Indiana University-Purdue University Indianapolis (IUPUI) / The incidence of Metabolic Syndrome (MetS) is increasing worldwide and accompanied by elevated risks for cardiovascular disease (CVD) and other subsequent comorbidities. MetS is associated with increased circulating triglycerides. A key enzyme involved in triglyceride (TG) clearance is lipoprotein lipase (LPL) whose activity is modulated by a variety of factors. Recent literature has identified the importance of angiopoietin-like proteins (ANGPTL) as regulators of LPL activity and has hypothesized a model in which three of these proteins interact with LPL to regulate the partitioning of TG metabolism from adipose to skeletal muscle. The work detailed in this dissertation adds to the model of ANGPTL regulation of LPL by establishing how ANGPTL8 modulates the ability of ANGPTL3 and ANGPTL4 to inhibit LPL activity in the bloodstream and localized environments, respectively. In the updated model, elevated insulin concentrations result in increased hepatic ANGPTL3/8 secretion and increased ANGPTL4/8 in adipose tissue. ANGPTL3/8 works as an endocrine molecule to inhibit skeletal muscle LPL from hydrolyzing circulating TG. Simultaneously, ANGPTL4/8 works in a paracrine mechanism to bind LPL on the endothelial vasculature adjacent to adipose tissue to alleviate ANGPTL4-mediated LPL inhibition and also prevent ANGPTL3/8 inhibition of localized LPL. Thus, in the postprandial state free fatty acids (FFA) from the hydrolysis of TG are directed into adipocytes for storage. Under fasting conditions, ANGPTL8 production is decreased in adipocytes and hepatocytes. This decreased production results in diminished ANGPTL4/8 and ANGPTL3/8 secretion from their respective tissues. As a result, ANGPTL4 inhibits adipocyte localized LPL activity while ANGPTL3 at physiological concentrations has minimal effect on LPL activity. Furthermore, any ANGPTL3/8 which is produced has its LPL-inhibitory ability diminished by the circulating apolipoprotein ApoA5. LPL is more active in skeletal muscle compared to adipose tissue where energy is shunted towards utilization in the muscle and away from storage in adipose tissue. A complete understanding of LPL regulation by ANGPTL proteins can potentially provide therapeutics targets for MetS.

<p> The Liver X receptor (LXR) is a type II nuclear receptor that is known to be a master regulator of cholesterol levels in the body through its transcriptional control of target genes involved in the handling of cholesterol. The regulation of LXR occurs at multiple levels including ligand and protein availability, post-translational modifications, protein-protein interactions with various cofactors and/or chaperones and a new concept of regulation that involves compartmentalization. This involves the establishment of regions where proteins can be active or inactive. Type II nuclear receptors have recently been found to shuttle between the cytoplasm and the nucleus, thus a compartmentalization component is likely to be involved. It was recently implicated that the centrosome-associated protein 350 (CAP350) can sequester PPARa. into nuclear bodies, and to regions in the cytoplasm. The significance of this appears to be the control of PPAR action. CAP350 is a large protein that has the ability to interact with nuclear receptors via an LXXLL motif, and with the cytoskeleton via a CAP-Giy motif. CAP350 is suggested to play a role in the organization of nuclear receptors in the nucleus, and their retention in compartments. In this report, LXRa. was confirmed to interact with CAP350 in vitro, using a GST-binding assay. Utilizing fluorescent protein chimeras with both nuclear receptors and CAP350 allowed the monitoring of this interaction in vivo. CAP350 was observed to form nuclear bodies that were capable of recruiting LXRa.. This recruitment was dependant on the integrity of the LXXLL motif. The mutated LXXLL motif of CAP350 was not able to colocalize with LXRa.. The significance of this interaction remains unknown. It is likely to be similar to that observed with PPARa., since the nuclear bodies formed by CAP350 seem to correspond to transcriptionally silent regions in the nucleus. </p> / Thesis / Master of Science (MSc)

The mechanisms causing luteolysis of the primate corpus luteum are unknown. There is an increase in expression of liver x receptor (LXR) target genes and reduced low density lipoprotein receptor (LDLR) during spontaneous luteolysis in primates. The LXRs belong to the nuclear receptor superfamily and increase cholesterol efflux by inducing transcription of their target genes. Uptake of cholesterol into primate luteal cells occurs primarily via LDL, and LDLR transcription is regulated by sterol regulatory element binding transcription factor 2 (SREBF2). Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) maintain luteal function by binding to the LH/CG receptor (LHCGR), which stimulates progesterone (P4) synthesis via protein kinase A (PKA). It has also been previously reported that there is an increase in 27-hydroxycholesterol (27OH) concentrations during spontaneous luteolysis in primates. Pregnenolone and P4 inhibit the enzyme activity of CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1), which converts cholesterol into 27OH, an oxysterol that is a natural LXR agonist and SREBF2 inhibitor. Therefore, the overall hypothesis is that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediate luteolysis of the human CL. The objective of study 1 is to determine the effects of LXR activation and SREBF2 inhibition on P4 production, cholesterol metabolism and gene expression; and how hCG signaling via PKA regulates these effects in human luteinized granulosa cells. Basal and hCG-stimulated P4 secretion were significantly decreased by the combined actions of the LXR agonist T0901317 (T09) and the SREBF2 inhibitor fatostatin, which was associated with alterations in cholesterol metabolism leading to reduced intracellular cholesterol storage. Expression of LXR target genes in the presence of T09 was significantly reduced by hCG, while hCG significantly increased LDLR expression. These effects of hCG were reversed by a specific PKA inhibitor. Chronic hCG exposure had similar effects on LXR target gene and LDLR expression without an exogenous LXR agonist. The objective of study 2 is to determine the effects of 27OH on P4 production and cholesterol metabolism; and to determine if inhibiting the conversion of cholesterol into pregnenolone increases LXR and decreases SREBF2 target gene expression via CYP27A1 in human luteinized granulosa cells. During luteolysis in primates and sheep, CYP27A1 expression significantly increased. 27OH significantly decreased hCG-stimulated P4 secretion and enhanced cholesterol efflux. Aminoglutethimide, which inhibits the conversion of cholesterol to pregnenolone, significantly increased ABCA1 and decreased LDLR. Knock-down of CYP27A1 resulted in a significant increase in P4 secretion, but did not prevent aminoglutethimide-induced effects on ABCA1 and LDLR. Knock-down of steroidogenic acute regulatory protein (STAR), which controls cholesterol transport into the mitochondria where CYP27A1 resides, significantly decreased LDLR transcription. Collectively, the data from study 1 support the hypothesis that LXR-induced cholesterol efflux and reduced LDL uptake via inhibition of SREBF2 activity mediates luteolysis in primates, which is reversed by hCG. Data from study 2 indicates that 27OH produced via CYP27A1 may contribute to reductions in P4 synthesis during luteolysis, partially by serving as a dual LXR agonist and SREBF2 inhibitor, although other oxysterols are also likely involved.

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Le cholestérol, élément nutritif indispensable mais toxique en excès, est naturellement converti en oxystérols. Les LXRs (Liver X Receptor) α et β sont les récepteurs nucléaires des oxystérols ayant un rôle hypocholestérolémiant et contrôlant plusieurs fonctions physiologiques. Notre but a été d'étudier leur rôle dans la reproduction chez la femelle. Dans l'ovaire, l'induction de l'ovulation chez des souris lxrα;β-/- provoque une hyperstimulation ovarienne (OHSS), caractérisée par une augmentation de la masse, des troubles vasculaires et de la sensibilité hormonale. Les LXRs contrôlent de plus l'oestradiolémie. Au niveau utérin, nous montrons un rôle spécifique de LXRβ. Les souris lxrβ-/- présentent une séquestration anormale d'esters de cholestérol dans les myocytes associée à un défaut de contractilité utérine. Cette étude montre l'importance des LXRs dans la reproduction chez la femelle et permettra de comprendre le lien entre déséquilibre alimentaire et troubles de la fertilité.

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Dispatched 3 (Disp3), a thyroid hormone-regulated gene, is studied extensively in our laboratory. Phenotype of cells with overexpressed Disp3 and its expression pattern make it a perfect candidate for a molecular link between thyroid hormone action and cholesterol homeostasis in the brain. Moreover, we hypothesize that it might play a role in certain neurodegenerative disorders and brain tumours. This thesis is aimed at the process of regulation of this gene via thyroid hormone receptor (TR), specifically identification of responsive elements of the thyroid hormone receptor that are necessary for the regulation. Also, we searched for elements recognized by liver X receptor (LXR), as LXR binds to the same arrangement of repeats as TR and there are a number of genes regulated by both of them. We combined in silico analysis of the Disp3 locus with reporter luciferase assays. A cluster of six elements identified around the first exon with two of them being conserved among human and mice draw our attention. In order to analyze this sequence in more detail, reporter vectors of various truncations of 3 kb region around exon 1 were constructed and tested in reporter assays. Reporter assays did not reveal any substantial element activated by TR or LXR; on the other hand, region containing repressor element(s)...