Oxidized Alkyl Phospholipids Are Specific, High Affinity Peroxisome Proliferator-activated Receptor γ Ligands and Agonists

Abstract

Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A 1 digestion, that PPAR␥ agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPAR␥. [ 3 H]azPC bound recombinant PPAR␥ with an affinity (K d(app) Ϸ40 nM) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket. azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nM. Overexpression of PPAR␣ or PPAR␥ revealed that azPC was a specific PPAR␥ agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPAR␥ that induce PPAR-responsive genes.The transcription factor PPAR␥, 1 in association with its 9-cis-retinoate-binding RXR partner, controls metabolic and cellular differentiation genes that contain variations of a cognate PPAR-response element (1). PPAR␥, like other members of the broad nuclear hormone receptor family, undergoes a conformational change when it binds specific lipid ligands. This structural reorganization alters its associated proteins, releasing transcriptional inhibitors and recruiting transcriptional co-activators. The regulation of PPAR␥ function is therefore controlled by lipid ligand binding.A number of synthetic ligands for PPAR␥ are known. One of these, rosiglitazone (BRL49653), binds with high affinity and is widely prescribed as an insulin sensitizer in type II diabetes. However, defining relevant biologic ligands has been problematic. Several oxidatively modified fatty acids bind and activate PPAR␥, including 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15-deoxy-PGJ 2 ), other arachidonate metabolites (2, 3), the linoleate derivatives 9-HODE and 13-HODE (4), and several free fatty acids (5, 6). However, none of these are particularly potent agonists, and for some their presence at concentrations sufficient to activate PPAR␥ can be questioned. For example, the oxygenated fatty acid products described above do not confer much advantage in potency over activation by free arachidonate (7) where a concentration of several micromolar is required to elicit a response. Moreover, the 9-and 13-HODEs and their peroxides found in oxidized LDL (8) or in skin exposed to the tumor promoter phorbol myristate ace...

Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects

“…This was first demonstrated by Davies et al 58 , who demonstrated that non-enzymatically oxidized alkyl phospholipids present in oxidized low density lipoproteins (LDL), are high affinity ligands for PPARγ. One of these species was identified as 1-hexadecyl-2-azelaoyl phosphatidylcholine (azPC), which had a Kd for binding to the PPARγ receptor of approx.…”

“…One of these species was identified as 1-hexadecyl-2-azelaoyl phosphatidylcholine (azPC), which had a Kd for binding to the PPARγ receptor of approx. 40 nM 58 .…”

“…Finally, given the ability of GPCs to serve as targets of photooxidation, how does this result in both PAF-R and PPARγ agonist production? Previous studies indicate that both PPARγ and PAF-R ligands are derived from intact oxidized alkyl phosphopholipids with a sn-1 ether linkage rather than the much larger pool of phospholipids with esterified fatty acids at the sn-1 position 58,90 . However, given the structure of PAF itself (sn-2 acetyl group), along with less potent succinoyl-PAF, 5-oxovaleroyl-PAF, butanoyl-PAF(C4-PAF) and butenoyl-PAF (C4:1-PAF), available evidence indicates that PAF-R agonists consist primarily of short chain sn-2 acyl groups terminating with either an ω-oxy function or a methyl group (Figure 1) 11,18,19 .…”

“…This idea is based on a number of observations: First, the structure of AzPC consists of a 9-carbon azelaic acid esterified at the sn-2 position 58 . Second, small chain products, like PAF, exhibit limited capacity to displace binding of the PPARγ ligand, rosiglitazone 58 .…”

“…This idea is based on a number of observations: First, the structure of AzPC consists of a 9-carbon azelaic acid esterified at the sn-2 position 58 . Second, small chain products, like PAF, exhibit limited capacity to displace binding of the PPARγ ligand, rosiglitazone 58 . Most importantly, we show that treatment of either AzPC or lipid extracts from UVB-irradiated KB cells with serum PAF acetylhydrolase (PAF-AH) results in loss of PPARγ-agonistic activities (Figure 3).…”