Expression of adiponectin in human placenta
Using RT-PCR analysis, we detected adiponectin mRNA in human term placenta, using adipose tissue as a positive control (Fig. 1a). Subsequent sequencing of the PCR products confirmed the gene identity. Using real-time RT-PCR, we showed that adiponectin mRNA was expressed at a significantly higher (p<0.01) level in s.c. adipose tissue compared with normal placenta from the same patients (Fig. 1b). Using Western blotting analysis, we also showed that these changes were also reflected at the protein level (Fig. 1b, insert).
Immunofluorescence analysis of human term placentae revealed that immunoreactive adiponectin staining was localised almost exclusively in the syncytiotrophoblast. We found no apparent expression in the cytotrophoblast and little staining was evident within the placental blood vessels (Fig. 1c, I). Adipose tissue was also used as a positive control (Fig. 1c, II). Negative serum controls confirmed the specificity of the positive immunoreactive staining (Fig. 1c, III, IV). Identical results were obtained from four independent experiments.
Secretion of placental adiponectin
Western blot analysis of conditioned media from placental explants (4 h) demonstrated the presence of adiponectin protein, with a molecular weight (around 33 kDa) identical to that found for placental (100 μg) and adipose tissue lysates (10 μg); there was no apparent protein expression of adiponectin detected in culture medium alone, whereas adiponectin was detectable in conditioned medium (Fig. 1d). Furthermore, RIA confirmed secretion of adiponectin in vitro from conditioned medium (n=10; 100±20 ng/ml), whereas in culture medium alone there was no detectable adiponectin present. Diluted serum from the same control subjects (n=10) was used as a positive control (Fig. 1e).
Expression of adiponectin and its receptors in GDM
Using real-time PCR and values standardised against β-actin, we showed that in GDM placentae (n=8) there was significant downregulation of placental adiponectin mRNA levels compared with normal (n=10), as can be seen from the delay in the amplification of the gene (Fig. 2a). Statistical analysis of these data revealed that this downregulation was significant (***p<0.001) (Fig. 2b). We also used real-time RT-PCR to assess the expression of ADIPOR1 and ADIPOR2 in GDM placenta. We showed that there was a significant increase in ADIPOR1 mRNA (**p<0.01; Fig. 2c) in GDM placenta (90% increase compared with normal), whereas no significant upregulation in the expression of ADIPOR2 was detected between the two groups (Fig. 2d).
Modulation of p38 and ERK1/2 MAP kinases by placental adiponectin
Following the detection of placental adiponectin, we used full-length adiponectin not to assess the biofunction of adiponectin receptors but to see whether the secreted placental adiponectin had affinity for the ADIPOR1 receptors. We did this by measuring its effect on the phosphorylation of MAPKs in HEK-293/ADIPOR1 cells. For this study, full-length adiponectin was used as a control.
Prior to this, we demonstrated the presence of functional ADIPOR1 in HEK-293 cells by using immunofluorescence and displacement studies. Immunofluorescent analysis, using a specific ADIPOR1 antibody, demonstrated expression of the receptor on the cell surface of HEK-293/ADIPOR1 cells, in accordance with its proposed localisation as a seven-transmembrane domain receptor (Fig. 2e, I). Preabsorption with blocking peptide for ADIPOR1 confirmed the specificity of the positive immunoreactive staining (Fig. 2e, II). Identical results were obtained from four independent experiments. The presence of functional ADIPOR1 receptors in HEK-293 cells was further confirmed by binding displacement studies. Human ‘cold’ adiponectin was able to displace its respective radiolabelled ligand from its binding sites in a concentration-dependent manner in HEK-293/ADIPOR1 cells (Fig. 2f).
Treatment of HEK-293/ADIPOR1 cells (n=10) with adiponectin induced significant (p<0.01) downregulation of phosphorylation of both p38 and ERK1/2 (Fig. 3a,b). This effect appeared to be dose-dependent (data not shown), with maximal effect at 100 nmol/l. Furthermore, this effect was maximal after 10 min of treatment and returned to basal levels after 45 min of treatment (data not shown). Despite decreased levels of both phospho-p38 and phospho-ERK1/2, the total amounts of both MAPKs were unchanged (Fig. 3a,b). Interestingly, conditioned medium exerted a very similar effect on p38 and ERK1/2 phosphorylation. However, when adiponectin was immunoprecipitated out of the conditioned medium, the adiponectin-free medium failed to alter the phosphorylation status of both MAPKs, indicating that the peptide exerting this specific effect was indeed placentally secreted adiponectin (Fig. 3a,b).
Modulation of placental adiponectin by cytokines
Placental explants from uncomplicated pregnancies (n=10) were treated with TNF-α, IFN-γ, TNF-α + IFN-γ, IL-6 or leptin at a concentration of 100 nmol/l for 4 and 24 h (Fig. 4).
Treatment of placental explants (Fig. 4a) with TNF-α or IFN-γ for 4 h did not exert any significant effect at the mRNA level, whereas at 24 h TNF-α reduced and IFN-γ induced adiponectin mRNA (p<0.05). Interestingly, a combination of TNF-α and IFN-γ led to greater downregulation of adiponectin mRNA levels at both 4 h (40% below basal; p<0.05) and 24 h (95% below basal; p<0.01). IL-6, at both time points, significantly reduced adiponectin mRNA levels (4 h, 72%, p<0.05; 24 h, 85%, p<0.01) compared with basal. Of interest, however, treatment with leptin for 4 h induced adiponectin mRNA (70% above basal, p<0.05), whereas at 24 h adiponectin mRNA levels were dramatically reduced (90% below basal, p<0.01).
Further to the mRNA changes, these cytokines also altered the secretion of placental adiponectin into culture medium (Fig. 4b). After 4 h of incubation, all cytokines reduced the secretion of placental adiponectin (p<0.01). After 24 h of incubation, the pattern of adiponectin secretion mirrored that seen at the mRNA level, with the exception that leptin did not exert any significant effect, while IL-6 increased adiponectin (80% above basal; p<0.05).
Modulation of placental adiponectin receptors by cytokines
We used the same mRNA from normal placental explants (n=10), treated with various cytokines, to assess their effects on both ADIPOR1 and ADIPOR2 mRNA (Fig. 5). Treatment of placental explants with TNF-α or IFN-γ or a combination of both significantly induced ADIPOR1 mRNA at both 4 and 24 h (Fig. 5a). Interestingly, a combination of TNF-α and IFN-γ at 4 h led to greater upregulation of ADIPOR1 mRNA levels at 4 h compared with 24 h. Similarly, IL-6 and leptin, at both time points, significantly reduced ADIPOR1 mRNA levels (4 h, p<0.01; 24 h, p<0.05) compared with the basal condition (Fig. 5a).
Interestingly, treatment of placental explants with TNF-α or IFN-γ or a combination of both did not have any apparent effect on adipoR2 at 4 h of treatment, whereas at 24 h changes in ADIPOR2 mRNA mirrored those of adiponectin (Fig. 5b). TNF-α significantly (p<0.05) downregulated ADIPOR2 mRNA and IFN-γ significantly (p<0.01) upregulated ADIPOR2, whereas a combination of both reduced ADIPOR2 mRNA levels (p<0.05). Moreover, both leptin and IL-6 significantly induced (p<0.05) ADIPOR2 mRNA at 4 h, whereas at 24 h leptin decreased ADIPOR2 mRNA and IL-6 had no effect on its gene expression (Fig. 5b).