Selective PPARγ modulators as an alternative treatment

To overcome side effects associated with the use of TZDs, a novel approach consists in developing new PPARγ ligands that have insulin-sensitizing properties, via selective action on beneficial pathways, without exacerbating fluid retention and obesity. Such compounds are called

“selective PPARγ-modulators” (SPPARγMs) analogous to selective estrogen receptor (ER)

modulators such as raloxifen, which spares the uterus functions, but acts as a partial ER agonist in bones (291). SPPARγMs would separate the effects of PPARγ on lipid and glucose metabolisms as well as on different organ systems (gastrointestinal, immune, cardiovascular). The selective action of such compounds is thought to depend on different structural configurations induced in the ligand-binding domain by their interaction with the receptor, allowing recruitment of different complexes of cofactors that impact on the activation or repression of specific sets of target genes in different tissues (167,183,211,284,292-295).

A variety of such new PPARγ ligands with differential pharmacologicalaffinities for PPARγ have been reported in recent years. One of the first SPPARγMs that was tested, and which validated this idea, is FMOC-L-Leucine (F-L-Leu), which separates insulin sensitivity from adipogenesis in vivo (200).

The non-TZD-selective PPARγ modulator (nTZDpa) was also shown to alter the conformational stability of the receptor when compared to TZDs. Chronic treatment of fat-fed C57BL/6J mice with nTZDpa improved hyperglycemia and hyperinsulinemia and promoted reductions in weight gain and adipose depot size, without causing cardiac hypertrophy. In WAT, nTZDpa produced a different in vivo expression pattern of a panel of PPARγ target genes when compared to a full agonist (211). Similarly, a series of metabolically robust N-benzyl-indole partial PPARγ agonists, with either a 3-benzoyl or 3-benzisoxazoyl moiety, also produced potent glucose reduction in db/db mice and attenuated increases in heart weight and brown adipose tissue mass, which are typically observed in rodents upon treatment with PPARγ full agonists (296,297). In addition, halofenate, one of the recently discovered SPPARγMs, displays the characteristics of an optimized modulator, retaining an insulin sensitization potential with minimal adipogenic activity in vitro and with less weight gain in vivo (ob/ob mouse and fa/fa Zucker rat models). At the molecular level, the partial agonism of halofenic acid may be explained in part by effective displacement of the corepressors NCoR and SMRT, coupled with inefficient recruitment of co-activators, such as p300, CBP, and TRAP 220 (195). Further characterization of SPPARγMs will most likely yield novel agents for the treatment of type 2 diabetes, which are as effective as current pharmacological compounds but without their side effects. These findings should encourage mechanism-based screens (291), which would capitalize on a still very incomplete knowledge of the ensemble of

INTRODUCTION-Role of PPARγγγγ in the white adipose tissue

PPARγ coactivators and corepressors and their expression profile in tissues that are pivotal for PPARγ action.

At this point, is worth mentioning the emergence of novel potential therapeutic targets for the treatment of metabolic-related diseases, among which is the cofactor sirutin 1 (SIRT1), which is a protein lysine deacectylase and 11-β-Hydroxysteroid dehydrogenase type 1 (Hsd11β1) which allows local conversion of inactive to active cortisol. The human SIRT1 regulates several transcription factors that govern metabolism among which is PPARγ. SIRT1 is induced by fasting in several tissues, such as the WAT where it represses PPARγ activity thus decreasing the amount of fat storage. Its activation by resveratrol was shown to improve mitochondrial function and to protect against metabolic related diseases (200,298-300). Glucocorticoids are important regulators of glucose, lipid and protein metabolism, acting mainly in the liver, adipose tissue and muscle. Chronic glucocorticoid excess is associated with clinical features, such as insulin resistance, visceral obesity, hypertension, and dyslipidemia, which represent the classical hallmarks of the metabolic syndrome.

Hsd11β1, a key intracellular enzyme which catalyses the conversion of inactive to active cortisol, has been implicated in the development of the metabolic syndrome. The shift of this reaction towards active cortisol generation may lead to tissutal overexposure to glucocorticoids even with normal circulating cortisol levels. The most robust evidence in support of a pathogenic role of this enzyme in the development of the metabolic syndrome has been reported in experimental animals where specific expression of this enzyme in WAT mediates critical features of the metabolic syndrome (301). Further, Hsd11β1 knockout mice are protected from these metabolic abnormalities (301). Moreover, treatment of diet-induced obese mice with Hsd11β1 inhibitors reduces their food intake and weight gain but maintains energy expenditure (302).

As illustrated in the introduction, the white adipose tissue plays an important role in the pathologies related to the metabolic syndrome. Deregulation of its functions, either in obesity or lipodystrophy affects not only its metabolism but also the systemic metabolism. We showed in the previous chapter that PPARγ is one of the key factors involved in the adipose tissue development, maintenance and metabolism. Besides, it is also one of the most important

pharmacological targets in the treatment of several metabolic related diseases such as type 2 diabetes.

Large amounts of publications in the last years underline the involvement of PPARγ in the treatment of metabolic related diseases, namely its mechanism of action principally in the white adipose tissues where it is highly expressed. However, several questions remain open. We therefore, aimed to (i) study the role of PPARγ in the metabolism and integrity of the adipose tissue;

questioning the established dogma of PPARγ being involved solely in lipid metabolism, and (ii) set up a lentivirus derived small interference RNA (siRNA) expression system allowing modulation of the expression of PPARγ and its partners in different cellular models related to metabolic functions, specially preadipocytes and adipocytes.

Although the homozygous deletion of PPARγ in a mouse model was shown to be embryonic lethal, the survival of PPARγ -/- mice by inactivation of PPARγ in all tissues except the trophoblasts was successful. These animals suffered from lipodystrophy, insulin resistance and hypotension, most likely due to blood vessel relaxation (244). Specific deletion of PPARγ in the white adipose tissue also induces a clear lipodystrophy of the tissue (248,250). Because of the lipodystrophic phenotype, we choose to address these questions in the context of a mouse model where only one of the PPARγ alleles was deleted (PPARγ +/-). Although PPARγ +/- mice had lower amount of WAT, they were not lipodystrophic (245). Diminution by half of the normal PPARγ in the context of the PPARγ +/- mice should already have an effect on its transcriptional program. Furthermore, this model allows a better separation between the effects of its deletion in WAT and the pathways triggered in this tissue by the accumulation of dead adipocytes, such as observed in models of specific deletion of PPARγ in WAT (lipodystrophy) (162).

Because of the early role of PPARγ in the development of the adipose tissue, we set up an innovative in vitro model where the expression of PPARγ could be modulated at different moments with the help of the lentivirus-mediated gene knock-down by siRNA. This model helps the investigation of the involvement of PPARγ in mechanisms important in different aspects of the mature adipose tissue functions. Furthermore, it gives a highly flexible tool for the in vitro study of metabolic pathways not only in adipocytes but also inhepatocytes and myocytes giving

INTRODUCTION-Role of PPARγγγγ in the white adipose tissue

us the opportunity to decipher their interconnection trough the adipokine and cytokine secretion.

Indeed, we can readily imagine models in which the knock-down of a target gene in adipocytes will influence the adipokine secretion program having a direct effect on the metabolic pathway, such as insulin induced glucose sensitivity, glycolytic and lipolytic pathways in hepatic and muscle cells.

RESULTS

I. Deletion of one PPARγγγγ allele affects the integrity of the adipose tissue and has further effects