Regulation of adiponectin secretion by adipocytes in the polycystic ovary syndrome: role of tumor necrosis factor-{alpha}

Abstract

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

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

Design and participants: Primary cultures of sc adipocytes and coculture of adipocytes and ATMs from 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 PCOS and control samples. The net change in adiponectin secretion in response to IL-6, monocyte chemoattractant protein-1, and TNF-alpha differed between PCOS (decreasing) and control (increasing) adipocytes, although the difference reached significance only for TNF-alpha (P < 0.04). Coculture of isolated 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 secretion in 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 and control women, favoring greater suppression of adiponectin in PCOS. The mechanisms underlying these defects and the role of concurrent obesity remain to be determined.

Figure 1

Characterization of isolated human adipose tissue adipocytes and macrophages. Untreated adipocytes from cell culture were fixed in 4% paraformaldehyde, and Oil Red O staining and nuclear DAPI labeling were performed to distinguish adipocytes from free lipid (A). Immunofluorescent labeling with 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 isolated cells are mature adipocytes (×100 magnification) (A), and lack of CD14 staining indicates that the isolated adipocytes fraction is free from adipocytes (C). ATMs isolated from adipose tissue were subjected to immunofluorescent labeling with antibodies to CD14 (green) (B) and S-100 (D) and mounted using DAPI after 3 d in culture. The cells are CD14(+), consistent with the identification of these cells as macrophages (B), and S-100(−), indicating that the ATM fraction is free from adipocytes (×200) (D).

Figure 2

Graphical depiction of the adipocyte-ATM coculture system and coculture of adipocytes and ATMs. Adipocytes and ATMs were isolated from adipose tissue as described in Materials and Methods. After a 16-h equilibration period, the adipocytes and ATMs are cocultured with direct contact between the cell types for 24 h. The adipocytes are then removed and cultured separately for an additional 48 h, after which the culture media were harvested for analysis by ELISA.

Figure 3

ATMs in adipose tissue from an obese control subject. Immunofluorescent staining of adipose tissue from an obese control subject demonstrating the presence of abundant ATMs. Fixed whole adipose tissues were immunolabeled with antibodies to S-100, a selective marker 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 by macrophages into the adipose tissue (C). Multiple direct contacts between the resident macrophages (green) and preadipocytes or adipocytes (red) are apparent (C). Similar results were observed in adipose tissues from a PCOS patient (data not shown).

Figure 4

Changes in adiponectin secretion by adipocytes in response to TNF-α and MCP-1. The graphs depict the log scale means of the basal levels of adiponectin secreted by PCOS and control adipocytes in picograms per milliliter (A). The graphs depict the fold change in log concentration of adiponectin (picograms per milliliter) in response to incubation with 1, 1.0, 10, and 100 ng/ml of the adipokines TNF-α (diamonds) and MCP-1 (squares). Each value was expressed as the mean of two separate experiments perform in different batches of human adipocytes. All samples were assayed in duplicate. All values were log transformed before analysis, and pairwise comparisons were performed using an ANOVA model. A concentration of 10 ng/ml of either MCP-1 or TNF-α was found to result in maximum suppression of adiponectin secretion by adipocytes. *, P < 0.05 compared with baseline (B).

Figure 5

Change in adiponectin secretion by adipocytes in response to treatment with adipokines/chemokines. The graphs depict the log scale means of the absolute adiponectin levels 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-α (C and F). All values were log transformed before analysis. Sample numbers (n) per group are indicated for each experiment.

Figure 6

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