Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma

Abstract

Peroxisome proliferator-activated receptors (PPARs) alpha and gamma are key regulators of lipid homeostasis and are activated by a structurally diverse group of compounds including fatty acids, eicosanoids, and hypolipidemic drugs such as fibrates and thiazolidinediones. While thiazolidinediones and 15-deoxy-Delta12, 14-prostaglandin J2 have been shown to bind to PPARgamma, it has remained unclear whether other activators mediate their effects through direct interactions with the PPARs or via indirect mechanisms. Here, we describe a novel fibrate, designated GW2331, that is a high-affinity ligand for both PPARalpha and PPARgamma. Using GW2331 as a radioligand in competition binding assays, we show that certain mono- and polyunsaturated fatty acids bind directly to PPARalpha and PPARgamma at physiological concentrations, and that the eicosanoids 8(S)-hydroxyeicosatetraenoic acid and 15-deoxy-Delta12,14-prostaglandin J2 can function as subtype-selective ligands for PPARalpha and PPARgamma, respectively. These data provide evidence that PPARs serve as physiological sensors of lipid levels and suggest a molecular mechanism whereby dietary fatty acids can modulate lipid homeostasis.

Figure 1

Structure of the fibrate GW2331.

Figure 2

[³H]GW2331 binds to PPARα and PPARγ. (A) Control bacterial extracts (c) or bacterial extracts containing GST-hPPARα (h), GST-mPPARα (m), or GST-xPPARα (x) were incubated with 60 nM [³H]GW2331 in either the absence or presence of 10 μM unlabeled GW2331 as indicated. The amount of GST included in the control experiments was comparable or exceeded the amount of GST-PPAR fusion proteins used in binding experiments as judged by SDS/PAGE analysis and Coomassie blue staining of bacterial extracts (data not shown). Data shown represent the mean of assays performed in triplicate ± SD. (B) Bacterial extracts containing GST-xPPARα were incubated with increasing concentrations of [³H]GW2331 in the absence (total binding; □) or presence (nonspecific binding; ▵) of 10 μM unlabeled GW2331. Specific binding is indicated (○). (Inset) Specific binding data were transformed by Scatchard analysis. (C) Bacterial extracts containing GST-xPPARγ were incubated with increasing concentrations of [³H]GW2331 in the absence (total binding; □) or presence (nonspecific binding; ▵) of 10 μM unlabeled GW2331. Specific binding is indicated (○). (Inset) Specific binding data were transformed by Scatchard analysis.

Figure 3

FAs are ligands for xPPARα and xPPARγ. (A) CV-1 cells were cotransfected with expression plasmids encoding either GAL4-xPPARα or GAL4-xPPARγ and the reporter plasmid UAS₅-tk-CAT. Cells were treated with vehicle (0.1% dimethyl sulfoxide; DMSO) alone, 1 × 10⁻⁴ M of the various FAs, or 1 × 10⁻⁶ M GW2331. Cell extracts were subsequently assayed for CAT activity. Data points represent the mean of assays performed in triplicate. Similar results were obtained in two independent experiments. (B) Competition binding assays were performed using bacterial extracts containing either GST-xPPARα or GST-xPPARγ and either 30 nM (xPPARα) or 60 nM (xPPARγ) [³H]GW2331 in the presence of 3 × 10⁻⁵ M of the indicated FAs or unlabeled GW2331. Data are presented as percent competition relative to addition of vehicle (1% DMSO) alone and represent the mean of assays performed in triplicate. Similar results were obtained in two independent experiments.

Figure 4

PUFAs are ligands for xPPARα and xPPARγ. Competition binding assays were performed with (A) GST-xPPARα and 30 nM [³H]GW2331 or (B) GST-xPPARγ and 60 nM [³H]GW2331 in the presence of increasing concentrations of linolenic acid (▵), linoleic acid (○), or arachidonic acid (□). Data represent the mean of duplicate points and were normalized to reactions performed with vehicle (1% DMSO) alone.

Figure 5

Eicosanoids are selective xPPARα and xPPARγ ligands. (A) CV-1 cells were cotransfected with expression plasmids encoding either GAL4-xPPARα or GAL4-xPPARγ and the reporter plasmid UAS₅-tk-CAT. Cells were treated with vehicle (0.1% DMSO) alone or 1 × 10⁻⁵ M of the eicosanoids 8(S)-HETE, 8(R)-HETE, or 15d-J₂. Cell extracts were subsequently assayed for CAT activity. (B and C) Competition binding assays were performed with (B) GST-xPPARα and 30 nM [³H]GW2331 or (C) GST-xPPARγ and 60 nM [³H]GW2331 in the presence of increasing concentrations of 8(S)-HETE (▵), 8(R)-HETE (○), or 15d-J₂ (□). Data represent the mean of duplicate points and were normalized to reactions performed with vehicle (1% DMSO) alone.