Physical Exercise Reduces the Expression of RANTES and Its CCR5 Receptor in the Adipose Tissue of Obese Humans
Figure 2
Expression of RANTES in obese subjects. (a) Western blot analysis of RANTES expression in PBMCs from lean and obese subjects. The bands reacting with anti-RANTES antibody were quantified as described in Section 2 and the relative intensity was determined after correction with Actin that was used as internal control to monitor loading efficiency. The blots shown are representative of three independent experiments with consistent results. The data are presented in the form of a bar graph on the right of the figure as fold changes of RANTES protein expression in obese compared to lean subjects. (b) Characterization of the monocyte subpopulations and T cells in peripheral blood from lean and obese participants. Monocytes subsets were defined by staining for CD14 (PE), T cells by CD3 (FITC), and expression of intracellular RANTES (APC) was analyzed. Left and right upper panels are representative dot plots of CD3 and RANTES expression on/in T cells from lean and obese participants, respectively. Left and right lowest panels are representative dot plots of CD14 and RANTES expression on/in monocyte subsets from lean and obese participants, respectively. The double-positive populations (i.e., CD3+RANTES+, CD14+RANTES+, and CD14++RANTES+) were analyzed for mean RANTES fluorescence intensity. (c) Analysis of RANTES expression by immunohistochemistry (IHC) in the subcutaneous adipose tissues from lean and obese nondiabetic participants. Aperio software was used to quantify positive staining (indicated by arrows) and quantified values relative to lean controls are plotted in a bar graph at the bottom. (d and e) Analysis of RANTES mRNA expression by quantitative real-time PCR (qRT-PCR) between lean and obese subjects. Total RNA was isolated from PBMCs (d) and adipose tissue biopsies (e). The data are presented as fold changes in obese compared to lean subjects after normalization with the GAPDH reference gene. (f and g) Analysis of TNF-α and IL-6 expression at the protein level by IHC (f) and at the mRNA level by qRT-PCR (g) in the adipose tissue from lean and obese subjects. In IHC experiments, Aperio software was used to quantify positive staining as indicated above and the values are illustrated at the bottom as fold changes compared to lean. Mann-Whitney test was used to determine significance of difference between the lean and obese subjects. For each experiment, the sample size from each group is indicated by .
