Fig 1.
Increased fibrosis and inflammation in obese adipose tissue.
(A) Representative photomicrographs (×200 magnification; scale bars: 200 μm) of omental white adipose tissue (WAT) sections stained with Sirius Red and Masson’n trichrome from non-obese control individuals (n = 8) and obese patients (n = 8). (B) Representative photomicrographs (×200 and x400 magnifications; scale bars: 100 μm) of obese WAT sections stained with Picrosirius Red (left panels) and visualized under polarized light (right panels). Arrows indicate accumulation of fibrosis around adipocytes. (C) Total and pericellular fibrosis expressed as percentage of the total cellular membranes in omental WAT from control and obese individuals. (D,E) mRNA expression for COLα1, TIMP1, αSMA and TGF-β in WAT from control and obese individuals. (F) mRNA expression of inflammatory markers IL-6 and CD68 in WAT. Results are expressed as mean ± SEM. *, P<0.01 and **, P<0.005 versus control.
Fig 2.
High-throughput transcriptomic analysis of adipose tissue from control and obese individuals.
(A) Differential gene expression by Affymetrix Human Genome U219 expression arrays in omental adipose tissue from non-obese control individuals (n = 4) and obese patients (n = 4). The results are expressed as a matrix view of gene expression data (heat map) in which rows represent genes and columns represent hybridized samples. The intensity of each colour denotes the standardized ratio between each value and the average expression of each gene across all samples. Red pixels correspond to an increased abundance of mRNA in the samples indicated, whereas green pixels indicate decreased mRNA levels. (B) Integrative IPA of transcriptomic data. Genes significantly down-regulated are indicated in green; those that were significantly up-regulated are indicated in red. (C) Cascade of top regulators of inflammation and fibrosis detected by IPA software.
Fig 3.
Targeted lipidomic analysis of COX products in adipose tissue from control and obese individuals.
(A) Levels of PGE2, 15d-PGJ2, 6-keto-PGF1α and PGF2α in adipose tissue from non-obese control individuals (n = 6) and obese patients (n = 7) were assessed by LC-ESI-MS/MS. (B) COX-2, mPGES-1, mPGES-2 and cPGES expression in omental adipose tissue from control (n = 7) and obese (n = 11) individuals. (C) Representative Western blot of mPGES-1 protein expression in omental adipose tissue. (D) Correlation between the expression of COX-2 and mPGES-1 in omental adipose tissue and the body mass index (BMI). The results are expressed as mean ± SEM. *, P<0.05 and **, P<0.01 versus control.
Fig 4.
Effects of PGE2 on adipose tissue remodeling.
(A) mRNA expression for COL1α1, TIMP1, COL1α2 and TGF-β in obese fat pads incubated with vehicle (Veh) or PGE2 (1 μM). (B) mRNA expression for COL1α1, TIMP1, COL1α2, TGF-β and αSMA in differentiated 3T3-L1 adipocytes growing in the presence of Veh, (0.19% DMSO) or TGF-β (10 ng/ml). (C) mRNA expression for COL1α1, TIMP1, COL1α2, TGF-β and αSMA in 3T3-L1 adipocytes growing in the presence of TGF-β (10 ng/ml) and incubated with vehicle (Veh) or PGE2 (0.1 μM). The results are expressed as mean ± SEM. *, P<0.05 and **, P<0.01 versus Veh.
Fig 5.
Anti-inflammatory effects of PGE2 on adipose tissue.
(A) mRNA expression for COX-2, IL-6 and MCP-1 in human obese fat pads incubated with vehicle (Veh) or PGE2 (1 μM). (B) COX-2, IL-6 and MCP-1 expression in differentiated 3T3-L1 adipocytes growing in the presence of LPS (100 ng/ml) and incubated with increasing concentrations of PGE2 (0, 1 and 5 μM). The results are expressed as mean ± SEM. #, P<0.05, ##, P<0.01 and ###, P<0.001 for LPS versus untreated cells. *, P<0.05 and **, P<0.01 versus Veh or LPS.
Fig 6.
Effects of PGE2 on beige/brite differentiation.
(A) Adipose tissue expression of UCP1 in adipose tissue from non-obese control individuals (n = 10) and obese patients (n = 11). (B) Expression of UCP1 and PRDM16 in obese fat pads incubated with vehicle (Veh, 0.19% DMSO) or PGE2 (1 μM) for 24 hours. (C) Representative photomicrographs of stromal vascular cell (SVC) and adipocyte cultures isolated from adipose tissue of obese patients (x200 magnification; scale bars; 200 μm). Right panel: Expression of COX-2 and mPGES-1 in SVC and adipocytes from obese adipose tissue. D) Expression of UCP1 and PRDM16 in differentiated pre-adipocytes isolated from obese patients growing in the presence of Veh (0.19% DMSO) or PGE2 (1 μM). (E) Expression of UCP1 and PRDM16 in adipocytes isolated from obese adipose tissue and incubated with Veh (0.19% DMSO) or PGE2 (1 μM) for 3 h. (F) Representative western blot of phosphorylated HSL (pHSL) protein expression in adipose tissue from non-obese control individuals (CT) (n = 7) and obese patients (Ob) (n = 8). (G) Glycerol levels released by adipocytes incubated with vehicle (V) (0.19% DMSO) or isoproterenol (1 μM) in the presence of V or PGE2 (1 μM) for 1.5 h. The results are expressed as mean ± SEM. *, P<0.05 versus control or Veh; a, P<0.05 and b P<0.01 versus SVC. #, P<0.05 versus isoproterenol-treated adipocytes.