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Biosynthesis of 15-deoxy-Δ12,14-PGJ2 and the ligation of PPARγ
L. Chastine Bell-Parikh, … , Muredach Reilly, Garret A. FitzGerald
L. Chastine Bell-Parikh, … , Muredach Reilly, Garret A. FitzGerald
Published September 15, 2003
Citation Information: J Clin Invest. 2003;112(6):945-955. https://doi.org/10.1172/JCI18012.
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Article Cell biology

Biosynthesis of 15-deoxy-Δ12,14-PGJ2 and the ligation of PPARγ

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Abstract

15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) has been identified as an endogenous ligand for PPARγ, inducing adipogenesis in vitro. Additional roles for this molecule in the propagation and resolution of inflammation, ligation of NF-κB, 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 PPARγ-dependent adipocyte activation and is unaltered in clinical settings in which PPARγ activation has been implicated.

Authors

L. Chastine Bell-Parikh, Tomomi Ide, John A. Lawson, Peter McNamara, Muredach Reilly, Garret A. FitzGerald

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Figure 1

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COX-dependent biosynthesis of 15d-PGJ2 by 3T3-L1 preadipocytes. (a) Cell...
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.
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