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, 112 (6), 945-55

Biosynthesis of 15-deoxy-delta12,14-PGJ2 and the Ligation of PPARgamma

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Biosynthesis of 15-deoxy-delta12,14-PGJ2 and the Ligation of PPARgamma

L Chastine Bell-Parikh et al. J Clin Invest.

Abstract

15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARgamma, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-kappaB, and mediation of apoptosis have been proposed. However, quantitative, physiochemical evidence for the formation of 15d-PGJ2 in vivo is lacking. We report that 15d-PGJ2 is detectable using liquid chromatography-mass spectrometry-mass spectrometry at low picomolar concentrations in the medium of 3T3-L1 preadipocytes. However, despite induction of COX-2, production of PGs, including 15d-PGJ2, does not increase during adipocyte differentiation, a process unaltered by COX inhibition. 15d-PGJ2 is detectable as a minor product of COX-2 in human urine. However, its biosynthesis is unaltered during or after COX activation in vivo by LPS. Furthermore, the biosynthesis of 15d-PGJ2 is not augmented in the joint fluid of patients with arthritis, nor is its urinary excretion increased in patients with diabetes or obesity. 15d-PGJ2 is not the endogenous mediator of PPARgamma-dependent adipocyte activation and is unaltered in clinical settings in which PPARgamma activation has been implicated.

Figures

Figure 1
Figure 1
COX-dependent biosynthesis of 15d-PGJ2 by 3T3-L1 preadipocytes. (a) Cellular incorporation of 15d-PGJ2. 3T3-L1 cells were incubated with 15d-PGJ2 mixed with [3H]15d-PGJ2 for 48 hours. Vertical bars represent dpm in the medium at the times indicated and in the cell wash and cell pellet after 48 hours. (b) Mass spectrum of standard 15d-PGJ2. Spectrum obtained by NI-ESI-LC-MS-MS. For LC-MS-MS conditions, see Methods section. (c) Substrate enhanced, COX-dependent formation of PGs. Stimulation of 15d-PGJ2 and PGE2 formation in 3T3-L1, 1-day preconfluent cells, pretreated with 15 μM arachidonic acid (AA) alone for 1 hour and in the presence of the nonselective COX inhibitor, indomethacin (Indo) (3 μM), or the COX-2 selective inhibitor, NS-398 (0.4–10 μM) in serum-free medium. Formation of PGE2 and 15d-PGJ2 were analyzed 3 hours after the stimulation. *P < 0.05 versus no inhibitors; †P < 0.01. (d) Substrate-enhanced COX proteins. Western blot analysis for COX-2 and COX-1 in 3T3-L1 1-day preconfluent cells after pretreatment with 15 μM AA.
Figure 2
Figure 2
3T3-L1 cells were treated with DM or ciglitazone (15 μM) to initiate differentiation and were evaluated on days 8 or 10. (a) COX inhibition fails to modulate DM-induced adipocyte differentiation. The extent of TAG formation was estimated in cells differentiated with DM, ciglitazone (Cig), or DM in combination with indomethacin (3 μM) or NS-398 (10 μM). (b) PG formation is not enhanced during adipogenesis. Products during adipogenesis, as determined by Northern blotting (PPARγ, aP2, GAPDH), RT-PCR (COX-1), ribonuclease protection assay (COX-2) analysis, and LC-MS-MS (PGE2, 15d-PGJ2) in the medium (med) and cells. (c) Inhibition of PG formation by indomethacin fails to modulate the induction of adipocyte protein expression by DM. (d) Inhibition of PG formation fails to influence accumulation of lipid induced by DM. Oil red O staining of cells illustrates lipid accumulation in untreated cells versus cells treated with DM ± indomethacin. (e) 15d-PGJ2 drives adipogenesis in a PPARγ-dependent manner. Cells were treated with 15d-PGJ2 (0.1–15 μM) for 2 days in the absence (open circle) or presence (filled circle) of the PPARγ antagonist BADGE (100 μM). (f) Low concentrations of 15d-PGJ2 do not amplify the effect of ciglitazone. Cells were differentiated with ciglitazone alone (filled circle) or in combination with 1 μM (open circle) or 0.1 μM 15d-PGJ2 (filled triangle). (g) COX inhibition fails to modulate the enhancement of ciglitazone-dependent differentiation by DM. Cells were incubated to differentiate by adding ciglitazone alone (filled triangle) or in combination with DM (filled circle) or DM + indomethacin (open circle).
Figure 3
Figure 3
15d-PGJ2 in human urine. (a) Mass chromatogram of 15d-PGJ2 in human urine. Representative chromatogram obtained by NI-ESI- MRM. Healthy human volunteers abstained from NSAID use for more than 5 days before urine collection. Asterisks denote isomers of [2H4]15d-PGJ2 present in the internal standard. The major detectable isomer is identified by shading (b) Formation of 15d-PGJ2 by dehydration of PGJ2 or PGD2. Unlabeled PGD2 or PGJ2 (0.2 ng/ml) was added to human urine at T = 0 hour. Aliquots (5.0 ml) were analyzed at the indicated time points for loss of spike and concomitant formation of 15d-PGJ2. The numbers above bars represent amount (%) of added PG remaining at the corresponding time point. (c) Failure to detect endogenous PGJ2 in freshly voided urine. Representative chromatogram of PGJ2 in urine. NI-ESI- MRM of urine samples (5.0 ml + 1.0 ng [18O2]-PGJ2) that were SPE extracted within 15 minutes of voiding. HPLC mobile phase was 30% CH3CN (isocratic). Other parameters were identical to those used for 15d-PGJ2. The PGJ2 peak is shaded. Estimation of the threshold of PGJ2 detection was based on the least integratable peak.
Figure 4
Figure 4
Biosynthesis of 15d-PGJ2 in humans. (a) Varied impact of COX inhibition on PG metabolites and a auto-oxidative product of arachidonic acid. Urine collections were obtained before and after aspirin (81 mg/d × 6 days) as described in the Methods section. The effects of a COX inhibitor on 15d-PGJ2 formation are compared with markers of COX-dependent (Tx-M and PGI-M) and COX-independent, free radical catalyzed isoprostane (8,12-iso-iPF-VI). Tx-M = 2,3-dinor TxB2. *P < 0.05. (b) COX-2 is the dominant source of 15d-PGJ2 biosynthesis in humans. 15d-PGJ2 levels in control volunteers (n = 21) were compared with those treated with the COX-2 selective NSAID celecoxib (n = 6) or nonselective ibuprofen (n = 5) (800 mg), administered acutely 30 minutes before urine collection. *P < 0.05; **P < 0.01. (c) Biosynthesis of PGI2, but not 15d-PGJ2, is altered during an acute inflammatory response in humans. Volunteers (n = 6) received LPS (4 ng/kg). PGI-M (2,3-dinor 6-keto-PGF) was measured at multiple time points and is plotted as the mean ± SD (dotted line). For 15d-PGJ2 measurements, urine was collected before LPS treatment (–2 to 0 hours) and at time points corresponding to peak inflammatory (4–6 hours) and resolution (16–24 hours) phases of response. Each individual is represented by a unique symbol that is conserved across time points. Median levels are indicated by horizontal lines. (d) Biosynthesis of 15d-PGJ2 is unaltered in diabetes or obesity. 15d-PGJ2 levels in healthy individuals are shown in comparison to obese (BMI ≥ 30) and nonobese (BMI < 30) patients with noninsulin-dependent type 2 diabetes. Median levels are indicated by horizontal lines.

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