Identification and mechanism of 10-carbon fatty acid as modulating ligand of peroxisome proliferator-activated receptors

Abstract

Peroxisome proliferator-activated receptors (PPARα, -β/δ, and -γ) are a subfamily of nuclear receptors that plays key roles in glucose and lipid metabolism. PPARγ is the molecular target of the thiazolidinedione class of antidiabetic drugs that has many side effects. PPARγ is also activated by long chain unsaturated or oxidized/nitrated fatty acids, but its relationship with the medium chain fatty acids remains unclear even though the medium chain triglyceride oils have been used to control weight gain and glycemic index. Here, we show that decanoic acid (DA), a 10-carbon fatty acid and a major component of medium chain triglyceride oils, is a direct ligand of PPARγ. DA binds and partially activates PPARγ without leading to adipogenesis. Crystal structure reveals that DA occupies a novel binding site and only partially stabilizes the AF-2 helix. DA also binds weakly to PPARα and PPARβ/δ. Treatments with DA and its triglyceride form improve glucose sensitivity and lipid profiles without weight gain in diabetic mice. Together, these results suggest that DA is a modulating ligand for PPARs, and the structure can aid in designing better and safer PPARγ-based drugs.

Figures

FIGURE 1.

Characterization of DA binding. a, AlphaScreen assay of different saturated and unsaturated fatty acid chains at 10 μm concentrations in the presence of 20 nm H6 PPARγ LBD and 20 nm biotinylated CBP-1 peptide. DMSO was used as a control. (MCFA, medium chain fatty acid; LCSFA, long chain saturated fatty acids; LCUFA, long chain unsaturated fatty acids) (n = 3, ± S.E.), *, petroselenic acid (cis-6); #, oleic acid (cis-9). b, AlphaScreen assay of DA with CBP-1 peptide and ligand binding domains of PPARα, -δ, and -γ. c, AlphaScreen assay of rosiglitazone and DA was performed in the presence of 20 nm biotinylated NCOR1 peptide. d, AlphaScreen® assay of the DA and Rosi with different biotinylated LXXLL cofactor peptide motifs. The ligands were used at 10 and 50 μm concentrations. H6 PPARγ LBD and peptides were used at 20 nm concentrations (n = 3, ± S.E.). e, LanthaScreenTM TR-FRET assay (Invitrogen) was used for measuring the binding affinities of the ligands OA, DA, and Rosi. The GST-PPARγ LBD was used at 0.5 nm concentration, and the ligands competed against 5 nm concentration of FluormoneTM. Terbium-coated GST antibody was used at 5 nm concentration. The concentration of the ligands used ranged from 1000 to 0.01 μm. Concentration is represented as log10 scale. The calculated ki values of DA and Rosi are 41.7 μm and 53 nm, respectively (n = 2, ± S.E.).

FIGURE 2.

Luciferase reporter assay of PPARγ activation in COS-7 cells. Luciferase reporter assay was performed in COS-7 cells. The luciferase activity was normalized against Renilla luciferase units. Fold activation was calculated against DMSO with no ligand treatment. Scr, siRNA-scrambled siRNA. a, fold activations of Rosi and DA were measured at 10 and 50 μm concentrations and in the presence of orlistat at 100 μm and 150 ng of Lpl plasmid. b, luciferase activity of glycerol triesters of DA was measured at 10 and 50 μm concentrations. Luciferase activity was also measured in the presence of orlistat at 100 μm, Lpl, Lpl siRNA, and nontargeted scr siRNA (n = 3, ± S.E.). c, quantitative PCR of lipoprotein lipase mRNA expression in COS-7 cells with GT at 10 and 50 μm concentration. The treatments were normalized to untreated COS-7 cells.

FIGURE 3.

3T3-L1 adipocyte differentiation assay and quantitative PCR. Oil Red O staining of 3T3-L1 fibroblast cells after differentiating with different ligands for 2 days and maintained in media with insulin for 12 days. Treatment groups include the following: a, DMI (1 μm dexamethasone (D); 0.5 mm 3-isobutyl-1-methylxanthine (IBMX) (M); and 167 nm insulin (I)); b, DMI + DA 300 μm; c, Rosi 10 μm; d, Rosi 10 μm + DA 300 μm; e, DA 300 μm; f, DMSO control. Quantitative PCR and relative mRNA expression of the adipocyte differentiation genes Pparg, Cebpa Srebp1, Fabp4, Lep, AdipoQ, Cox7a1, and Pgc1a on mRNA extracted from differentiating cells after 2 days (g) and 8 days (h). (n = 2, ± S.E.)

FIGURE 4.

3T3-L1 adipocyte differentiation assay using OA, OLA, and Rosi. Oil Red O staining of 3T3-L1 fibroblast cells after differentiating with different ligands for 2 days and maintained in media with 167 nm insulin for 12 days. Treatment groups include the following: a, DMSO; b, Rosi 10 μm; c, OA 300 μm; d, Rosi 10 μm + OA 300 μm; e, OLA 300 μm; f, Rosi 10 μm + OLA 300 μm.

FIGURE 5.

Crystal structure of PPARγ LBD with DA (PDB code 3U9Q). a, schematic representation of PPARγ LBD bound to DA. Helices are represented in red, sheets in yellow, and loops in green. DA is presented as blue dots. Helices are abbreviated as H. b, superimposition of DA (green), rosiglitazone (blue), and nitrolinoleic acid (LNO2) (yellow) with respect to H3. Oxygen atoms are represented as red, nitrogen atoms as dark blue, and sulfur as orange. c, polar interactions of the TZD group of rosiglitazone (blue) and carboxyl headgroup of DA (green) with residues with Ser-289 from H3, His-323 from H5, His-449 from H10, and Tyr-473 from the AF-2 helix. d, side-on superimposition of the PPARγ H10 from the crystal structures bound to DA (red) and rosiglitazone (cyan) (PDB 3CS8) shows a kink at the His-449 position in case of the rosiglitazone (cyan)-bound structure. e, hydrogen/deuterium exchange (HDX) data plotted over the structures of PPARγ LBD bound with rosiglitazone (left, PDB 2PRG) and DA (right, 3U9Q). Percentage reduction in hydrogen/deuterium exchange relative to unliganded receptor is colored according to the key. f, electron density map (2Fo − Fc at 1σ) of DA in the ligand binding pocket of PPARγ and the residues that form polar interactions. g, stick representation of the PPARγ crystal structure bound to DA showing the displacement of F282. h, stick representation of Phe-282 residue of the PPARγ structure 2HFP bound to rosiglitazone.

FIGURE 6.

Profiling of DA and its triglyceride form GT in db/db mice. In one set of experiments, male db/db mice were randomly divided into three groups of seven each and were given regular chow diet or modified chow diet containing GT (10 g/kg diet) and pioglitazone (pio) (100 mg/kg diet), respectively, for 4 weeks. In another set of experiments, mice were subcutaneously injected with vehicle DMSO or 5 mg/kg rosiglitazone or 250 mg/kg DA for 4 weeks. a, blood glucose levels of mice fed control chow diet and modified diet with GT and pioglitazone after 0, 2, and 4 weeks. b, blood glucose levels of mice injected with vehicle DMSO and treatments DA and Rosi 0, 2, and 4 weeks. c, body weight change in mice fed control chow diet and modified diet with GT and pioglitazone after 0, 2, and 4 weeks. d, body weight change in mice injected with vehicle DMSO and treatments DA and Rosi after 0, 2, and 4 weeks. e, GTT of different mice fed control chow diet and modified diet with GT and pioglitazone. f, total serum cholesterol after 4 week treatment. g, total serum triglycerides after 4 weeks of treatment. (Two-way analysis of variance was performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n = 7, ± S.E.

FIGURE 7.

Quantitative PCR of different tissues from db/db mice fed DA-containing chow diet. Mice were randomly divided into three groups of seven each, which were given regular chow diet, chow diet containing glyceryl tridecanoate (10 g/kg diet), or pioglitazone (Pio) (100 mg/kg diet), respectively, for 4 weeks. Results were quantitated by using ΔΔCT method and vehicle treatment as reference. Gapdh gene was used as an internal reference. a, relative mRNA expression of Pparg, Fabp4, AdipoQ, Pgc1a, and Pgc1a in WAT. b, relative mRNA expression of Pparg, Fabp4, AdipoQ, Pgc1a, and Pgc1a in BAT. c, relative mRNA expression of Pparγ, Pparα, and Fabp1 in liver tissue (two-way analysis of variance was performed. *, p < 0.05; **, p < 0.01; ***, p < 0.001) (n = 5, ± S.E.) d, total ion chromatogram for adipose tissue from GT-treated mice. After incubation of adipose tissue with methanol/acetyl chlorides (20:1 v/v) at 70 ºC for 90 min, all forms of DA were trans-esterified in the form of methyl ester. DA shows a distinct peak when compared with the internal standard. e, Ser-273 phosphorylation of PPARγ in mouse adipose tissues. Phosphorylation status of Ser-273 in mouse adipose tissues was analyzed using Ser-273-specific antibodies. Tissues from four mice from each treatment (vehicle, GT, and pioglitazone) were analyzed for Ser-273 phosphorylation as well as total PPARγ. Cross-section of liver from mouse with different treatments. f, vehicle; g, glyceryl tridecanoate; h, pioglitazone (pio).