Polyacetylenes from Notopterygium incisum--new selective partial agonists of peroxisome proliferator-activated receptor-gamma

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of glucose and lipid metabolism and therefore an important pharmacological target to combat metabolic diseases. Since the currently used full PPARγ agonists display serious side effects, identification of novel ligands, particularly partial agonists, is highly relevant. Searching for new active compounds, we investigated extracts of the underground parts of Notopterygium incisum, a medicinal plant used in traditional Chinese medicine, and observed significant PPARγ activation using a PPARγ-driven luciferase reporter model. Activity-guided fractionation of the dichloromethane extract led to the isolation of six polyacetylenes, which displayed properties of selective partial PPARγ agonists in the luciferase reporter model. Since PPARγ activation by this class of compounds has so far not been reported, we have chosen the prototypical polyacetylene falcarindiol for further investigation. The effect of falcarindiol (10 µM) in the luciferase reporter model was blocked upon co-treatment with the PPARγ antagonist T0070907 (1 µM). Falcarindiol bound to the purified human PPARγ receptor with a Ki of 3.07 µM. In silico docking studies suggested a binding mode within the ligand binding site, where hydrogen bonds to Cys285 and Glu295 are predicted to be formed in addition to extensive hydrophobic interactions. Furthermore, falcarindiol further induced 3T3-L1 preadipocyte differentiation and enhanced the insulin-induced glucose uptake in differentiated 3T3-L1 adipocytes confirming effectiveness in cell models with endogenous PPARγ expression. In conclusion, we identified falcarindiol-type polyacetylenes as a novel class of natural partial PPARγ agonists, having potential to be further explored as pharmaceutical leads or dietary supplements.

Figure 1. PPARγ activation by N. incisum extracts and chemical structures of the isolated polyacetylenes.

(A) HEK-293 cells were transiently co-transfected with a plasmid encoding full-length human PPARγ, a reporter plasmid containing PPRE coupled to a luciferase reporter and an EGFP plasmid as internal control. After re-seeding, cells were treated as indicated for 18 h. Since the positive control pioglitazone (5 µM), as well as the dried DCM- and MeOH-extract were reconstituted in DMSO, cells were treated with equal amount of the solvent vehicle DMSO (0.1%) as negative control. The luciferase activity was normalized to the EGFP-derived fluorescence, and the result is expressed as fold induction compared to the solvent vehicle control. The data shown are means ± SD of three independent experiments each performed in quadruplet. ***p<0.001, *p<0.05 (compared to the solvent vehicle group; ANOVA/Bonferroni). (B) Chemical structures of the PPARγ-activating polyacetylenes isolated from N. incisum.

Figure 2. PPARγ-mediated transactivation activity as well as receptor binding activity of falcarindiol.

(A) HEK-293 cells, transiently transfected with a human PPARγ expression plasmid, a luciferase reporter plasmid (tk-PPREx3-luc) and EGFP as internal control, were treated with different concentrations of pioglitazone or falcarindiol (0.1–30 µM) for 18 h. Luciferase activity was normalized by the EGFP-derived fluorescence, and the result is expressed as fold induction compared to the solvent vehicle control (DMSO, 0.1%). The data points shown are means ± SEM of three independent experiments each performed in quadruplet. (B) Cells were transfected and treated as indicated above. Pioglitazone was applied at 5 µM, falcarindiol at 10 µM, and the PPARγ antagonist T0070907 at 1 µM. The data shown are means ± SD of six independent experiments each performed in quadruplet. ***p<0.001 (ANOVA/Bonferroni). (C) The cells were prepared as indicated above and treated for 18 h with different concentrations of falcarindiol, always in the presence of 1 µM pioglitazone. The data shown are means ± SD of three independent experiments each performed in quadruplet. (D) Dilutions of the two investigated compounds were prepared in DMSO and mixed with a buffer containing the hPPARγ LBD tagged with GST, terbium-labelled anti-GST antibody, and fluorescently-labelled PPARγ agonist. After 6 h of incubation, the ability of the test compounds to bind to the PPARγ LBD and thus to displace the fluorescently labelled ligand was estimated from the decrease of the emission ratio 520 nm/495 nm upon excitation at 340 nm. Each data point represents the mean ± SEM from four independent experiments performed in duplicate.

Figure 3. Proposed molecular binding mode of falcarindiol in the PPARγ LBD.

Predicted binding mode of falcarindiol shown as 3D depiction in which predicted hydrogen-bonds are shown as dashed lines (A), and 2D depiction including chemical features of the interaction pattern derived from the docking pose (B). Chemical features in the 2D depiction are color-coded: green arrow – hydrogen-bond donor; magenta – hydrophobic contacts.

Figure 4. Assessment of adipogenicity and glucose uptake-enhancing properties of falcarindiol.

(A) 3T3-L1 preadipocytes were differentiated to adipocytes as described in the Materials and Methods section for 10 days in the presence of pioglitazone, falcarindiol, solvent vehicle (0.1% DMSO), or a standard differentiation mixture (MDI; containing 10% FBS, 500 µM IBMX, 500 nM dexamethasone, and 1 µg/ml insulin) as a positive control. For an estimate of accumulated lipids, and thus for the adipogenic potential of the test compounds, Oil Red O staining and photometric quantification at 550 nm was performed. The data shown are means ± SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05 (ANOVA/Bonferroni). (B) 3T3-L1 mature adipocytes were incubated with falcarindiol (10 µM) or solvent vehicle (0.1% DMSO) for 48 h and 2-deoxy-D-(1H³)-glucose cellular uptake was determined for 10 min in the presence or absence (basal uptake) of insulin (500 pM). The data shown are means ± SEM of five independent experiments. *p<0.05 (compared to the solvent vehicle group; two-tailed paired t-test).