Fig 1.
Effects of the phosphatidylcholine (PPC)/deoxycholate (DC) formula or DC alone on the viability of preadipocytes and adipocytes.
A. 3T3L1 preadipocyte and B. adipocyte cell viability was measured eight hours after treatment with various doses of the PPC/DC formula or DC only using the MTT assay. Protein samples from C. 3T3L1 preadipocytes and D. adipocytes following 0.01% or 0.1% PPC/DC formula or DC treatment for eight hours, respectively, were prepared and analyzed by Western blotting.
Fig 2.
Effect of phosphatidylcholine (PPC) or deoxycholate (DC) on the viability of preadipocytes and adipocytes.
A. 3T3L1 preadipocyte and B. adipocyte viability was measured eight hours after treatment with various doses of PPC or DC using the MTT assay. Live confocal images of C. 3T3L1 preadipocytes and D. adipocytes captured before (0 h) and eight hours of treatment with 0.03% of PPC or DC. The white dots indicate the lipid vacuoles of mature adipocytes, and the eight-hour treatment with PPC decreased the number of lipid vacuole-positive cells to a greater extent than DC treatment. Protein samples from E. 3T3L1 preadipocyte and F. adipocytes treated with 0.01% or 0.03% of PPC or DC respectively for eight hours, were prepared and the expression of preadipocyte (C/EBPα, PPARγ, and HSL), and mature adipocyte (C/EBPα, PPARγ, HSL, and FABP4) markers, apoptotic markers (cleaved-caspase3 and cleaved-parp), as well as the amount of loading controls (HSP90 and α-tubulin) were analyzed using Western blotting. The scale bar indicates 20 μm.
Fig 3.
In vivo analysis of the inguinal adipose tissue area before and after phosphatidylcholine (PPC) or deoxycholate (DC) injection.
MR images were acquired to measure the inguinal fat pad area of Sprague-Dawley rats, before (Pre) and 7, 14, and 28 days after injection of 25 mg of PPC (n = 6), 12.5 mg of PPC (n = 6), 25 mg of DC (n = 5), or 12.5 mg of DC (n = 5) into one side of the bilateral inguinal fat pad and control solution (ethanol for PPC and PBS for DC) into the other side. Data are presented as the percentage of the inguinal fat pad area injected with PPC or DC to the control-injected fat pad area.
Fig 4.
Tissue weights of Sprague-Dawley rats 30 days after phosphatidylcholine (PPC) or deoxycholate (DC) injection.
Weights of bilaterally excised tissue, including inguinal adipose tissue (IngWAT), epididymal adipose tissue (EpiWAT), quadriceps muscle, and tissue from the kidneys, were measured 30 days after injection of 25 mg PPC (n = 6), 12.5 mg PPC (n = 6), 25 mg DC (n = 5), or 12.5 mg DC (n = 5). Data are presented as the percent tissue weight resulting from PPC or DC injection to tissue weight resulting from injection of the control solution.
Fig 5.
Effects of phosphatidylcholine (PPC) and deoxycholate (DC) on inguinal adipose tissue in the rat.
A. Hematoxylin and eosin staining of inguinal adipose tissue sections from rats 30 days after control, PPC, or DC injection. The arrow indicates macrophage infiltration and the scale bar indicates 50 μm. B. Gene expression analysis of inguinal adipose tissue injected with control or 25 mg or 12.5 mg of PPC or DC, respectively. Using real-time qRT-PCR, the relative expression levels of FABP4, a marker for mature adipocytes and EMR1, NOS2, CD68, CD80, and IL1β markers for macrophages, in inguinal adipose tissue injected with PPC or DC were compared with the expression in inguinal adipose tissue injected with ethanol or PBS control solutions, respectively.