P2 Sphingosine kinase 1/sphingosine-1-phosphate receptor 2 axis modulates macrophage-mediated inducible nitric oxide synthase expression and nitric oxide production in adipocytes

Background

Obesity is a major global health problem and an important risk factor for cardiovascular diseases. Chronic inflammation due to macrophage infiltration into adipose tissue is a hallmark of obesity and is associated with increased expression of inducible nitric oxide synthase (iNOS) which contributes to the development of insulin resistance and other metabolic dysfunction. Sphingosine kinase (SphK1) phosphorylates sphingosine into sphingosine-1-phosphate (S1P), a bioactive lipid implicated in the regulation of adipose tissue inflammation and insulin resistance. These effects may be attributed to the activation of the S1P-specific G protein-coupled receptor, S1PR2. However, the role of the SphK1/S1P/S1PR2 axis in macrophage-mediated iNOS expression in adipocytes remains to be fully elucidated. Therefore, the objective of the present study was to investigate the role of the axis in mediating iNOS expression and nitric oxide (NO) production in adipocytes.

Methods

RAW 267.4 cells were stimulated with LPS 100 ng/ml for 8 hours in the presence and absence of PF543 (SphK1 inhibitor-100 nM) and JTE 013 (S1PR2 antagonist-10μM). Conditioned media (CM) was collected and stored at -80°C until used. SphK1 phosphorylation in RAW cells was measured by western blot. Cultured 3T3-L1 differentiated adipocytes were stimulated with 1:4 dilution of macrophage CM for 24h and iNOS expression and NO production measured. To investigate the involvement of SphK1/S1PR2, adipocytes were stimulated with CM treated with PF543 or JTE 013 for 24h. In some experiments, adipocytes were pre-treated with JTE013 prior to stimulation with CM. iNOS protein expression was determined using western blotting and NO production in the supernatant was measured using a Sievers 280 analyzer. Data are expressed as mean ± SEM; statistical analysis was carried out by one way ANOVA.

Results

CM from LPS-treated RAW 267.4 cells induced upregulation of iNOS and NO production in 3T3-L1 adipocytes. LPS increased SphK1 phosphorylation in RAW 267.4 cells. SphK1/S1PR2 axis inhibition using PF543 and JTE 013 in LPS-activated macrophages decreased iNOS expression and NO production in 3T3-L1 adipocytes co-cultured with activated RAW 267.4 conditioned media (p< 0.05, n = 3). S1PR2 antagonism in 3T3-L1 adipocytes decreased iNOS expression and NO production following co-culture with activated RAW 267.4 CM (p< 0.05, n = 3).

Conclusion

Activated macrophage conditioned media increases iNOS expression and NO production in 3T3 adipocytes. These results also show that macrophage-derived S1P activates S1PR2 on macrophages and adjacent adipocytes, contributing to iNOS regulation within adipose tissue. In conclusion, the SphK1/S1PR2 axis may be a promising target for the development of treatments aimed at ameliorating adipose inflammation and insulin resistance associated with obesity and type 2 diabetes.