Understanding The Role Of PPARá In The Regulation Of Lipid And Carbohydrate Metabolism In Skeletal Muscle Cells

Nuclear hormone receptors (NRs) are agonist-dependent transcription factors that translate nutritional and physiological signals into gene regulation. The significance of NRs in human health is highlighted by the variety of medicinal drugs associated with dysfunctional hormone signalling, in the context of inflammation, endocrine and metabolic diseases (including dyslipidemia and diabetes). A subgroup of the NR family controls metabolism in a tissue/cell specific manner. For example, the Peroxisome Proliferator Activated Receptor (PPAR) subgroup comprising PPARá, â/ä and ã isoforms, regulate lipid storage, adipogenesis and lipid catabolism in an organ specific manner. For example, agonists such as the hypolipidemicfibrates and insulin sensitizer-thiazolidinedione (TZD), that modulate PPARá and PPARã respectively, have utility in the treatment of dyslipidemia and diabetes, respectively. Many NRs are expressed in skeletal muscle, a major mass tissue that accounts for ~40% of the total body mass and energy expenditure. This lean tissue is a major site for lipid mobilization and catabolism, cholesterol efflux, and insulin-stimulated glucose disposal. Moreover, this tissue expresses cytokines that regulate adiposity, and energy expenditure, in a process that involves reciprocal signalling between adipose tissue and muscle. Therefore, this peripheral organ plays a critical role in insulin sensitivity, the blood lipid profile, and energy balance. Accordingly, skeletal muscle has an important role in the progression of dyslipidemia, diabetes and obesity. Further, several studies demonstrate that NRs in muscle regulate carbohydrate, lipid and energy homeostasis. Therefore, NR and skeletal muscle are therapeutic targets in the battle against metabolic disorders. PPARá is abundantly expressed in skeletal muscle, surprisingly, the precise role and function of PPARá in skeletal muscle cell metabolism has not been examined. Nevertheless, given the utility of hypolipidemic fibrate drugs, and the contribution of muscle to lipid homeostasis, the role of PPARá in skeletal muscle needs to be further investigated. Correspondingly, the objective of this study was to examine the differential effects of clinically utilized fibrates on metabolic gene expression in skeletal muscle cells. Furthermore, we were interested in identifying the molecular mechanisms that mediated the agonist specific effects on gene expression. Consequently, we utilized a number of clinically used hypolipidemic fibrates including fenofibrate, clofibrate, ciprofibrate and gemfibrozil to demonstrate that PPARá, can functionally regulate genes involved in lipid and carbohydrate homeostasis in skeletal muscle cells. We demonstrate that fenofibrate induces genes involved in fructose uptake and glycogen metabolism in skeletal muscle cells. Interestingly, fenofibrate represses the mRNA expression of SREBP1c and ABCA1, classical LXR target genes. Moreover, we demonstrate that the different PPARá agonists have similar, but distinct effects on gene expression. Each drug appears to have a unique regulatory footprint. These studies also suggest that the differential effects of fibrate esters and acids in skeletal muscle cells involves cross talk with the oxy-sterol dependent LXR signaling pathway. In addition, we show overlapping, and distinct effects of selective ligands for PPAR-á, -â/ä and ã isoform in skeletal muscle cells, where PPARá regulates expression of genes implicated in, TG hydrolysis, fructose uptake and glycogen synthesis, PPARâ/ä isoform regulates expression of genes implicated in preferential lipid utilization, FA catabolism and energy uncoupling and finally, PPARã regulates genes involved in glucose uptake, FA synthesis and lipid storage. We utilized cDNA microarray expression profiling technology to rigorously characterize the genes that were upregulated or downregulated in response to the PPARá agonists. Unfortunately, due to several technical shortcomings we were unable to extensively elucidate the transcriptional program induced in response to the PPARá agonists. This is elaborately discussed in Chapter-4. However, we identified two genes implicated in lipid metabolism, Melanocortin 2 receptor (MC2R) and Glutathione-S-Transferase (GST), which were differentially expressed in response to fenofibrate. In addition we observed Sortilin and Insig1 were differentially expressed in response to wyeth14643. MC2R belongs to the melanocortin pathway and is implicated in the process of lipolysis, while GST is an oxidative stress response gene that prevents the free radical formation, that are linked to the development of insulin resistance. Additionally, Sortilin and Insig1 are implicated in the insulin mediated glucose uptake and antilipogenic activity respectively. The differential expression of these genes provided us with a new direction by which PPARá may be regulating the pathogenesis of metabolic diseases as a manifestation of lipid imbalance. However, due to limited time for this study, validation of these results could not be performed. We had observed that fenofibrate repressed the expression of the LXR target genes, SREBP1c and ABCA1 in the presence and absence of the LXR agonist, T0901317. Therefore, we endeavored to investigate the molecular mechanism underlying the regulatory cross talk between PPARá and LXR signaling pathways. Our studies utilizing the GAL4 hybrid assay suggest that fenofibrate antagonizes LXR agonist mediated activation. We hypothesized that fenofibrate antagonizes LXR activity by attenuating LXR mediated corepressor displacement and/or cofactor recruitment. However, in a mammalian two-hybrid assay, we observed that fenofibrate (in the absence of LXR agonist) efficiently induced corepressor displacement from LXR. In contrast, fenofibrate had an additive effect on LXR dependent cofactor recruitment. These studies presents us with a paradox, that although fenofibrate represses LXR activity, it does not prevent corepressor displacement or inhibit coactivator recruitment by LXR. These data suggest fenofibrate-mediated attenuation of LXR activity and LXR dependent gene expression does not involve cofactor displacement/recruitment. Further studies are therefore required to elucidate the precise mechanism of fenofibrate-mediated modulation of LXR activity and LXR dependent gene expression. In conclusion, the activation of PPARá in skeletal muscle and the transcriptional regulation of the genes involved in the lipid and carbohydrate homeostasis, suggests PPARá activation in skeletal muscle is an important site for the therapeutic effect of fibrates