Figure 1.
Proposed pathway for PGD3 metabolism
(adapted from Ref Shibata et al., 2002 for PGD2).
Table 1.
Nutrient composition and fatty acid composition of standard (A03) and EPA-rich diet.
Figure 2.
(A) Plasma levels of adiponectin from mice fed a standard diet or an EPA-rich diet.
Plasma levels are expressed in µg/mL. Results are means ± sem (n = 8). *P<0.05 as compared to the control group. (B) Body weight gain (g) of mice fed a standard diet or an EPA-rich diet. Mice were killed on days 0 and 4. Results are means ± sem (n = 8). *P<0.05 as compared to the control group. (C) Effects of eicosapentaenoic acid on adiponectin secretion by 3T3-L1 adipocytes. Cells were incubated for 2 h (gray) or 4 h (black) with 1 µM or 10 µM EPA complexed with bovine serum albumin. Adiponectin in the medium was determined by ELISA. Results are means ± sem (n = 4 in triplicate), expressed as percentage of the control (□). Statistical significance is represented as *P<0.05 vs control.
Figure 3.
HPLC profile of metabolites formed from PGD2 (A) or PGD3 (B).
1 mM of PGD2 or PGD3 was incubated in phosphate-buffered saline at 37°C for 24 h. PGD2/PGD3 and their metabolites were chromatographed on a Waters Xbridge C18 column (4.6×250 mm, 3.5 µm) at a flow rate of 1 ml/min starting at 100% solvent A (acetonitrile/water acidified to pH 3, 2/8 v/v) to 100% solvent B (acetonitrile) from 1 to 30 min. The elution profiles were monitored by UV absorbance at 195 nm.
Figure 4.
GC-MS (A) and GC-MS/MS (B) spectra of 15d-PGJ3 generated during incubation of PGD3 in phosphate-buffered saline.
The anion at m/z 313 [M-CH2C6F5]- generated by NICI was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 313 are shown in the figure.
Figure 5.
Effects of PGD3 and 15d-PGJ3 on adiponectin secretion by 3T3-L1 adipocytes.
Cells were incubated for 2 h with 1 µM PGD3 or 0.1 µM 15d-PGJ3. Adiponectin in the medium was determined by ELISA. Results are means ± sem (n = 4 in triplicate), expressed as percentage of the control. Statistical significance is represented as **P<0.01 vs control.
Figure 6.
GC-MS and GC-MS/MS analysis of 15d-PGJ3 in the culture medium following incubation of cells with EPA.
3T3-L1 were incubated with 10 µM EPA for 4 h. Culture medium was extracted. 15d-PGJ3 was purified by HPLC. The 15d-PGJ3 HPLC peak was collected and was analyzed by GC-MS as a pentafluorobenzyl ester derivative (m/z 313) (Fig. 6A). B. GC-MS/MS spectrum of 15d-PGJ3. The molecular ion [M-CH2C6F5]- of m/z 313 was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 313 are shown in the figure.
Figure 7.
GC-MS/MS analysis of d5-15d-PGJ3 in the culture medium following incubation of cells with d5-EPA.
3T3-L1 cells were incubated with d5-EPA for 4 h. Culture medium was extracted. d5-15d-PGJ3 was purified by HPLC. The d5-15d-PGJ3 HPLC peak was collected and was analyzed by GC-MS/MS. The anion at m/z 315 [M-CH2C6F5]- was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 315 are shown in the figure.
Figure 8.
GC-MS/MS analysis of 15d-PGJ3 in epididymal adipose tissue of mice fed a EPA-rich diet.
15d-PGJ3 was purified by HPLC. The 15d-PGJ3 HPLC peak was collected and was analyzed by GC-MS as a pentafluorobenzyl ester derivative (m/z 313) (Fig. 8A). B. GC-MS/MS spectrum of 15d-PGJ3. The molecular ion [M-CH2C6F5]- of m/z 313 was subjected to CID. The structure of the derivative and the proposed structure of the product ion produced by CID of m/z 313 are shown in the figure.
Figure 9.
Effect of a PPAR-γ antagonism on the PGD3 or 15d-PGJ3 increase in adiponectin secretion by cells.
3T3-L1 adipocytes were incubated for 2 h with 1 µM PGD3 or 100 nM 15d-PGJ3 in the presence or absence of 10 µM GW9662 or with GW9662 alone. Adiponectin in the medium was determined by ELISA. Results are means ± sem (n = 3), expressed as percentage of the control. Statistical significance is represented as *P<0.05, **P<0.01.
Figure 10.
Effect of 15d-PGJ3 on FAS, FABP4, adiponectin, PPAR-γ and PDK4 gene expression in 3T3-L1 adipocytes.
Cells were incubated for 2 h with or without 100 nM 15d-PGJ3. FAS, FABP4, adiponectin, PPAR-γ and PDK4 mRNA levels were quantified by qPCR. Results are means ± sem (n = 3). Statistical significance is represented as *P<0.05 vs control.