Novel mechanisms in PPARy-mediated adipogenesis

Abstract

Obesity-induced type 2 diabetes mellitus (T2DM) has grown to epidemic proportions in the last decades. The number of adults with T2DM in the United States has increased rapidly by almost 50% between 1991 and 2000 according to the Center for Disease Control and Prevention (CDC). Furthermore, the World Health Organisation (WHO) predicts that the number of T2DM patiens worldwide will grow to as much as 300 million in the year 2025. T2DM is not characeterised by the lack of insulin production found for the much less common type 1 diabetes mellitus, but manifests itself by decreased sensitivity towards insulin. In both types of diabetes similar complications arise as a result of impaired insulin signalling and lack of response to increased glucose levels. Serious complications, mostly resulting from vascular problems, like kidney failure and blindness are often found in untreated diabetic patients. Both types of diabetes can be treated and, in the case of T2DM, this often involves administration of insulin sensitizing drugs. A commonly used group of insulin sensitizers are the thiazolidinediones (TZDs). In 1995 it was found that TZDs function as ligands for the transcription factor PPAR a member of the nuclear receptor superfamily and the TZDs rosiglitazone and pioglitazone are now well known compounds, both in clinical and experimental settings. The importance of the different chromatin remodelling complexes in adipogenesis and PPARmediated gene expression has become evident from various studies. Ribozyme mediated decrease of the histone acetyltransferases CBP and p300 expression, inhibited PPARmediated gene expression and adipogenesis. In accordance with these findings CBP-deficient as well as heterozygous mice show a reduced fat mass due to undeveloped white adipose tissue. Furthermore expression of dominant negative BRG-1 or BRM, components of the ATP-dependent chromatin remodelling SWI/SNF complex, also decreased PPAR mediated gene activation. The main role of the TRAP complex, which is related to the yeast mediator proteins, lies in direct communication between the various coactivators and the general transcription complex. TRAP220 is an essential subunit of the TRAP complex, interacting with various nuclear receptors including PPAR. By using TRAP220-/- fibroblasts it was shown that the TRAP complex plays an essential role in adipogenesis, acting as a PPARmediator complex. These data underscore the important function of coactivator complexes in PPAR signalling and thereby regulation of energy metabolism. Therefore, further understanding of PPAR activity is important since novel PPAR modifiers could offer the possibility of therapeutic modulation and provide new ways of improving insulin sensitivity (see also chapter 2). Therefore, as described in this thesis, we set out to investigate the different levels at which coactivators function in PPAR2 activation and also how mutant forms of PPARare affected in their associations with coactivator proteins (chapter 3). In an effort to identify novel regulators of PPAR we have developed an immunoprecipitation assay coupled to mass spectrometry analysis (chapters 4 and 5) as well as a novel screening method based on PPAR2 driven miRNA expression (chapter 6) for the use of high troughput siRNA library screens. Using these assays, several novel PPAR2 interactors were identified.