Faculty of Science; School of molecular and Cell Biology; MSC Dissertation / Background: Cancer of the oesophagus ranks as the ninth most common malignancy in the world, and recent evidence shows that its incidence is increasing. Apoptosis is a process of programmed cell death, which is as essential as cell growth, for the maintenance of homeostasis. When these processes lose integration, such as cancer, then uncontrolled cell growth occurs. There are at least five ACBP subgroups and the two being focused on in this study is B-ACBP (brain specific) and 1-ACBP (found in nearly all tissues). ACBPs act as intracellular carrier-proteins for medium to long chain acyl-coA, mediating fatty acid transport to the mitochondrion for ß-oxidation. ACBPs are also believed to be putative ligands of PBR (Peripheral Benzodiazepine Receptor), and bound to this receptor facilitates mitochondrial membrane permeabilization giving the notion that it favours apoptosis. Aim: To establish the expression patterns of 1-ACBP, B-ACBP, and PBR in oesophageal cancer, and to characterize their roles in this disease. Methodology: Paraffin-embedded sections of normal and malignant oesophageal tissues were utilized for localization studies. RNA probes was synthesized and labelled using Digoxigenin for colorimetric and fluorescent detection during the in situ hybridization (ISH) technique for localization. Real time quantitative RT-PCR was performed to determine the expression levels of the three genes in oesophageal cancer RNA using the Roche Lightcylcer .Results: All three genes showed substantial upregulation within the malignant tissue sections compared to normal oesophageal sections, all three transcripts localized specifically to plasma cells and lymphocytes in diseased and normal tissue section. In the diseased tissue B-ACBP and 1-ACBP mRNA localized to endothelial cells of blood vessels in the submucosa. B-ACBP also localized to the nucleus of squamous epithelium cells. PBR localization occurred in tumour islands in invasive tissue sections. Quantitative RT-PCR also illustrated PBR expression level was the highest compared to the ACBP genes expression in tumours. Conclusion: These results show that 1-ACBP, B-ACBP and PBR play a role in the pathogenesis of oesophageal cancer as well as immunology. Further experiments are still required to determine the function of these genes and the role they play in apoptosis and oesophageal cancer.

Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

Unique among the intracellular lipid binding proteins, acyl CoA binding protein (ACBP) exclusively binds long chain fatty acyl CoAs (LCFA-CoAs). To test if ACBP is an essential protein in mammals, the ACBP gene was ablated by homologous recombination in mice. While ACBP heterozygotes appeared phenotypically normal, intercrossing of the heterozygotes did not result in any live homozygous deficient (null) ACBP^(-/-) pups. Heterozygous and wild type embryos were detected at all postimplantation stages, but no homozygous ACBP null embryos were obtained– suggesting that an embryonic lethality occurred at a preimplantation stage of development, or that embryos never formed. While ACBP null embryos were not detected at any blastocyst stage, ACBP null embryos were detected at the morula (8- cell), cleavage (2-cell), and zygote (1-cell) preimplantation stages. Two other LCFACoA binding proteins, sterol carrier protein-2 (SCP-2) and sterol carrier protein-x (SCPx) were significantly upregulated at these stages. These findings demonstrate for the first time that ACBP is an essential protein required for embryonic development and its loss of function may be initially compensated by concomitant upregulation of two other LCFA-CoA binding proteins only at the earliest preimplantation stages. The fact that ACBP is the first known intracellular lipid binding protein whose deletion results in embryonic lethality suggests its vital importance in mammals.

Cholesterol and fatty acyl-coenzymeA thioesters are signalling molecules with role in regulation of genes involved in lipid and glucose transport and metabolism. The studies described herein focused on three proteins that bind lipids and have different cellular functions: steroidogenic acute regulatory protein (StAR), hepatocyte nuclear factor-4a (HNF-4a) and acyl-CoA binding protein (ACBP). First, StAR mediates delivery of cholesterol to inner mitochondrial membrane in steroidogenesis by a poorly understood mechanism. In our studies, fluorescent NBD-cholesterol binding assays demonstrate that StAR binds cholesterol at two binding sites with 32 nM Kds and circular dichroism spectra show that cholesterol binding results in changes of StAR secondary structure. Fluorescent sterol exchange assays between donor and acceptor mitochondrial membranes indicate that StAR significantly increased the formation of rapidly transferable cholesterol domains. Second, HNF-4a, a nuclear receptor, had been shown to bind fatty acyl-CoAs as natural ligands with apparent low affinities obtained with radiolabeled ligand binding assays. Our fluorescence spectroscopy studies demonstrate that HNF-4a ligand binding domain (HNF-4aLBD) binds acyl-CoAs at a single binding site with Kds of 1.6-4 nM. Fluorescence resonance energy transfer (FRET) between HNF-4aLBD tryptophan residues and cis-parinaroyl-CoA yielded an intermolecular distance of 42 Â thus pointing to direct molecular interaction. Third, although ACBP has been detected in the nucleus, it is not known whether ACBP may directly and/or functionally interact with a nuclear acyl-CoA binding protein such as HNF-4a to regulate transcription. Our present studies in vitro and in intact cultured cells, including circular dichroism of HNF-4a in the presence of ACBP, coimmunoprecipitation of HNF-4a/ACBP complexes, ACBP and HNF-4a colocalization in nuclei of cells by confocal microscopy demonstrate a physical association of ACBP and HNF-4a. FRET microscopy data indicated an intermolecular distance of 53 Â between ACBP and HNF-4a in rat hepatoma cells. Functional assays (transactivation of an HNF4a-dependent reporter gene) showed significant increase in the presence of ACBP in two different cell lines. Expression of ACBP anti-sense RNA decreased HNF-4a-mediated transactivation, pointing to a role of ACBP in co-regulating HNF-4a-dependent transcription.

Un déséquilibre de la balance énergétique constitue la principale cause du développement des pathologies métaboliques telles que l’obésité et le diabète de type 2. Au sein du cerveau, l’hypothalamus joue un rôle primordial dans le contrôle de la prise alimentaire et du métabolisme périphérique via le système nerveux autonome. Ce contrôle, repose sur l’existence de différentes populations neuronales au sein de l’hypothalamus médio-basal (MBH), neurones à neuropeptide Y (NPY)/Agouti-related peptide (AgRP), et neurones a proopiomelanocortine (POMC), dont l’activité est directement modulée par les variations des taux circulants des nutriments tels que le glucose et les acides gras (FA). Alors que les mécanismes de détection et le métabolisme intracellulaire du glucose ont été largement étudiés, l’implication du métabolisme intracellulaire des FA dans leurs effets centraux, est très peu comprise. De plus, on ignore si le glucose, module le métabolisme intracellulaire des acides gras à longue chaine (LCFA) dans le MBH. Le but de notre première étude est, de déterminer l'impact du glucose sur le métabolisme des LCFA, le rôle de l’AMP-activated protein kinase (AMPK), kinase détectrice du statut énergétique cellulaire, et d'établir s’il y a des changements dans le métabolisme des LCFA en fonction de leur structure, du type cellulaire et de la région cérébrale. Nos résultats montrent que le glucose inhibe l'oxydation du palmitate via l’AMPK dans les neurones et les astrocytes primaires hypothalamiques, in vitro, ainsi que dans les explants du MBH, ex vivo, mais pas dans les astrocytes et les explants corticaux. De plus, le glucose augmente l'estérification du palmitate et non de l’oléate dans les neurones et les explants du MBH, mais pas dans les astrocytes hypothalamiques. Ces résultats décrivent le devenir métabolique de différents LCFA dans le MBH, ainsi que, la régulation AMPK - dépendante de leur métabolisme par le glucose dans les astrocytes et les neurones, et démontrent pour la première fois que le métabolisme du glucose et des LCFA est couplé spécifiquement dans les noyaux du MBH, dont le rôle est critique pour le contrôle de l'équilibre énergétique. Le deuxième volet de cette thèse s’est intéressé à déterminer les mécanismes intracellulaires impliqués dans le rôle de la protéine de liaison ACBP dans le métabolisme central des FA. Nous avons démontré que le métabolisme de l’oléate et non celui du palmitate est dépendant de la protéine ACBP, dans les astrocytes hypothalamiques ainsi que dans les explants du MBH. Ainsi, nos résultats démontrent qu’ACBP, protéine identifiée originellement au niveau central, comme un modulateur allostérique des récepteurs GABA, agit comme un régulateur du métabolisme intracellulaire des FA. Ces résultats ouvrent de nouvelles pistes de recherche liées à la régulation du métabolisme des acides gras au niveau central, ainsi que, la nouvelle fonction de la protéine ACBP dans la régulation du métabolisme des FA au niveau du système nerveux central. Ceci aiderait à identifier des cibles moléculaires pouvant contribuer au développement de nouvelles approches thérapeutiques de pathologies telles que l’obésité et le diabète de type 2. / An imbalance of energy balance is the main cause of the development of metabolic diseases such as obesity and type 2 diabetes. Within the brain, the hypothalamus plays an important role in the control of food intake and peripheral metabolism, via the autonomic nervous system. This control relies on the existence of different neuronal populations in the medio-basal hypothalamus (MBH), including neuropeptide Y (NPY), agouti-related peptide (AgRP) and proopiomelanocortin (POMC) neurons, the activity of which, is directly modulated by changes in the circulating levels of nutrients such as glucose and fatty acids (FA). While mechanisms governing the detection and the intracellular metabolism of glucose have been extensively studied, the involvement of FA intracellular metabolism, in their central effects is poorly understood. It is currently unknown if glucose regulates long chain fatty acids (LCFA) metabolism in the MBH. The aim of our first study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), a sensor of cellular energy status, and to establish if changes in LCFA metabolism, and its regulation by glucose, vary as a function of LCFA type, cell type and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures and MBH slices, ex vivo, but not in cortical astrocytes and slice preparations. In addition, our results show that glucose increases palmitate but not oleate esterification into neutral lipids, in neurons and MBH slices, but not in hypothalamic astrocytes. These findings reveal the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons and established for the first time the metabolic coupling of glucose and LCFA as a specific feature of the MBH, whose role is critical for the control of energy balance. During the second part of this thesis, we were interested to determine the intracellular mechanisms involved in the role of Acyl-CoA binding protein (ACBP), in the central metabolism of FA. We have shown that the metabolism of oleate but not palmitate is ACBP -dependent in hypothalamic astrocytes and MBH slices. Thus, our results demonstrate That ACBP, a protein originally identified as an allosteric modulator of GABA receptor peptide, acts as a regulator of intracellular metabolism of FA. These results open a new avenues of research related to the central regulation of fatty acid metabolism and the new function of ACBP protein in the regulation of FA metabolism in the central nervous system, which could help to identify molecular targets that may contribute to the development of new therapeutic approaches of diseases such as obesity and type 2 diabetes.

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