Changes in COX-2, iNOS and GCL gene expression in 3T3-L1 preadipocytes incubated in macrophage conditioned medium

Abstract

PURPOSE: Obesity is recognized as a major risk factor for a number of diseases, including metabolic syndrome and type 2 diabetes mellitus. It is characterized with increased macrophage infiltration in adipose tissue. Macrophages secrete numerous inflammatory mediators and contribute to the increased inflammatory status of adipose tissue which is associated with increased reactive oxygen species generation and oxidative stress. In cell biology conditioned nutrient mediums (CMs) are used to assess the complex impact of the secretory products of a particular cell type on the behaviour of other cells. The aim of the present study was to examine the effect of CM obtained from J744A.1 macrophage cell culture on expression in 3T3-L1 preadipocytes of some enzymes related to antioxidant defense and the inflammatory response. MATERIAL AND METHODS: mRNA levels of inducible cyclooxygenase (COX-2), inducible NO synthase (iNOS) and glutamate-cysteine-ligase (GCL) were measured. 3T3-L1 cells were treated with increasing CM concentrations (10-30%) for 24 hours. Changes in gene expression levels were analyzed by real-time qPCR method. Calculations were performed using 2-ΔΔCt method. RESULTS: The higher CM concentration contributed to increased expression levels of the genes examined. Treatment with CM in concentrations of 10, 20 and 30% resulted in 2.3-, 2.6- and 2.7-fold increase in COX-2 expression (p<0.01), respectively. Both 10% and 20% CM up-regulated iNOS by 1.7 times (p<0.001). The highest CM concentration of 30% stimulated the enzyme by two times (p<0.01). GCL mRNA levels were by 2.5 times higher than these of the untreated controls when stimulated with 30% CM (p<0.05). CONCLUSION: CM from J744A.1 cell culture may induce inflammatory and oxidative stress-related response in 3T3-L1 preadipocytes thus demonstrating a possible link between macrophage infiltration and local inflammation in adipose tissue. Scripta Scientifica Medica 2013; 45(2): 32-35.