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

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 (ITT) show that the compound improves glucose homeostasis and insulin sensitivity; LT175 decreases plasma glucose, insulin, Not Esterified Fatty Acids (NEFA), triglycerides and cholesterol and increases adiponectin and FGF21 circulating levels. I determined the levels of cholesterol and triglycerides in the liver and their distribution in lipoprotein fractions. As expected, LT175 decreased total cholesterol in mice, and lowered VLDL triglycerides, possibly by increasing triglyceride catabolism. Interestingly, LT175 decreases total body weight, reducing visceral fat as assessed by in vivo Magnetic Resonance Imaging (MRI) and increasing brown adipose tissue mass. The analysis of the gene expression of different PPAR targets in the liver and white adipose tissue shows an increased expression for Fibroblast Growth Factor 21 (Fgf21), HMG-CoA Synthase 2 (Hmgcs2), Acyl-CoA Oxidase 1 (Acox1), Acyl-CoA Dehydrogenase, long chain (Acadl), Acyl-CoA Dehydrogenase, medium chain (Mcad), Transcription Factor A, mitochondrial (Tfam), Carnitine Palmitoyltransferase 1α (Cpt1α) in the liver and Fatty Acid Binding Protein 4 (Fabp4), Glucose Transporter type 4 (Glut4), Adiponectin (Acrp30) in white adipose tissue after treatment with LT175. Moreover, PPARγ full agonists enhance the expression of ENaCγ, the gene encoding for renal sodium transporter, whose increase is involved in fluid retention. In this experimental model rosiglitazone enhances the expression of this gene, while LT175 does not elicit any effect. Collectively, these results show that LT175 behaves differently as compared to the PPARγ full agonist rosiglitazone in this experimental model, mainly concerning weight gain, the most important side effect induced by the thiazolidinediones. To better understand how LT175 works, i focused on the mechanisms involved in the decreased lipid accumulation observed in vitro and in vivo. These effects could be due to differential induction of PPARγ targets by the ligands. The ability of the ligands to stimulate the 3T3-L1 preadipocytes differentiation to adipocytes was investigated, and no differences between the treatment with LT175 or with rosiglitazone on differentiation was observed; moreover, the expression of genes involved in lipid catabolism and mitochondrial oxidative metabolism was investigated by measuring mRNA levels of Acadl, Cytochrome C (CytC), Succinate CoA Ligase alpha subunit (Suclg1), Isocitrate Dehydrogenase 3 alpha (Idh3α), showing no influences on these parameters induced by LT175. In addition, LT175 did not change mitochondrial DNA content, a parameter reflecting mitochondrial biogenesis. These results rule out the hypothesis whereby increased mitochondrial oxidative metabolism underlies the decreased lipid content in adipocytes. Therefore, to explain the decreased lipid accumulation in adipocytes treated with LT175, the expression of genes involved in lipid uptake and storage was tested. Rosiglitazone exerts its action by enhancing the expression of genes for fatty acid uptake (Cd36) and glycerol 3- phosphate formation (Phosphoenolpyruvate Carboxykinase Pck1 and Glycerol Kinase Gk), while LT175 does not increase the expression of these genes. These results suggest that decreased expression of genes for glycerol 3-phosphate production, required for fatty acid esterification and storage in adipose tissue, may explain the lower lipid accumulation in adipocytes in vitro. Finally, to explain at the molecular level the different behaviour of the partial agonist LT175 as opposed to the full agonist rosiglitazone, i focused on the different interaction of the ligand with the receptor. The consequent differential structural conformation induced by the ligand may allows a different profile of coregulator recruitment and ultimately dictates the selectivity of the response to the ligand. As mentioned above, LT175 recruits differential set of coregulators when compared to rosiglitazone, as assessed by coregulator recruitment by FRET assay. Therefore, based on these results, i decided to investigate the quantity and the balance of PPAR coregulators in different murine tissues. This analysis, coupled with the biochemical data of in vitro recruitment of PPAR coregulators, can predict the behavior of different PPAR ligands depending on the absolute content and the ratio of coactivators/corepressors in different tissues. All together, these results allowed to elucidate the mechanisms underlying PPAR activation by different ligands. LT175 has been characterized as a new dual PPAR α/γ ligand with improved therapeutic profile in diabetic mice. This integrated experimental approach will be used in the future to design scaffold molecules for the treatment of obesity and type 2 diabetes.