STUDY OF THE MOLECULAR MECHANISM UNDERLYING PPAR ACTIVATION BY LT175, A NOVEL DUAL PPARA/G LIGAND THAT AMELIORATES THE METABOLIC PROFILE AND INSULIN SENSITIVITY IN A MOUSE MODEL OF INSULIN RESISTANCE

Abstract

The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily, ligand-dependent transcription factors that play a key role in the regulation of lipid, glucose and energy metabolism. Recent works aim at developing new PPAR agonists devoid of the side effects of the marketed antidiabetic agents thiazolidinediones and the dual PPAR α/γ agonists glitazars. To this purpose, it’s fundamental to understand the molecular mechanism underlying PPAR activation induced by different ligands. The aim of my project is to study the peculiar mechanism of action of LT175, a novel dual PPAR α/γ ligand, to better understand how the ligand-receptor complex works. In the first part of my doctorate project i reported that LT175 binds and activates both PPARα and PPARγ. LT175 is a more potent human PPARα agonist than Wy 14,643, whereas his efficacy is comparable; the potency and efficacy of this ligand to activate PPARγ is significantly lower than that of rosiglitazone. FRET experiments were set to investigate the coregulator recruitment induced by the ligand; differently from rosiglitazone, LT175 does not allow the recruitment of the coactivator CREB Binding Protein (CBP) on PPARγ, while the corepressor NCoR still interacts with the receptor. These results support the hypothesis that the compound is a full PPARα agonist and a partial PPARγ agonist. Next, i tested the biological activity of LT175 incubating confluent 3T3-L1 preadipocytes with insulin and different PPAR ligands for 7 days. The analysis of gene expression indicates that LT175 activates the PPARγ-dependent program of differentiation in adipocyte cultures; however, adipocytes differentiated in the presence of LT175 accumulate less lipids as compared to those treated with rosiglitazone. Based on these promising results, i decided to study the effect of the administration of this compound in vivo using different animal models. To evaluate the in vivo bioavailability and transcriptional activity, LT175 was orally administered (100 mg/kg body weight/day) for 3 days to PPRE-LUC reporter mice. The luciferase activity was evaluated by ex-vivo imaging in the chest and by enzymatic assay in the liver, white (WAT) and brown (BAT) adipose tissue, bone, myocardium, intestine and brain. The results show that LT175 reaches the liver, WAT, BAT and in vivo switches on the PPAR-dependent transcription program. In a different experiment, PPRE-LUC reporter mice were treated for 19 days with different PPAR ligands, and the magnitude and distribution of luminescence induced by PPAR activation was evaluated by ex-vivo imaging every day in chest and abdomen areas. The results show a different spatial PPAR activation induced by LT175 during the days of treatment, compared to the reference PPARα agonist Wy 14,643. To test the in vivo influence on metabolic profile induced by LT175, the compound was orally administered for 2 weeks to C57Bl/6 mice fed with a high fat diet (DIO) for 16 weeks. The Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test…