Regulation of Adiponectin Secretion by Adipocytesin the Polycystic Ovary Syndrome: Role of TumorNecrosis Factor-�

Gregorio Chazenbalk, Bradley S. Trivax, Bulent O. Yildiz, Cristina Bertolotto,Ruchi Mathur, Saleh Heneidi, and Ricardo Azziz

Departments of Obstetrics/Gynecology and Center for Androgen Related Disorders (G.C., B.S.T., R.M.,R.A.) and Pediatrics (C.B., S.H.), Cedars-Sinai Medical Center, and Departments of Obstetrics/Gynecology and Medicine (G.C., R.M., R.A.) and Pediatrics (C.B.), David Geffen School of Medicine,University of California, Los Angeles, Los Angeles, California 90048; and Department of InternalMedicine (B.O.Y.), Endocrinology and Metabolism Unit, Hacettepe University School of Medicine,Hacettepe, 06100 Ankara, Turkey

Context: Adipose tissue dysfunction associated with low-grade chronic inflammation and dys-regulation of adipokine secretion might significantly contribute to the pathogenesis of polycysticovary syndrome (PCOS).

Objective: The objective of the study was to determine whether the effect of TNF-�, IL-6, monocytechemoattractant protein-1, or coculture of adipocytes and adipose tissue macrophages (ATMs), onthe secretion of adiponectin by adipocytes, differs in PCOS compared with controls.

Design and Participants: Primary cultures of sc adipocytes and coculture of adipocytes and ATMsfrom overweight and obese patients with PCOS and healthy control women were used.

Main Outcome Measures: Adiponectin secretion by adipocytes was measured.

Results: The baseline secretion of adiponectin by isolated adipocytes did not differ between PCOSand control samples. The net change in adiponectin secretion in response to IL-6, monocyte che-moattractant protein-1, and TNF-� differed between PCOS (decreasing) and control (increasing)adipocytes, although the difference reached significance only for TNF-� (P � 0.04). Coculture ofisolated adipocytes and ATMs resulted in a decrease in adiponectin secretion by PCOS (P � 0.05)but not control adipocytes, and the difference between the net change in adiponectin secretionin PCOS vs. control samples was significant (P � 0.03).

Conclusions: Our results suggest that adiponectin secretion by adipocytes in response to cytokines/chemokines and most notably in response to coculturing with ATMs differs between PCOS andcontrol women, favoring greater suppression of adiponectin in PCOS. The mechanisms underlyingthese defects and the role of concurrent obesity remain to be determined. (J Clin Endocrinol Metab95: 935–942, 2010)

Polycystic ovary syndrome (PCOS) is a common en-docrine-metabolic-reproductive disorder, affect-

ing about 6–10% of reproductive-aged women (1). Insu-lin resistance, and the development of compensatoryhyperinsulinemia, is a frequent finding in PCOS (2–5).

The resulting compensatory hyperinsulinemia stimu-lates androgen secretion from the ovarian theca cells,acting synergistically with LH (6, 7) and inhibits the he-patic production of SHBG (8, 9), accounting for much ofthe hyperandrogenic features and ovulatory dysfunction

ISSN Print 0021-972X ISSN Online 1945-7197Printed in U.S.A.Copyright © 2010 by The Endocrine Societydoi: 10.1210/jc.2009-1158 Received June 1, 2009. Accepted November 30, 2009.First Published Online January 20, 2010

Abbreviations: ATM, Adipose tissue macrophage; BMI, body mass index; DAPI, 4�,6�-diamino-2-phenylindole; DHEAS, dehydroepiandrosterone and sulfate; MCP, monocytechemoattractant protein; mFG, modified Ferriman-Gallwey; PCOS, polycystic ovary syn-drome; T, testosterone.

O R I G I N A L A R T I C L E

E n d o c r i n e R e s e a r c h

J Clin Endocrinol Metab, February 2010, 95(2):935–942 jcem.endojournals.org 935

of these patients. Likewise, evidence of subclinical inflam-mation is present in PCOS, regardless of the degree ofobesity (10–12). These features result in an increased riskfor developing type 2 diabetes (13) and cardiovasculardisease (14) in PCOS.

Insulin resistance in PCOS is in part due to the highprevalence of obesity in the disorder. Nonetheless, weshould note that approximately 40% of PCOS women,even in the United States, are not obese (1), that obesity inPCOS appears to primarily reflect environmental factorsand to have a modest impact on the prevalence of thedisorder (15), and that PCOS women are insulin resistantabove and beyond that determined by their body massindex (BMI) (5). However, whereas obesity per se may notbe the primary driver of PCOS, it is possible that adiposetissue dysfunction may play a role in the insulin resis-tance and the subclinical inflammation and conse-quently the metabolic and cardiovascular consequencesof the disorder.

Adipose tissue is an endocrinologically active organproducing a number of peptides, notably adipokines(which includes cytokines, chemokines, growth factors,and neurally active hormones among others), lipids, andsteroids (16–19). Adipokines act in an autocrine and para-crine fashion on adipose tissue itself and in an endocrinemanner to affect other tissues and organs, includingskeletal muscle, adrenal cortex, and the central andsympathetic nervous systems (19), modulating a num-ber of functions, including insulin action and subclinicalinflammation (20–22). Overall, adipose tissue, throughits endocrine role, is an important determinant of totalbody in vivo insulin sensitivity (18, 19).

Some adipokines are synthesized only by adipocytes, in-cluding the antiinflammatory molecule adiponectin. Adi-ponectin demonstrates insulin-sensitizing and antiin-flammatory properties, and dysregulation of adiponectinhas been implicated in the pathogenesis of obesity-relatedinsulin resistance and increased subclinical inflammation(23). Other adipokines, such as TNF-�, are mainly secretedby adipose tissue macrophages (ATMs), whereas IL-6 andmonocyte chemoattractant protein (MCP)-1 are producedby both adipocytes and ATMs (19, 22, 24); TNF-� andMCP-1 have proinflammatory properties that lead insulinresistance in adipocytes (25, 26), whereas IL-6 may havedual properties (27–31).

A recent metaanalysis of 16 studies in PCOS womenreported that circulating adiponectin levels are lower inPCOS than controls after controlling for BMI, albeit withsignificant heterogeneity across individual studies (32).Furthermore, adiponectin mRNA levels appear to be de-creased in both visceral and sc fat in PCOS women com-pared with weight-matched controls (33), although Fain

et al. (34) noted that adiponectin is mainly produced in sc,as opposed to visceral, fat. Adiponectin secretion in PCOSis potentially regulated by several mechanisms, includingthe paracrine effects of adipokines and direct interactionsbetween adipocytes and ATMs (35). In the present study,we aimed to determine in PCOS and matched controlwomen: 1) the effects of individual adipokines (TNF-�,IL-6, and MCP-1) on adiponectin secretion by adipo-cytes; and 2) the direct effect of ATMs on the secretionof adiponectin by adipocytes.

Subjects and Methods

Study subjectsTen patients with PCOS (BMI range 26–40 kg/m2) were re-

cruited from the Center for Fertility Reproductive Medicine andthe Center for Androgen-Related Disorders at Cedars-SinaiMedical Center. Twelve healthy women were recruited asmatched controls (BMI range 26–40 kg/m2). The diagnosis ofPCOS was made according to the National Institutes of Health1990 criteria (1). These include: 1) clinical evidence of hyperan-drogenism and/or hyperandrogenemia; 2) oligoovulation; and 3)the exclusion of related disorders, including nonclassic 21-hy-droxylase-deficient adrenal hyperplasia, hyperprolactinemia,thyroid dysfunction, Cushing’s syndrome, or androgen-produc-ing tumors (1). The criteria for defining hirsutism, hyperandro-genemia, ovulatory dysfunction, and exclusion of related disor-ders have been previously reported (36). Controls were womenwith regular menstrual cycles and without family history of en-docrine abnormality or hirsutism. These women had no evidenceof hirsutism, acne, or alopecia or endocrine dysfunction.

All subjects underwent a brief physical examination, in-cluding hirsutism scoring using a modification of the modifiedFerriman-Gallwey method (mFG) (37). Subjects were deemedhirsute if their mFG score was 6 or greater (38); controls had anmFG score of 3 or less. Blood sampling was performed in thefasting state between d 3 and 8 (follicular phase) after a spon-taneous menstrual cycle or a progesterone-induced withdrawalbleed. No subjects had used hormonal preparations, includingoral contraceptives, for 3 or more months preceding the study,and none were pregnant. All had normal TSH and prolactinlevels. All subjects gave informed written consent, according tothe guidelines of the Cedars-Sinai Medical Center InstitutionalReview Board.

Hormonal analysesHormonal measures, including total and free testosterone

(T), dehydroepiandrosterone and sulfate (DHEAS) were ob-tained. Total T was measured using high-turbulence liquid chro-matography tandem mass spectrometry and free T determinedby equilibrium dialysis (Quest Diagnostics, San Juan Capistrano,CA). Insulin was assayed by chemiluminescence (ADVIA Cen-taur chemiluminescent immunoassay system; Siemens Health-care, Deerfield, IN). DHEAS analysis was performed by a com-petitive immunoassay (Modular E170; Roche Diagnostics,Indianapolis, IN). Glucose levels were measured using the hex-okinase/glucose-6-phosphate dehydrogenase method (RocheApplied Sciences, Indianapolis, IN).

936 Chazenbalk et al. Adiponectin Secretion by Adipocytes in PCOS J Clin Endocrinol Metab, February 2010, 95(2):935–942

Isolation of human adipocytesAdipose tissue samples (�5 gm) were obtained from sc adi-

pose tissue through a small incision in the lower abdomen. Spec-imens were transported immediately to the laboratory in aHEPES salts buffer containing 4% BSA and 2 mM pyruvate (pH7.4) and were finely minced. A small amount of tissue was fixedin 4% PBS-buffered paraformaldehyde for immunofluorescence(see below), and the remainder was used for adipocyte and mac-rophage isolation. The minced adipose tissue was treated withcollagenase at a ratio of 3.5 mg/g of tissue and incubated for 60min at 37 C in a rotary shaking bath at 100 rpm. The cell sus-pension was then filtered through a premoistened 400-�m nylonmesh (Small Parts, Inc., Miami Lakes, FL) to isolate the adipo-cytes, and the cells were washed twice for 2 min at 50 � g at roomtemperature. After the second wash, the cells were refilteredthrough nylon mesh. At this point the cells were ready forexperimentation.

To confirm the purity of the adipocyte isolates, Oil Red Ostaining, nuclear 4�,6�-diamidino-2-phenylindole (DAPI) label-ing, and immunostaining with an antibody to the macrophagemarker CD14 were performed. Untreated adipocytes were fixedin 4% paraformaldehyde, rinsed twice for 10 min in PBS, andthen stained with 0.35% Oil Red O (Sigma Aldrich, St. Louis,MO) in a 3:2 isopropanol-water solution for 10 min and rinsedin PBS. Adipocytes (positive Oil Red O and DAPI staining) wereclearly distinguishable from free lipid (positive Oil Red O andnegative DAPI staining). Little free lipid remained in the adipo-

cyte isolates (Fig. 1A). Furthermore, immuno-staining of the isolates with a CD14 antibody dem-onstrated that the adipocyte fraction was freefrom contamination with macrophages (Fig. 1C).

Adipocyte culture and determination ofsecreted adiponectin levels

Isolated adipocytes (107 cell/ml) were main-tained in 24-well clusters in the presence of DMEMcontaining 10% fetal calf serum and antibiotics andincubated at 37 C in humidified 5% CO2-95% airatmosphere for 16 h. For all assays, duplicate wellswere plated for each sample and assay condition.Cells were treated with adipokines or left untreatedas appropriate, and 48 h after treatment, the cellculture media were collected for the detection of adi-ponectin. The concentrations of adiponectin (pico-grams per milliliter) in the culture media were de-termined using a solid phase ELISA (Dousset; R&DSystems, Minneapolis, MN). Sample concentrationswere determined using an ELISA reader (Sunrise,T-can, Vmax kinetic microplate reader; MolecularDevices, Sunnyvale, CA). Duplicates were preparedand read for every sample. Sample concentrationswere interpolated from a standard curve calculatedby linear regression of the color development ofknown concentrations of known recombinant hu-man standards (ranging from 62.5 to 4000 pg/ml,prepared fresh for each assay). Initially, three differ-ent sample dilutions were made and each dilutionwas assayed in duplicate. The sample concentrationswere then compared with the standard curve, andthe results of the dilution with values closest to themiddle (straightest) portion of the control curvewere used. Log transformation of the adiponectin

concentrations was used to facilitate the use of parametric dataanalysis methods.

To validate the ELISA itself, intra- and interassay compari-sons were performed. Intraassay percent coefficients of varia-tion, obtained by measuring three different samples 30 timeseach, ranged from 4.2 to 5.8%. Interassay percent coefficients ofvariation, obtained by measuring three different samples in 30different experiments, ranged from 4.7 to 5.7%. Assay specific-ity was tested using three different human recombinant adipo-kines (C-reactive protein, IL-6, MCP-1) at concentrations rang-ing from 50 to 50,000 pg/ml, all of which failed to yield positivemeasurements for adiponectin. We determined the linearity ofthe assay by diluting human recombinant adiponectin (Dousset;R&D Systems) in either assay dilution buffer �PBS containing1% BSA (Sigma) or adipocyte culture medium (see above)�. Thelinearity of standard curves obtained using dilution buffer andconditioned medium were very similar (y � 0.0028 � �0.0166,r2 � 0.999; and y � 0.0028 � �0.0156, r2 � 0.999, respec-tively). In addition, we determined the percentage of recoveryby measuring known concentrations of recombinant adi-ponectin (ranging from 62.5 to 4000 pg/ml) by ELISA. Theadiponectin concentrations detected by ELISA were approx-imately the same as those of the standard curve at each dilu-tion, with a range of recovery between 91 and 106 (n � 6)using either dilution buffer or conditioned medium (data notshown).

FIG. 1. Characterization of isolated human adipose tissue adipocytes andmacrophages. Untreated adipocytes from cell culture were fixed in 4%paraformaldehyde, and Oil Red O staining and nuclear DAPI labeling wereperformed to distinguish adipocytes from free lipid (A). Immunofluorescent labelingwith an antibody to CD14 was performed to test for the presence of macrophages(C). Oil Red O (red) and DAPI (light blue) colabeling demonstrate that the isolatedcells are mature adipocytes (�100 magnification) (A), and lack of CD14 stainingindicates that the isolated adipocytes fraction is free from adipocytes (C). ATMsisolated from adipose tissue were subjected to immunofluorescent labeling withantibodies to CD14 (green) (B) and S-100 (D) and mounted using DAPI after 3 d inculture. The cells are CD14(�), consistent with the identification of these cells asmacrophages (B), and S-100(), indicating that the ATM fraction is free fromadipocytes (�200) (D).

J Clin Endocrinol Metab, February 2010, 95(2):935–942 jcem.endojournals.org 937

Incubation of adipocytes in the presence orabsence of adipokines

To identify the optimal concentrations of adipokines to use,TNF-� or MCP-1 was added to adipocyte cell culture medium atconcentrations ranging from 0.1, 1.0, 10, and 100 ng/ml, andafter 48 h the medium was harvested for the detection of adi-ponectin by ELISA as described above. Pair-wise comparisons ofthe results were performed under an ANOVA model.

Isolation of human ATMsATMs were partially purified from the resuspension of the

stromal pellet fraction obtained during adipocyte isolation asdescribed above, using a Ficoll gradient. This ATM fraction wasthen diluted in RPMI 1640 medium supplemented with 10%fetal calf serum, 100 U/ml penicillin, 100 �g/ml streptomycin, 2mM L-glutamine, 1% non-essential amino acids, 1% sodiumpyruvate, and 10 ng/ml granulocyte macrophage-stimulatingfactor, and kept in culture for up to 96 h. Cells in the macrophagefraction expressed CD14, also labeled with DAPI (Fig. 1B), andalso expressed CD68, another macrophage marker (data notshown). To confirm that the ATM fraction was free fromcontamination with adipocytes, immunostaining with S-100,a marker for preadipocytes and newly formed adipocytes (39,40), was performed. The results indicated that the isolatedmacrophage fraction was free from contaminating adipocytes(Fig. 1D).

Coculturing of adipocytes and ATMsTo determine the role that ATMs play in regulating adiponec-

tin secretion from adipocytes, we devised a coculture system (Fig.2). Isolated adipocytes and ATMs (see above) were allowed toequilibrate overnight in their respective cell culture media. Thefollowing day, adipocytes were added to the wells containingATMs in a 1:1 ratio. Again, duplicate wells were plated for eachsample and assay condition. The cells were then cocultured for24 h in a coculture plate (Corning Costar Co., Lowell, MA). Thisis a direct coculture system that allows contact between the celltypes. After 24 h of coculture, the adipocytes were removed andplaced in new wells containing adipocyte medium, and the adi-pocytes were cultured in isolation again for an additional 48 h.At the end of this 48-h time period, the media from the adipocyteswas collected for measurement of adiponectin levels. We inves-tigated the effects of different ATM to adipocyte ratios in ourcoculture studies (e.g. 1:1, 1:3, 1:6) and found that a 1:1 ratio ofadipocytes and ATMs maximized the production of adiponectin(data not shown).

Statistical analysisStatistical analysis was performed using the Statistical Anal-

ysis System program (SAS Institute, Inc., Cary, NC). All valueswere log transformed to achieve a more normal distribution. Forthe dose-response curves performed for TNF-� and MCP-1,pair-wise comparisons were made using an ANOVA model. Formeasurements of the effects of individual adipokines or coculturewith ATMS on adiponectin secretion, comparisons were carriedout parametrically using t tests. We chose to use log scale analysisfollowed by t tests, rather than nonparametric analysis becausethe SDs increase systematically as the means increase on the orig-inal scale, which is evidence that a log transformation is needed.Residual quantile-quantile plots confirmed that there were nomajor violations of the parametric normality assumptions. Dueto the small amount of adipose tissue isolated from each subject,not all of the subjects contributed to each of the experimentsperformed. The number of samples per group used for each ex-periment is indicated in the figures and figure legends. Insulinresistance and �-cell function were estimated using the ho-meostasis model assessment (41).

FIG. 2. Graphical depiction of the adipocyte-ATM coculture systemand coculture of adipocytes and ATMs. Adipocytes and ATMs wereisolated from adipose tissue as described in Materials and Methods.After a 16-h equilibration period, the adipocytes and ATMs arecocultured with direct contact between the cell types for 24 h. Theadipocytes are then removed and cultured separately for anadditional 48 h, after which the culture media were harvested foranalysis by ELISA.

TABLE 1. Baseline data comparing controls vs. PCOS

Controls(n � 12)

PCOS(n � 10)

Pvalue

Age (yr) 35.8 7.1 27.7 5.6 0.013BMI (kg/m2) 30.5 5.1 33.5 3.2 NSmFG hirsutism score 1.5 1.8 7.9 3.8 0.001Waist to hip ratio 0.8 0.1 0.9 0.1 0.018Total T (ng/ml) 19.6 5.7 49.1 40.1 NSFree T (pg/ml) 2.5 1.5 5.9 2.9 0.013DHEAS (�g/dl) 198.7 91.1 253.0 97.0 NSFasting insulin (�IU/ml) 12.3 10.1 36.6 40.6 NSFasting glucose (mg/dl) 85.0 8.2 89.1 11.3 NSHOMA-IR 2.2 1.5 7.6 9.5 NSHOMA-%�-cell 165 55 600 629 NS

Values expressed as mean SD. HOMA-IR and HOMA-%�-cell arecalculations of insulin resistance and �-cell function estimated by thehomeostasis model assessment, respectively. HOMA-IR, Homeostasismodel assessment insulin resistance index; HOMA-%� -cell, homeostasismodel of percent � -cell; NS, P � 0.05. NS, No significant difference.

938 Chazenbalk et al. Adiponectin Secretion by Adipocytes in PCOS J Clin Endocrinol Metab, February 2010, 95(2):935–942

Results

Hormonal and biochemical features of the PCOSpatients

Of the 12 control women studied, 70% were white,whereas 80% of the 10 PCOS subjects were white. Therewere no significant differences in BMI between PCOS andcontrols (Table 1), although controls were slightly olderthan PCOS women. As expected, waist to hip ratio, mFGscore, and free T were greater in PCOS women; althoughfasting insulin, homeostasis model assessment insulin re-sistance index, and homeostasis model assessment of per-cent �-cell function tended to be higher in PCOS womenthan controls (Table 1), these differences did not reachsignificance, likely a reflection of sample size.

Adipose tissue contains resident or ATMsTo identify cell types present in human adipose tissue,

we performed immunofluorescent staining with specificantibodies. We demonstrated the presence of preadipo-cytes and adipocytes, identified by positive staining forS-100, a marker found in preadipocytes and recently ma-tured adipocytes (Fig. 3A), and also identified cells positivefor CD14, a macrophage/monocyte-specific marker, clearlyindicating the presence of macrophages in the adipose tis-sue (Fig. 3B). Areas of CD14/S100 overlap (Fig. 3B, yellow

in the merged image) indicate potentialregions of interaction between adipo-cytes and ATMs (Fig. 3C).

Adiponectin secretion byadipocytes in response to IL-6,MCP-1, and TNF-�

To assess the effect of cytokines/che-mokines on adiponectin secretion byadipocytes, we first determined the basallevels of adiponectin secretion by adipo-cytes from PCOS subjects and BMI-matched controls. No significant differ-ences were observed in the basal levels ofadiponectin secretion between the twogroups after 48 h in culture (Fig. 4A). Wethen performed dose-response experi-ments with TNF-� and MCP-1. Adi-ponectin secretion in response to TNF-�and MCP-1 was measured in duplicatein two different adipocyte samples afterpreincubation with 0.1, 1.0, 10, and 100ng/ml of TNF-� or MCP-1. TNF-� andMCP-1 at concentrations of 10 ng/mlwere found to produce maximal inhibi-tion of adiponectin secretion (P � 0.05,Fig. 4B). These results are consistentwith the findings of other researchers,

who also found adipokine concentrations of 10 ng/ml tobe optimal for use (42, 43). We then tested the effects oftreatments with individual adipokines on adiponectin se-cretion, performing group-to-group (aggregate) analyses.The baseline secretion of adiponectin by isolated adipo-

A B

C

FIG. 3. ATMs in adipose tissue from an obese control subject. Immunofluorescent stainingof adipose tissue from an obese control subject demonstrating the presence of abundantATMs. Fixed whole adipose tissues were immunolabeled with antibodies to S-100, a selectivemarker of preadipocytes and newly formed adipocytes (red) (A) and CD14, a macrophage/monocyte-specific marker (green) (B), and counterstained with the nuclear label DAPI.Combined S-100/CD14/DAPI staining (�200) demonstrates significant infiltration bymacrophages into the adipose tissue (C). Multiple direct contacts between the residentmacrophages (green) and preadipocytes or adipocytes (red) are apparent (C). Similar resultswere observed in adipose tissues from a PCOS patient (data not shown).

FIG. 4. Changes in adiponectin secretion by adipocytes in response toTNF-� and MCP-1. The graphs depict the log scale means of the basallevels of adiponectin secreted by PCOS and control adipocytes inpicograms per milliliter (A). The graphs depict the fold change in logconcentration of adiponectin (picograms per milliliter) in response toincubation with 1, 1.0, 10, and 100 ng/ml of the adipokines TNF-�(diamonds) and MCP-1 (squares). Each value was expressed as themean of two separate experiments perform in different batches ofhuman adipocytes. All samples were assayed in duplicate. All valueswere log transformed before analysis, and pairwise comparisons wereperformed using an ANOVA model. A concentration of 10 ng/ml ofeither MCP-1 or TNF-� was found to result in maximum suppression ofadiponectin secretion by adipocytes. *, P � 0.05 compared withbaseline (B).

J Clin Endocrinol Metab, February 2010, 95(2):935–942 jcem.endojournals.org 939

cytes did not differ between PCOS and control samples(data not shown). The net change in adiponectin secre-tion (treated minus baseline) in response to incubationwith 10 ng/ml of IL-6, MCP-1, and TNF-� was found todiffer between PCOS (decreasing) and control (increasing)adipocytes, although the difference in the response of

PCOS vs. control adipocytes reachedsignificance only for TNF-� (P � 0.04)(Fig. 5, A–F).

Adiponectin secretion byadipocytes in response tococulturing with ATMs

At the end of coculture period (Fig. 2),the culture media were collected foranalysis by ELISA. We found that directcoculture of isolated adipocytes andATMs resulted in a decrease in adi-ponectin secretion by PCOS adipocytes(P � 0.05) but not control adipocytes(Fig. 6A) and that the difference betweenthe net change in adiponectin secretionin PCOS vs. control samples was signif-icant (P � 0.03) (Fig. 6B).

Discussion

Available data on adiponectin secretionby adipocytes suggest that adiponectinproduction in adipose tissue is regulatedby various paracrine and endocrine fac-tors and that inflammation-related adi-pokines TNF-� (19) and IL-6 (44, 45)are involved in the decreased expressionof adiponectin in adipocytes and in thedevelopment of insulin resistance. Un-

derstanding the cellular and molecular mechanisms in-volved in regulating adiponectin secretion in PCOS adi-pocytes is critical to understanding the etiology of theinsulin resistance in PCOS. Our results suggest that adi-ponectin secretion by adipocytes in response to cytokines/

chemokines, and most notably in re-sponse to TNF-� and direct coculturewith ATMs, differs between PCOS andcontrol women, favoring greater sup-pression of adiponectin in PCOS.

Circulating adiponectin levels andadiponectin mRNA levels in both vis-ceral and sc fat are decreased in PCOSwomen compared with healthy women(8, 10), whereas adiponectin receptors 1and 2 expression are increased in both ofthese adipose compartments. Thesedata, together with our findings that adi-ponectin secretion by adipocytes is sup-pressed to a greater extent in PCOS thanin controls in response to cytokines/chemokines, or direct coculturing with

FIG. 5. Change in adiponectin secretion by adipocytes in response to treatment withadipokines/chemokines. The graphs depict the log scale means of the absolute adiponectinlevels in picograms per milliliter (A–C) and net changes in log adiponectin concentration(D–F) secreted by PCOS and control adipocytes in response to incubation with 10 ng/ml IL-6(A and D), MCP-1 (B and E), and TNF-� (C and F). All values were log transformed beforeanalysis. Sample numbers (n) per group are indicated for each experiment.

FIG. 6. Change in adiponectin secretion by adipocytes in response to adipocyte-ATMcoculture. The graphs depict the log scale means of the absolute adiponectin levels inpicograms per milliliter (A) and net change in log adiponectin concentration (B) in responseto ATM-adipocyte coculture for control (n � 6) and PCOS (n � 5) adipocytes. All values werelog transformed before analysis.

940 Chazenbalk et al. Adiponectin Secretion by Adipocytes in PCOS J Clin Endocrinol Metab, February 2010, 95(2):935–942

ATMs, suggest that the regulation of adiponectin secre-tion by adipose tissues differs between PCOS and controlwomen. Because adiponectin is an important insulin-sen-sitizing hormone (40), the greater suppression of adi-ponectin by ATM-secreted factors (e.g. TNF-�) in PCOSsuggests that this mechanism may account, at least in part,for the decreased insulin sensitivity of PCOS women.

These results are consistent with recent data indicatingthat, of the cytokine/chemokines secreted by macrophages,TNF-� is a crucial determinant of adipokine dysregu-lation in the metabolic syndrome, and that immuno-neutralization of TNF-� abrogates adipokine dysregula-tion in adipocytes of obese subjects (46). TNF-� exerts itseffects through two distinct receptors, TNF receptor-1 and -2.Through complex signaling cascades and networks, theseeffectors lead to the activation of caspases and two transcrip-tion factors, activation protein-1 (C-Jun-C-Fos complex)and nuclear factor �B, which may inhibit the secretion ofadiponectingeneexpressionandsecretion(26,47,48).Thus,dysregulation of these effectors may be a potential molecularmechanism by which TNF-� acts to overly decrease adi-ponectin secretion from PCOS adipocytes.

In conclusion, our results suggest that adiponectin se-cretion by adipocytes in response to cytokines/chemo-kines, and most notably in response to TNF-�, differs be-tween PCOS and control women, favoring greatersuppression of adiponectin in PCOS. In addition, adipo-cyte-macrophage cross talk has an even greater inhibitoryeffect on adiponectin secretion by adipocytes from womenwith PCOS, suggesting that other ATM-secreted factorsmay also play important, and possibly synergistic, effectsin regulating adiponectin secretion, both in general and inPCOS in particular. Further research into the molecularmechanisms underlying the dysregulation of adiponectinsecretion in PCOS will provide vital clues to the etiologyof insulin resistance in PCOS and possibly other metabolicdisorders and to the nature of paracrine signaling betweenadipocytes and macrophages in adipose tissue. These stud-ies may eventually lead to the identification of therapeutictargets that may decrease obesity-induced inflammationand diminish the metabolic dysfunction of obesity, PCOS,and related disorders.

Acknowledgments

Address all correspondence and requests for reprints to: Dr.Ricardo Azziz, Department of Obstetrics and Gynecology andCenter for Androgen-Related Disorders, Cedars-Sinai MedicalCenter, 8635 West Third Street, Suite 160W, Los Angeles, Cal-ifornia 90048. E-mail: azzizr@cshs.org.

This work was supported in part by the Helping Hand of LosAngeles and Grants RO1-DK073632 (to R.A.) and M01-

RR00425 (to the Cedars-Sinai Medical Center General ClinicalResearch Center) from the National Institutes of Health.

Disclosure Summary: G.C., B.S.T., B.O.Y., C.B., R.M., andS.H. have nothing to declare. R.A. has received consulting feesfrom Bionovo and Merck & Co.

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