were altered in women with PCOS. However, there were no significant associations between gonadal and/or adrenal steroids and cartonectin. In addition, we found that treatment of human omental AT explants with gonadal or adrenal steroids did not significantly alter cartonectin production in AT. It therefore remains to be clar-
ified as to whether the differences in cartonectin levels are attributable to altered gonadal and/or adrenal steroids. Further studies are needed to elucidate the role of the effects of gonadal and adrenal steroids as well as other factors that regulate cartonectin production. In a recent publication, Schmid et al (22) had emphasized the important role of cartonectin in adipocyte biology, ie, lipolysis and adipokine secretion. Of relevance, catecholamine-induced lipolysis is elevated in visceral adipocytes of women with PCOS (23). This may lead to increased portal fatty acid levels culminating in liver dysfunction causing glucose intolerance, hyperinsulinemia, and dyslipidemia, metabolic aberrations that are often found in women with PCOS (1, 2). The factors driving increased catecholamine-induced lipolysis in visceral adipocytes of women with PCOS remain unexplained (23). We hypothesize that cartonectin may play a pivotal role in this context. Future studies are needed to elucidate this point. To the best of our knowledge, the effects of metformin treatment on cartonectin levels have not been investigated in any human/animal studies. We report for the first time that metformin (6 months treatment; 850 mg twice daily) is associated with a significant increase in serum cartonectin and a parallel decrease in insulin resistance in women
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Metformin Increases Cartonectin
J Clin Endocrinol Metab, December 2013, 98(12):E1891–E1900
Table 5. Linear Regression Analysis of Variables Associated With Changes in Serum Cartonectin Levels (Before and After Metformin Treatment), ⌬Cartonectin, in Women With PCOS (n ⫽ 21) Simple Variable ⌬BMI, kg/m ⌬WHR ⌬Glucose, mmol/L ⌬Insulin, pmol/L ⌬HOMA-IR ⌬Total cholesterol, mmol/L ⌬HDL-cholesterol, mmol/L ⌬LDL-cholesterol, mmol/L ⌬Triglycerides, mmol/L ⌬E2, pmol/L ⌬T, nmol/L ⌬Androstenedione, nmol/L ⌬DHEA-S, mol/L ⌬SHBG, nmol/L ⌬FAI ⌬hs-CRP, mg/L ⌬SBP, mm Hg ⌬DBP, mm Hg ⌬IMT, mm ⌬Leptin, ng/mL ⌬Adiponectin, g/mL ⌬Visfatin, ng/mL ⌬TNF-␣, pg/mL 2
Multiple
Estimate
P
⫺0.120 ⫺0.476 ⫺0.447 0.131 0.046 ⫺0.074 ⫺0.003 ⫺0.460 ⫺0.498 ⫺0.160 ⫺0.152 ⫺0.107 ⫺0.241 0.322 ⫺0.401 ⫺0.675 0.398 ⫺0.176 ⫺0.450 0.013 0.214 ⫺0.230 ⫺0.248
.605 .029a .042a .570 .845 .749 .989 .036a .022a .489 .511 .645 .294 .155 .072 .001b .074 .447 .041a .955 .101 .077 .057
Estimate
P
⫺0.263 0.266
.536 .503
⫺0.231 ⫺0.388
.539 .207
⫺0.550
.033a
0.216
.624
In multiple linear regression analysis, values included were ⌬WHR, ⌬glucose, ⌬LDL-cholesterol, ⌬triglycerides, ⌬hs-CRP, and ⌬IMT. a
P ⬍ .05.
b
P ⬍ .01.
with PCOS. More importantly, we observed that cartonectin production was significantly increased by metformin in human omental AT explants. The profound effect of metformin on serum cartonectin levels could be due to its suppressive effect on hepatic glucose production (24 –26). Of relevance, cartonectin has been shown to have glucose-lowering effects through suppression of hepatic gluconeogenesis (11). However, as mentioned above, we did not find a significant difference in AT cartonectin production after D-glucose treatment. Lihn et al (27) and Sell et al (28) had reported that stimulating the AMP-activated protein kinase signaling pathway promotes adiponectin (the foremost adipokine of the C1q complement/TNF-related protein superfamily) expression in human adipose tissue. Because metformin mainly acts through the AMP-activated protein kinase pathway, this could be the mechanism wherein metformin increases cartonectin production and secretion in AT. Our results may account for the elevated cartonectin levels in type 2 diabetes observed by Choi et al (18) as discussed above and highlights metformin therapy as a confounding factor with regard to the regulation of circulating cartonectin levels. This should alert investigators who are studying cartonectin biology to consider this in their analysis. This may also apply to other forms of an-
tidiabetic therapy, and hence, caution needs to be exercised appropriately. In addition, we observed a significant decrease in IMT after metformin treatment in keeping with a recent report by Palomba et al (29). Furthermore, although the change in serum cartonectin was significantly negatively associated with changes in WHR, glucose, LDL-cholesterol, triglyceride, hs-CRP, and IMT, only hsCRP was predictive of the change in serum cartonectin associated with metformin therapy. This suggests that changes in serum cartonectin after metformin treatment observed in our PCOS subjects are more robustly linked to changes in inflammatory rather than metabolic parameters. As such, numerous studies have championed the antiinflammatory action of cartonectin (12–14). Therefore, the elevated serum cartonectin associated with metformin treatment may be an important mechanism through which metformin exerts its antiinflammatory effects (20). Further studies are needed to clarify this concept. In study A, a limitation may relate to the number of subjects studied and hence we would advise caution with regard to our findings; further studies with larger number of study subjects are needed to reaffirm our results. However, obtaining BMI/WHR comparable and menstrual cycle-synchronized blood and AT samples from two AT depots impeded subject recruitment. Nevertheless, our
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doi: 10.1210/jc.2013-2227
observations are highly consistent and significant and raise interesting questions on the mechanisms regulating cartonectin production. In study B, given ethical constraints, ie, blood samples, could and were obtained only at baseline and after 6 months of metformin treatment. It is therefore difficult to be certain as to whether changes in serum cartonectin precede or follow changes in clinical and hormonal indices as a consequence of metformin treatment. Moreover, because diet and lifestyle modifications were only subjectively assessed, the changes in serum cartonectin may be partially attributable to diet and lifestyle modifications. Further studies are needed to elucidate this point. Given the paucity of research into cartonectin, it is still not entirely clear as to how this novel adipokine fits into the homeostatic mechanisms that modulate the various functions of the human body in both health and disease, in particular, with respect to the metabolic syndrome and its associated complications such as type 2 diabetes. The current lack of a definitive receptor for cartonectin and an understanding of its molecular and cellular dynamics have only made the task of understanding the physiological actions of cartonectin more difficult and challenging. Given that cartonectin is by and large expressed in AT stromal vascular cells (13), its well-documented antiinflammatory actions (12–14) as well as the novel data presented in this manuscript, we postulate that cartonectin, potentially, may have a predominant role as an antiinflammatory molecule and that its cardiometabolic actions plausibly are intertwined in the most part via inflammatory pathways. In conclusion, circulating and omental AT cartonectin levels are significantly lower in insulin resistant women with PCOS compared with non-insulin-resistant subjects. Metformin treatment significantly increases circulating cartonectin and it appears that it does this (at least in part) by stimulating cartonectin production and secretion from AT. Because circulating cartonectin is significantly correlated with various cardiometabolic factors, cartonectin may be a useful biomarker of cardiometabolic disease in humans.
Acknowledgments Address all correspondence and requests for reprints to: Dr Bee K. Tan and Dr Harpal S. Randeva, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom. E-mail: [email protected]; or [email protected]. This work was supported by the General Charities of the City of Coventry. Disclosure Summary: The authors have nothing to declare.
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