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Obesity is a major epidemic in the US and abroad, and therefore, uncovering compelling, and novel methods for addressing this issue is paramount. We have previously shown that bilirubin activates the nuclear receptor transcription factor peroxisome proliferator-activated receptor α (PPARα) to induce β-oxidation, which reduces lipid accumulation in adipocytes and hepatocytes. RNA-sequencing in HepG2 hepatocytes showed that bilirubin-induced transcriptome responses are predominantly PPARα-dependent (~95%). To further elucidate this relationship, we studied the direct interaction of bilirubin and PPARα. We found that bilirubin was specific for the PPARα, and did not interact with the other PPAR isoforms, PPARβ/δ or PPARγ. Further analysis showed that the interaction of bilirubin-PPARα lowered lipid accumulation and increased mitochondrial respiration in both brown and white adipocytes cell lines. We found that mice with hyperbilirubinemia by either treatment or genetically, had increased mitochondria function and reduced white adipocyte size, but brown adipocytes were unaffected. Using the Pamgene Pamstation nuclear hormone receptor (NHR) chip technology, we found that bilirubin-induced coregulator recruitment to PPARα similar to other ligands fenofibrate and WY-14,643, and that this also occurred in animals with hyperbilirubinemia. Next, using CRISPR technology, we excised the gene that generates bilirubin, Blvra, in both mouse kidney and liver cells. The loss of the Blvra gene (BVRA protein) in kidney cells caused lipid buildup and lipotoxicity, which was reversed when cells were treated with bilirubin. The lipotoxic state in the kidney cells dysregulated phosphorylation of BAD and subsequent cell apoptotic pathways. In hepatocytes, the loss of Blvra diminished mitochondrial respiratory function. Here, using a plethora of techniques, we found that bilirubin is a metabolic hormone that binds directly to PPARα. Also, the BVRA enzyme is essential in the regulation of lipotoxicity via bilirubin production. We complete this dissertation with a discussion of the fundamental findings of the aforementioned studies and the novel concept of bilirubin binding PPARα as a hormone to mitigate metabolic disease.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with chronic inflammation affecting multiple organ systems, as well as accelerated atherosclerosis as a major complication. Prior studies by our lab have shown beneficial effects of PPARgamma agonists towards preventing SLE in two different mouse models: the well-established lupus mouse model, MRL.lpr, and the gld.apoE^-/- model of accelerated lupus and atherosclerosis. Retinaldehyde is a retinoic acid precursor that has recently been shown to inhibit PPARgamma signaling in adipose tissue. We proposed that abnormal accumulation of retinaldehyde in lupus promotes autoimmunity by inhibition of PPARgamma signaling. We measured the serum retinaldehyde levels in both lupus mouse models using reversed-phase high-performance liquid chromatography. We also examined the mRNA expressions of genes involved in retinaldehyde metabolism and PPARgamma signaling in white adipose tissues using real-time quantitative PCR. We observed a higher level of circulating retinaldehyde in the MRL.lpr mouse model on a chow diet. The circulating retinaldehyde levels in both .gld.apoE^-/- and C57 increased when maintained on a high-cholesterol Western diet. Within visceral and subcuntaneous adipose tissue, we saw several changes to expression of the genes responsible for retinaldehyde synthesis and catabolism, however further study is required to definitively assess the role of these genes. Importantly, the expression levels of genes involved in PPARgamma signaling decreased in the subcutaneous fat of gld.apoE^-/- mice on a Western diet. Our data suggest that retinaldehyde may play a role in SLE pathogenesis and could be a potential therapeutic target for SLE.

The identification of beige fat within the last decade, its ability to burn energy in adult humans, and its great potential for therapeutic applications has motivated many to work towards understanding how beige fat is made. Although several proteins have been identified as important for beige fat differentiation, it is clear that the differences that distinguish beige fat from other types of fat cannot be explained by the presence of these proteins alone. Additional regulatory proteins, including transcription factors and their co-factor proteins, must be involved.I took advantage of two important developments in the field of fat differentiation to develop two high throughput approaches that identified new transcription factors involved in beige fat: (1) our ability to culture beige fat cells by differentiating white fat pre-adipocytes in the presence of Rosiglitazone and (2) the established role for PRDM16 as required for beige fat differentiation. In brief, I combined RNA-seq data from beige fat cells and proteomics data from Rosiglitazone-dependent PRDM16 protein complexes to identify a set of candidate transcription factors involved in beige fat differentiation. The most promising candidate among this pool of putative beige fat regulatory transcription factors was GTF2IRD1.I determined that GTF2IRD1 is a PRDM16-interacting transcription factor that is enriched in beige and brown fat cells. In vivo, GTF2IRD1 is enriched in brown adipose tissue and is increased in beige and brown fat in response to beta-3-adrenergic stimulus. In the presence of the potent PPARgamma agonist Rosiglitazone, GTF2IRD1 overexpression enhances and shRNA-mediated knockdown reduces beige fat differentiation. GTF2IRD1 represses TGF-beta-mediated inhibition of beige fat differentiation. In summary, my data strongly supports that GTF2IRD1 is an essential regulator of beige fat differentiation through interaction with PRDM16 and inhibition of TGF-beta-mediated repression of differentiation.

The study of fat tissue or adipose over the last half century has dramatically altered the perception of the fat cell, also known as the adipocyte. Once only thought to store fat, the adipocyte is currently recognized to be a highly critical and important metabolically active cell. Adipogenesis, the process of adipocyte formation, has now become an intense subject of basic scientific research in large part due to the up-surge in obesity and metabolic disease in modern day society. PPARgamma is the master regulator of adipogenesis and is also a validated target of anti-diabetic drug therapy. However, a renewed sense of urgency in discovering novel factors and mechanisms that regulate the expression and/or activity of PPARgamma now exists due to recent scrutiny in the clinical use of molecules that directly activate PPARgamma. Therefore, this thesis attempts to address this need by investigating and uncovering entirely new modes of regulation in the differentiating adipocyte. Part one of this thesis presents the construction of a transgenic mouse line that carries a reporter vector containing a cloned set of highly conserved non-coding genomic sequences endogenously found within and adjacent to the PPARgamma locus in the mouse genome. This examination represents an effort to identify a genomic regulatory sequence that confers tissue selective expression of adipose-specific genes such as PPARgamma. Identification of such an enhancer would undoubtedly shed new light on the regulation of gene expression in adipocytes and usher in a new paradigm in the study of adipose tissue. Part two of this thesis presents the identification of Vestigial-like 3 (Vgll3) as an inhibitor of adipocyte differentiation. Vgll3 is a conserved transcriptional co-activator that is down-regulated during adipogenesis. This gene was initially observed to be differentially regulated between pre-adipocyte cell lines that exhibit contradictory potential to become lipid-laden adipocytes. When overexpressed in differentiating adipocytes in vitro, Vgll3 induces a potent block in PPARgamma expression and adipocyte formation. Furthermore, ectopic expression of Vgll3 was observed to up-regulate the expression of genes previously determined to be inhibitors of adipogenesis and genes associated with other mesenchymal-derived cellular differentiation programs. These results point to Vgll3 as a gene whose expression must be carefully modulated during the formation of fully differentiated, mature adipose tissue.

Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by the progressive loss of cognition and memory. The pathological hallmarks of AD include extracellular amyloid deposits and intra-neuronal neurofibrillary tangles. Progression of the disease is associated with a disruption of Aβ homeostasis and accumulation and deposition of Aβ in the brain parenchyma, initiating a robust microglial-mediated immune response that leads to the production of pro-inflammatory cytokines, chemokines and reactive nitrogen and oxygen species which are deleterious to the CNS. Despite the abundance of activated microglia surrounding plaques, they are inefficient in clearing fibrillar Aβ deposits. It is thought that activating mechanisms that facilitate Aβ clearance will alleviate disease related pathophysiology and be of great therapeutic utility for the treatment of Alzheimer’s disease.The peroxisome proliferator activated receptor-ɣ (PPARɣ), is a ligand activated nuclear receptor, which regulates lipid and glucose homeostasis in the body and exhibits potent anti-inflammatory actions. Activation of PPARγ has been shown to reduce brain Aβ levels and ameliorate AD related cognitive deficits in animal models of AD. However, the mechanisms through which PPARγ attenuates AD related pathophysiology have yet to be elucidated. We have shown that activation of another nuclear receptor, LXRα, facilitates the proteolytic degradation of Aβ in an ApoE-dependent mechanism through induction of target genes, ABCA1 and ApoE. ABCA1 regulates cholesterol efflux via lipidation of ApoE and this process augments the degradation of soluble Aβ species. Importantly, PPARγ activation induces the expression of LXRα and its targets ApoE and ABCA1, metabolically linking these pathways.We demonstrate that PPARɣ activation using the synthetic agonist, Pioglitazone (Actos™) enhances the proteolytic degradation of Aβ in microglia and astrocytes by utilizing the PPAR-LXR-ApoE linked pathway in the brain. PPARɣ mediated intracellular degradation of Aβ is dependent on expression of ApoE and LXR. Furthermore, an acute (9 day) treatment of APP/PS1 mice with pioglitazone, rapidly increased brain levels of ABCA1 and ApoE, decreased amyloid deposition and ameliorated AD-related cognitive deficits. The reduction in plaque deposition was paralleled with an increase in amyloid-laden microglia and astrocytes in the parenchyma of treated animals. Significantly, pioglitazone treatment of APP/PS1 animals polarized CNS microglia from a “classical” to an “alternative” activation state, reducing glial activation, re-engaging the phagocytic machinery and facilitating the clearance of fibrillar Aβ deposits. Together our data demonstrates an important role for PPARɣ activation in facilitating the clearance of both soluble and fibrillar species of Aβ and provides a mechanistic explanation for how PPARɣ agonists reduce AD-related pathophysiology.

The ligand-dependent transcription factor, peroxisome proliferator activated receptor, (PPAR-gamma), is a member of the nuclear hormone receptor superfamily and a target of antidiabetic thiazolidinediones (TZDs). Initially characterized for its effects in glucose and lipid metabolism, PPAR-gamma activation has been shown to play critical roles in the inhibition of inflammatory vascular diseases such as atherosclerosis. Since PPAR-gamma agonists have receptor-independent actions, knockout studies are required to determine whether PPAR-gamma is necessary for the inhibitory effects of the PPAR-gamma agonists. Due to the pleiotropic effects of the PPAR-gamma activation, establishing which cell types are responsible for the anti-inflammatory effects observed in whole organism studies is difficult without the use of the cell-specific knockouts. To unravel the complex role of PPAR-gamma in inflammation and to begin categorizing relative contributions of PPAR-gamma activity in different cell types, we created two PPAR-gamma knockout models: pancreatic epithelial cell knockout and vascular endothelial cell knockout. These knockout models not only provided insight into the important role PPAR-gamma plays in the control of cell inflammation but also showed that TZDs act directly through PPAR-gamma in suppressing inflammation in an in vivo model of acute pancreatitis and in an in vitro models of endothelial cell inflammation. We show that PPAR-gamma in acinar cells, and not in inflammatory cells, is the target of the TZD anti-inflammatory activity in the model of acute pancreatitis. In vascular endothelial cells, PPAR-gamma is required for TZD activity and PPAR-gamma activation inhibits the function of a pro-inflammatory transcription factor NF-kappaB by decreasing the formation of functional transcriptional complexes at the promoters of NF-kappaB target genes.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a nutrient-sensing transcription factor that associates with cardiovascular diseases and metabolic disorders. Its agonists, the thiazolidinediones (TZDs), are clinically used to treat type 2 diabetes. This thesis investigated the multifaceted functions of PPAR-gamma and the pharmacology of TZDs in insulin sensitivity, glucose homeostasis, adipogenesis, blood pressure regulation, tumorigenesis, and cardiac growth. We rescued the embryonic lethality of generalized PPAR-gamma deficiency using Cre-LoxP technology to preserve PPAR-gamma expression in the placenta (MORE-PGKO mice). These mice demonstrated that PPAR-gamma is required to maintain normal adiposity, insulin sensitivity, and blood pressure. Administration of rosiglitazone, a TZD, to these PPAR-gamma deficiency mice improved glucose homeostasis in both males and females. In females, rosiglitazone stimulated fat growth and was sufficient to improve insulin sensitivity. Vaspin, but not likely leptin or adiponectin, is a candidate adipokine that is responsible for this insulin-sensitizing effect. In males, glucose homeostasis was improved independently of adipose tissue and insulin-sensitivity likely by improving beta-cell function. Surprisingly, rosiglitazone treatment of male MORE-PGKO mice led to the development liver tumors, suggesting a tumor suppressor function of PPAR-gamma. To directly determine the role of PPAR-gamma in cardiac growth, we developed a cardiomyocyte-specific PPAR-gamma knockout (CM-PGKO) mouse model. Cardiomyocyte PPAR-gamma was required to suppress cardiac growth, embryonic gene expression, and nuclear-factor-kappa-B activity in vivo. Rosiglitazone induced cardiac hypertrophy at least partially independent of PPAR-gamma in cardiomyocytes and through different mechanisms from CM-PGKO. These data may provide guidance for designing new drugs with fewer cardiac side effects. In order to distinguish TZDs' intrinsic effects on cardiomyocytes from their blood pressure-lowering effects, we monitored pressure overload (POL) and found that systolic pressure at later stages was the major determinant of cardiac hypertrophy. In contrast to pioglitazone and troglitazone, rosiglitazone did not inhibit POL-induced cardiac hypertrophy. Future studies comparing multiple TZDs are needed to understand the pharmacology of this effect and to explore the role of PPAR-gamma in POL-induced cardiac hypertrophy.