Effects of co-incubation of LPS-stimulated RAW 264.7 macrophages on leptin production by 3T3-L1 adipocytes: A method for co-incubating distinct adipose tissue cell lines.

Adipose tissue is a major endocrine organ capable of releasing inflammatory adipokines. Inflammatory adipokine release is linked to the changes occurring in adipose tissue in the overfed state, where tissue remodeling results in hypertrophic adipocytes that recruit monocytes to infiltrate the tissue and take on an inflammatory phenotype. As the proportion of inflammatory macrophages increases there is a concurrent increase in release of macrophage-specific inflammatory mediators, further contributing to the inflamed state and setting an inflammatory loop between the macrophages and adipocytes. Although most inflammatory adipokines are released by macrophages, adipocytes can also release immunomodulatory adipokines, such as leptin. The objective of this research was to determine if co-incubation of activated macrophages with mature adipocytes, using Transwell inserts, affected leptin release by mature adipocytes. We also examined if there were differences in the amount of cell-secreted products quantified in cell-conditioned media collected from macrophage-containing (Transwell insert) and adipocyte-containing (well) compartments. Mature adipocytes (differentiated 3T3-L1 murine fibroblasts) were co-incubated with control and lipopolysaccharide-stimulated (0.01 g/ml) murine macrophages (RAW264.7), and nitric oxide, interleukin-6, and leptin levels were quantified in the cell-conditioned media from the two compartments. Activation status of the macrophages did not affect leptin release by the adipocytes. We observed higher amounts of leptin in wells compared to Transwells. Nitric oxide and interleukin-6 levels were similar between Transwells and wells, suggesting that these adipokines are traveling through the Transwell inserts and reaching equilibrium between the two compartments. There was a weak negative relationship between nitric oxide release by macrophages and leptin release by adipocytes. Our results suggest that co-incubating activated macrophages and adipocytes using Transwell inserts can result in distinct microenvironments in the different cellular compartments and that separate sampling of these compartments is required to detect the subtle signaling dynamics that exist between these cells.