Chondroitin Sulfate Inhibits Monocyte Chemoattractant Protein-1 Release From 3t3-L1 Adipocytes: A New Treatment Opportunity For Obesity-Related Metabolic Syndromes?

Purpose: Monocyte chemoattractant protein-1(MCP-1) overproduction from inflamed adipose tissue is a major contributor to obesity-related metabolic syndromes. We have recently published that chondroitin sulphate (CS) can attenuate the MSU crystal mediated THP-1 macrophage inflammatory response reflected by reduced release of pro-inflammatory cytokines IL-1β and TNFα. We have also recently determined that the CS inhibitory effect is not acting at the inflammasome but upstream, most likely by inhibiting activation of NF-κB. We sought to determine whether CS had a similar inhibitory effect on MCP-1 release from LPS stimulated adipocytes. Methods: We cultured 3T3-L1 embryonic fibroblasts and induced their differentiation into adipocytes using an established protocol. We then treated the adipocytes with lipopolysaccharide (LPS) to induce inflammation and thus MCP-1 release. At the same time we added varying concentrations of CS (Bioibérica, Spain) in a physiologically relevant range (10–200 μg/ml) and 24h after we measured MCP-1 release (R&D Systems, Minneapolis, MN, USA). All experiments were run a single time with each treatment group run in triplicate. We also cultured THP-1 monocytes and tested whether CS at the highest physiologically relevant concentration (200 μg/ml) could inhibit cell migration induced by human recombinant MCP-1. Monocyte chemotaxis in response to 24hrs exposure to varying concentrations (0, 3.125–100 ng/ml) of recombinant human MCP-1 (R&D Systems) was tested using the CytoSelect 96-well cell migration assay (Cell Biolabs, San Diego, CA, USA). All experiments were run a single time with each treatment group run in triplicate. After normalization for cell viability, cell culture results were expressed as fold change from the media only negative control (no CS, no LPS). One-way ANOVA with Bonferroni’s post-hoc test and post-hoc linear trend were performed on cell culture results using Graphpad Prism software (La Jolla, CA, USA). Results: We found that LPS (1μg/ml) caused a significant rise in MCP-1 release (<0.0001) from 3T3-L1 adipocytes. CS in physiologically achievable concentrations (100–200 μg/ml) produced a dose dependent reduction (<0.01 at 100 μg/ml and <0.001 at 200 μg/ml) of MCP-1 release from 3T3-L1 adipocytes in response to LPS. Recombinant MCP-1(25–100 ng/ml) caused a dose dependent increase (<0.001 at 25ng/ml and <0.0001 at 100ng/ml) in cell migration of THP-1 monocytes. CS at the highest test concentration (200 μg/ml) had no effect on MCP-1 mediated THP-1 migration. Conclusions: Our data demonstrate that CS inhibits the release of MCP-1 from 3T3-L1 adipocytes that have been stimulated with LPS, but has no effect on the chemotactic action of MCP-1 on THP-1 monocytes. It has recently been demonstrated that CS can inhibit the release of MCP-1 from human coronary artery endothelial cells (HCAECs) inflamed by TNF-α; in this model system, CS also inhibited the migration of THP-1 monocytes towards the inflamed HCAEC. Our data strongly suggest that it is the inhibition of MCP-1 release by CS that underlies this effect and not a direct inhibition of the chemotactic action of MCP-1 by CS. Given the importance of MCP-1 over-production in obesity-related metabolic syndromes, inhibiting the release of MCP-1 from adipocytes by CS, and thus blocking the recruitment of macrophages to adipose tissue, could provide a new treatment opportunity for these syndromes.