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Cellular cholesterol homeostasis ensures that cells are able to acquire enough cholesterol for cellular needs including membrane renewal, but avoid excess accumulation. Cholesterol homeostasis is maintained by two transcription factors, the Sterol Regulatory Element-Binding Protein-2 (SREBP-2) which regulates the genes involved in cholesterol biosynthesis and uptake; and the Liver X Receptor (LXR), which regulates the genes involved in maintaining Reverse Cholesterol Transport (RCT). Both LXR and SREBP-2 are regulated by oxidized cholesterol derivatives, referred to as oxysterols. 24(S),25-epoxycholesterol (24,25EC) is unique amongst the physiologically produced oxysterols as it is synthesized de novo in the mevalonate pathway. The overall aim of this thesis was to investigate the role of 24,25EC in cellular cholesterol homeostasis. In Chapter 3, using the statin class of HMG-CoA reductase inhibitors, we showed that 24,25EC synthesis correlates with the expression of the LXR-target gene, the ATP-binding cassette transporter, A1 (ABCA1) in human macrophages. In Chapters 4 and 5, we investigated this further showing that the effect of statins on ABCA1 transcription can be modulated by cellular cholesterol status and the extent of macrophage differentiation. Our results also indicated a critical role of SREBP-2 as a positive regulator of ABCA1 transcription by enabling the generation of 24,25EC. In Chapter 6, we showed that 24,25EC synthesis paralleled cholesterol synthesis, suggesting that this oxysterol may protect against the accumulation of newly-synthesized cholesterol. In Chapter 7, we investigated this contention further using a novel strategy of overexpressing the enzyme 2,3-Oxidosqualene Cyclase (OSC) in Chinese Hamster Ovary cells to selectively inhibit 24,25EC synthesis. In cells lacking 24,25EC, fine-tuning of the acute regulation of cholesterol homeostasis was lost, supporting the hypothesis that 24,25EC functions to protect the cell against the accumulation of newly-synthesized cholesterol. In Chapter 8, we focused on 24,25EC in a neurological context since cholesterol is an essential component of the central nervous system. Both astrocytes and neurons had the capacity to synthesize 24,25EC. Furthermore, both added 24,25EC and stimulated cellular production of 24,25EC modulated expression of LXR and SREBP-2 target genes. The evidence present in this thesis demonstrates the importance of 24,25EC as an endogenously synthesized regulator of cholesterol homeostasis.

Nuclear Hormone Receptors (NRs) are hormone dependent DNA binding proteins that translate physiological signals into gene expression. Gene products have been identified that belong to the NR superfamily on the basis of homology. However, the endogenous and /or synthetic ligands that regulate their activity remain unknown, consequently, this subgroup of proteins are designated as orphans). Retinoic acid receptor related orphan receptors alpha and gamma(RORα and γ) are orphan NRs, and are preferentially expressed in skeletal muscle a major metabolic tissue and other tissues including pancreas, thymus, prostate, liver, adipose and testis. Surprisingly, the specific roles of ROR α and γ in skeletal muscle, a peripheral tissue, have not been examined. Muscle is one of the most energy demanding tissues which accounts for ~40% of the total body mass and energy expenditure, ~75% of glucose disposal and relies heavily on β-oxidation of fatty acids. We hypothesize that ROR α and γ regulates metabolism in this major mass lean tissue. Initially, this hypothesis was examined by “gain and loss” of function studies in an in-vitro mouse skeletal muscle cell culture model. Previous in vitro studies analyzed the role of RORα in the regulation of lipid homeostasis in skeletal muscle cells. We similarly conducted in vitro RORγ gain and loss of function studies in skeletal muscle cells to understand the role of this isoform in metabolism. We utilized stable ectopic over-expression of VP16-RORγ (gain of function), native RORγ and RORγΔH12 (loss of function) vectors to modulate RORγ mRNA expression and function. Candidate driven expression profiling of lines that ectopically express the native and variant forms of RORγ suggested that this orphan NR has a function in regulating the expression of genes that control lipid homeostasis (fatty acid-binding protein 4), CD36 (fatty acid translocase), lipoprotein lipase and uncoupling protein 3), carbohydrate metabolism (GLUT5 (fructose transporter), adiponectin receptor 2 and interleukin 15 (IL-15)) and muscle mass (including myostatin and IL-15). Interestingly, our study revealed a function for RORγ in the pathway that regulates production of reactive oxygen species which was also correlated with increased expression of UCP3 mRNA. Subsequently, we conducted in vivo studies with mouse models displaying global and muscle specific perturbation in RORα expression and function to elucidate the physiological role of this orphan NR in the context of metabolism.Along these lines, we characterized homozygous staggerer mice (sg/sg) in the context of lipid, carbohydrate and energy homeostasis. Staggerer mice were characterized by decreased and dysfunctional retinoic acid receptor-related orphan receptor alpha (RORα) expression. We observed decreases in serum (and liver) triglycerides and total and high density lipoprotein serum cholesterol in staggerer mice. Moreover, the staggerer mice were associated with reduced adiposity, decreased fat pad mass and adipocyte size. Candidate-based expression profiling demonstrated that the dyslipidemia in staggerer mice was associated with decreased hepatic expression of SREBP-1c, and the reverse cholesterol transporters, ABCA1 and ABCG1. This was consistent with the reduced serum lipids. Furthermore, the lean phenotype in staggerer mice was also characterized by significantly increased expression of PGC-1α, PGC-1β, and lipin1mRNAin liver and white and brown adipose tissue from staggerer mice. In addition, we observed a significant 4-fold increase in β2-adrenergic receptor mRNA in brown adipose tissue. Finally, dysfunctional RORα expression protects against diet-induced obesity. Following a 10-week high fat diet, wild-type (but not sg/sg) mice exhibited a ~20% weight gain, increased hepatic triglycerides, and notable white and brown adipose tissue accumulation. In summary, these changes in gene expression (that modulate lipid homeostasis) in metabolic tissues were involved in decreased adiposity and resistance to diet induced obesity in the sg/sg mice, despite hyperphagia. Finally, we specifically modulated RORα signaling in skeletal muscle by the targeted over-expression of truncated RORαΔDE (lacking the ligand binding domain) driven by a myogenic specific promoter, to investigate the contribution of this peripheral tissue to the RORα phenotype. Interestingly, transgenic heterozygous animals exhibit increased fasting blood glucose levels and mild glucose intolerance. Expression profiling (and western analysis) identified perturbations in the insulin signaling cascade. For example, we observed attenuation of p85alpha (PI3K) and Akt2 (mRNA and protein) expression; and insulin dependent induction of phospho-Akt2. In concordance, significantly increased levels of active phospho-AMPK were detected in the muscle of transgenic mice (relative to wt littermates). The increase in phospho-AMPK correlated with: (i) the suppression of lipogenic gene expression; and (ii) increased phospho-ACC and activation of genes involved in fatty acid oxidation in the skeletal muscle of transgenic animals. In conclusion, we suggest these orphan nuclear receptors (RORα and γ) are key modulators of fat and carbohydrate homeostasis in skeletal muscle tissue. Specifically, we propose that, RORα plays vital role in fat accumulation in adipose tissue and insulin mediated glucose homeostasis in skeletal muscle. Therefore we suggest that selective muscle specific RORα modulators may have utility in the treatment of type2 diabetes and obesity.

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published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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