Establishmentof An In Vitro PPARγ-ligand Screening System For Identifying Novel Compounds With PPAR-partial Agonist Activity

Background: Type2diabetes (T2D) is an insulin resistant state ofbody tissues caused by a variety of factors which lead to decreased insulinsensitivity. With the changing of lifestyle, excess energy intakes, sedentaryhabits and persisting work stress led to a dramatic increase of diabetes inChina. According to the International Diabetes Federation (IDF), there are371million adults living with diabetes, the number of diabetic in China hasmore than92million. More than90%of diabetes are Type2diabetes in theworld. Now diabetes has become the third largest chronic diseases thatendanger human health，coming after tumor and cardiovascular diseases.Peroxisome proliferator-activated receptor γ (PPARγ) is aligand-activated transcription factor that belongs to a superfamily ofnuclear hormone receptors and plays a pivotal role in adipocytedifferentiation. Its ligands, thiazolidinedione (TZDs), such as rosiglitazone,have been proved to ameliorate insulin resistance and widely used to treat type2diabetes. Based on cell transactivation assays, PPARγ agonists areclassified as either full or partial agonists depending upon differentialphysical interaction with the receptor and maximal efficacy on activatingPPARγ.Compared with full PPARγ agonists, several partial PPARγ agonistsshow similar effect of insulin-sensitizing, however with lower risk causingside effects. Therefore it is necessary to identify more safer and effectivePPARγ part agonists.Methods: Human PPARγ plasmid (pIRES2-PPARγ), firefly luciferasereporter plasmid (pPPRE×3-TK-Luciferase) and an internal referenceplasmid (pRL-TK) were cotransfected into HEK293cells by usingLipofectamineTM2000. The optimal ratio of three plasmids wasdetermined. Luciferase expression intensity was determined to assess thePPARγ agonist activity after addition of potential ligand into cotransfectedHEK293cell culture. Specificity of the model was examined by treatmentof PPARγ agonist, PPARγ specific antagonist, PPARα agonist and severalother nuclear receptor agonists. Repeatability was also determined by Z’value. Time dependent activation of PPARγ agonist was also observed.CMHX008exhibited ideal agonist activity on PPARγ.3T3-L1cellswere used to examin insulin sensitizer effects of CMHX008.3T3-L1celldifferentiation was induced typically in the presence of CMHX008orrosiglitazone. Oil red O staining, cell morphology observation, and determination of intracellular TG content were performed on the seventhday of differentiation. Finally, adiponectin and aP2mRNA levels and thesecretion of adiponectin were assessed by Q-PCR and ELISA afterCMHX008or rosiglitazone treatment.Results: PPARγ plasmid, reporter gene vector and internal controlplasmid ratio of2:6:1was proved to be suitable for highest relativeluciferase activity. PPARγ agonist rosiglitazone can significantly increasethe relative luciferase activity and co-treatment with GW9662resulted insignificantly reduced expressions of luciferase and increased relativeluciferase activity in the HEK293cells in a time dependent manner.Dexamethasone, all-trans retinoic acid,17β-estradiol and fenobrate did notalter the relative luciferase activity. Z’ Value was0.70based on the resultsfrom9repeating experiments.CMHX008exihibited ideal PPAR γ agonist activity and the potencywas lower than that of rosiglitazone. Results of Oil red O staining, cellmorphology and TG contents showed that CMHX008can effectivelypromote3T3-L1pre-adipocyte differentiation and lipid deposition, but itsintensity was lower than rosiglitazone. Q-PCR and ELISA assays show thatCMHX008increased the expression of adiponectin and secretion with thesimilar potency as rosiglitazone, but the induction to aP2significantlylower than rosiglitazone.Conclusions: An in vitro PPARγ ligand screening system was established, with relatively high specificity and stability. CompoundCMHX008was a novel PPARγ partial agonist, showing the similarinsulin-sensitizing effects as rosiglitazone, however with lower adipogeniccapacity.