Dissertations / Theses on the topic 'HSOD1'

Glucocorticoid production by the adrenal gland is regulated by hypothalamicpituitary- adrenal (HPA) axis activity. Within cells, glucocorticoid levels are modulated by 11β-hydroxysteroid dehydrogenase (11β-HSD), which interconverts active and intrinsically inert glucocorticoids. Glucocorticoids have widespread physiological effects and, in the cardiovascular system, they play a crucial role in heart development and maturation, blood pressure control, and myocardial calcium cycling. Mice which are unable to regenerate the physiological glucocorticoid, corticosterone, from 11-dehydrocorticosterone due to deletion of the type 1 11β-HSD isozyme (11β-HSD1) have previously been shown to have smaller, lighter hearts but unaltered systolic function. Moreover, a single nucleotide polymorphism (SNP) in the Hsd11b1 gene has been associated with reduced left ventricular mass in humans, suggesting a role for 11β-HSD1 in regulating cardiac size. After myocardial infarction (MI), 11β-HSD1 deficient mice have an augmented inflammatory response, increased numbers of pro-reparative alternatively-activated macrophages in the heart, enhanced peri-infarct angiogenesis and improved cardiac function compared to C57BL/6 controls. However, the role of ‘cardiovascular’ 11β-HSD1 in the development of these phenotypes, both basally and after MI, is unknown. It was hypothesised that ‘cardiovascular’ 11β-HSD1 deficiency would result in smaller hearts, and that this selective deletion would lead to altered calcium handling protein expression and diastolic abnormalities. Furthermore, it was hypothesised that ‘cardiovascular’ 11β-HSD1 deletion would reproduce the beneficial post-MI phenotype previously seen in global 11β-HSD1 deficient mice. The first aim was to characterise the cardiac phenotype of mice with global deletion of 11β-HSD1 (DelI mice), and mice in which deletion is restricted to cardiomyocytes and vascular smooth muscle cells (SMAC mice). SMAC mice have ‘floxed’ 11β- HSD1 alleles and a Cre recombinase transgene inserted into the Sm22α gene. Sm22α is expressed in vascular smooth muscle cells, and transiently in cardiomyocytes during development. Thus, Cre expression in these cells results in deletion of exon three of the Hsd11b1 gene and gives rise to a non-functional protein. Controls for DelI mice were C57BL/6 mice, and controls for SMAC mice were their Cre- littermates. DelI, but not SMAC, mice have smaller, lighter hearts, which may be explained by their shorter cardiomyocytes measured following isolation using a Langendorff preparation. Cardiomyocyte cross-sectional area is unchanged. In vivo measurement of cardiac function using ultrasound imaging showed systolic function is comparable between DelI mice and SMAC mice and their respective controls. However, there is mild diastolic dysfunction in both DelI and SMAC mice, characterised by reduced E wave deceleration and an increased mitral valve deceleration time. This phenotype occurred following pharmacological inhibition of 11β-HSD1, by administration of UE2316, a selective 11β-HSD1 inhibitor, to adult C57BL6/SJL mice. While ventricular collagen content is unaltered in DelI, SMAC and UE2316-treated mice compared to their respective controls, expression of sarcoplasmic reticulum Ca2+ ATPase (SERCA) is reduced, suggesting that altered calcium handling, rather than changes in stiffness, may underlie this phenotype. The second aim was to determine whether the beneficial acute outcomes seen previously in 11β-HSD1 deficient mice after MI could be reproduced by selective cardiovascular deletion of the enzyme. Seven days after MI, compared to Cre- littermate controls, SMAC mice have similar peri-infarct angiogenesis, total macrophage and alternatively-activated macrophage infiltration into the heart, infarct size, ventricular dilatation and systolic function. This suggests 11β-HSD1 deletion in another cell type, or types, is responsible for the phenotype seen in global 11β-HSD1 deficient mice. The final aim was to assess the impact of global 11β-HSD1 deficiency and ‘cardiovascular’ 11β-HSD1 deletion on the development of heart failure, using magnetic resonance imaging to determine structure and function. Eight weeks after MI, mice globally deficient in 11β-HSD1 have attenuated expression of ANP and β- MHC, RNA markers of heart failure, and show attenuated pulmonary oedema, reduced chamber dilatation, preserved systolic function and smaller infarcts compared to control. None of these parameters are altered in SMAC mice relative to control. In conclusion, the data presented in this thesis shows that cardiovascular 11β-HSD1 influences physiological cardiac function, potentially through regulation of calcium handling. 11β-HSD1 in other cells influences cardiomyocyte length, resulting in smaller hearts in its absence. Despite this, global 11β-HSD1 deficiency prevents heart failure development after MI, suggesting that pharmacological inhibition of 11β-HSD1 may be of benefit in treating this condition. Cardiovascular 11β-HSD1 does not, however, account for the changes in infarct healing or remodelling associated with this beneficial outcome, therefore these effects must be related to 11β-HSD1 deficiency elsewhere, such as fibroblasts or myeloid cells.

The present PhD Thesis evolves around the design, synthesis and pharmacological evaluation of novel 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors. Given that the enzyme active site includes a hydrophobic pocket to accommodate bulky lipophilic scaffolds, the main objective was focused on the study of new 11β-HSD1 inhibitors exploring different hydrophobic polycyclic substituents. 11β-HSD1 catalyzes the cortisol regeneration from its inactive form cortisone in tissues mainly expressing glucocorticoid (GC) receptors, such as liver, adipose and brain. GCs are well known hormones that play a major role in our organism. It is well accepted that the GC concentration in peripheral tissues not only depends on the adrenal secretion but also on the intracellular metabolism in these tissues, namely by 11β-HSD1. During the last years, both academia and industry have made great efforts to develop 11β-HSD1 inhibitors to target diseases such as type 2 diabetes and Alzheimer’s. The general structure of these molecules consists on a bulky lipophilic group –usually an adamantyl- linked by an amide core to a right-hand side (RHS) substituent. The first goal was the development of a new polycyclic amine, the 2-oxaadamantan-5- amine, to add to our library of polycyclic substituents (Chapter 3). The target amine was envisioned to contain an oxygen atom in its hydrophobic skeleton to mimic the structure of some hydroxylated adamantyl derivatives in development. Its synthesis involved consecutive Criegee rearrangements on 2-methyl-2-adamantanol to deliver the 2- oxaadamantane, which was then functionalized by C-H activation using phase-transfer catalysis. Finally, a Ritter reaction followed by deprotection with thiourea delivered the desired 2-oxaadamantan-5-amine. The second objective of the present thesis was the synthesis of a small series of 1- and 2-adamantyl-based 11β-HSD1 inhibitors, as most of the 11β-HSD1 inhibitors evaluated are 2-adamantyl substituted derivatives and no comparison with their C-1 isomers was available. Moreover, considering that very few heteroadamantanes have been studied in 11β-HSD1 inhibitors, we also evaluated the introduction of the previously synthesized 5-substituted 2-oxaadamantane (Chapter 4). 1 Focusing on the main goal, it is reported the exploration of other hydrophobic polycyclic substituents as replacement of adamantane with a design supported by molecular modeling studies in order to optimize the filling of the hydrophobic pocket in the binding site (Chapter 5). This work let us to a new family of potent 11β-HSD1 inhibitors featuring unexplored pyrrolidine-based polycyclic substituents. The in vitro biological profiling of the compounds permitted us to select a proper candidate for an in vivo study in a rodent model of cognitive dysfunction. The results supported the neuroprotective effect of 11β- HSD1 inhibition in cognitive decline related to the aging process, since the treatment prevented memory deficits through a reduction of neuroinflammation and oxidative stress, and an increase of the abnormal proteins degradation in the brain. An additional in vivo study in a model of cognitive dysfunction and metabolic disease is currently ongoing to study how 11β-HSD1 inhibition can modulate these two linked disorders, as so-called type 3 diabetes. Finally, the focus was on the exploration of different substituents in the RHS of the molecule to further improve potency, selectivity and metabolic stability. The endeavour started integrating different aromatic, heteroaromatic, heterocycloalkyl and branched alkyl substituents generating diversity to build some structure-activity relationship (SAR) information (Chapter 6). From this work we obtained potent nanomolar inhibitors but still without the needed selectivity and stability properties. In light of these results, we started a rational design of new substitution patterns in order to establish additional interactions that would deliver more potent and selective inhibitors (Chapter 7). The pharmacological tests revealed some low nanomolar activities together with good metabolic stabilities, although selectivity over the isoenzyme 11β-HSD2 remains a challenge to be accomplished.

Die 11beta-HSD1 reguliert intrazellulär die Cortisolkonzentration durch Regeneration von Cortison z.B. aus dem Blutkreislauf, zu Cortisol. Daher stellt diese ein wichtiges Element in der Glucocorticoid-vermittelten Genregulation dar. Die 11beta-HSD1 wird ubiquitär exprimiert, auf hohem Niveau besonders in Leber, Fettgewebe und glatten Muskelzellen. Insbesondere die Bedeutung der 11beta-HSD1 in Leber und Fettgewebe konnte mehrfach nachgewiesen werden. In der Leber führte eine erhöhte Aktivität aufgrund einer Überexpression in Mäusen zu einer verstärkten Gluconeogeneserate. Des Weiteren konnte gezeigt werden, dass eine erhöhte Expression und erhöhte Enzymaktivität der 11beta-HSD1 im subkutanen und viszeralen Fettgewebe assoziiert ist mit Fettleibigkeit, Insulinresistenz und Dyslipidämie. Über die Regulation ist jedoch noch wenig bekannt. Zur Untersuchung der Promotoraktivität wurde der Promotorbereich von -3034 bis +188, vor und nach dem Translations- und Transkriptionsstart, der 11beta-HSD1 kloniert. 8 Promotorfragmente wurden mittels Dual-Luciferase-Assay in humanen HepG2-Zellen sowie undifferenzierten und differenzierten murinen 3T3-L1-Zellen untersucht. Anschließend wurde mittels nicht-radioaktiven EMSA die Bindung des TATA-Binding Proteins (TBP) sowie von CCAAT/Enhancer-Binding-Proteinen (C/EBP) an ausgewählte Promotorregionen analysiert. Nach der Charakterisierung des Promotors wurden spezifische endogene und exogene Regulatoren untersucht. Fettsäuren modifizieren die Entstehung von Adipositas und Insulinresistenz. Ihre Wirkung wird u.a. PPARgamma-abhängig vermittelt und kann durch das Inkretin (Glucose-dependent insulinotropic Peptide) GIP modifiziert werden. So wurden die Effekte von unterschiedlichen Fettsäuren, vom PPARgamma Agonisten Rosiglitazon sowie dem Inkretin GIP auf die Expression und Enzymaktivität der 11beta-HSD1 untersucht. Dies wurde in-vitro-, tierexperimentell und in humanen in-vivo-Studien realisiert. Zuletzt wurden 2 Single Nucleotide Polymorphismen (SNP) im Promotorbereich der 11beta-HSD1 in der Zellkultur im Hinblick auf potentielle Funktionalität analysiert sowie die Assoziation mit Diabetes mellitus Typ 2 und Körpergewicht in der MeSyBePo-Kohorte bei rund 1.800 Personen untersucht. Die Luciferase-Assays zeigten basal eine zell-spezifische Regulation der 11beta-HSD1, wobei in allen 3 untersuchten Zelltypen die Bindung eines Repressors nachgewiesen werden konnte. Zudem konnte eine mögliche Bindung des TBPs sowie von C/EBP-Proteinen an verschiedene Positionen gezeigt werden. Die Transaktivierungsassays mit den C/EBP-Proteinen -alpha, -beta und -delta zeigten eben-falls eine zellspezifische Regulation des 11beta-HSD1-Promotors. Die Aktivität und Expression der 11beta-HSD1 wurde durch die hier untersuchten endogenen und exogenen Faktoren spezifisch modifiziert, was sowohl in-vitro als auch in-vivo in unterschiedlichen Modellsystemen dargestellt werden konnte. Die Charakterisierung der MeSyBePo-Kohorte ergab keine direkten Assoziationen zwischen Polymorphismus und klinischem Phänotyp, jedoch Tendenzen für eine erhöhtes Körper-gewicht und Typ 2 Diabetes mellitus in Abhängigkeit des Genotyps. Der Promotor der 11beta-HSD1 konnte aufgrund der Daten aus den Luciferaseassays sowie den Daten aus den EMSA-Analysen näher charakterisiert werden. Dieser zeigt eine variable und zell-spezifische Regulation. Ein wichtiger Regulator stellen insbesondere in den HepG2-Zellen die C/EBP-Proteine -alpha, -beta und -delta dar. Aus den in-vivo-Studien ergab sich eine Regulation der 11beta-HSD1 durch endogene, exogene und pharmakologische Substanzen, die durch die Zellkulturversuche bestätigt und näher charakterisiert werden konnten.
The enzyme 11beta-HSD1 regulates intracellular the cortisol concentration by regeneration of cortisone to cortisol. Hence, 11beta-HSD1 is an important factor in glucocorticoid-mediated gene expression. It is ubiquitously expressed, but high levels have been specifically described in liver, adipose tissue and smooth muscle cells. A pivotal role for 11beta-HSD1 has been demonstrated with respect to metabolism in liver and adipose tissue. Thus, a liver-specific overexpression results in an elevated gluconeogenesis and hepatic glucose output. Furthermore, a fat-specific overexpression was associated with obesity, insulin resistance and dyslipidemia. Despite these intriguing data, the regulation of the human 11beta-HSD1 gene is still in its infancies. 8 promoter fragments from -3034 to +188 of 11beta-HSD1-gene were cloned to analyze promoter activity. Dual-Luciferase-Assay was used in humane HepG2 cells and in undifferentiated and differentiated 3T3-L1 cells. Furthermore, the region close to the transcription start was studied with a non-radioactive EMSA for binding of TATA-binding protein (TBP) and CCAAT/enhancer-binding-protein (C/EBP). The role of the endogenous and exogenous regulators fatty acids, PPARgamma and the incretin (Glucose-dependent insulinotropic Peptide) GIP was investigated in-vitro and in-vivo. Finally, the functional consequences of 2 Single Nucleotide Polymorphisms (SNP) within the promoter region were studied in cell culture and the MeSyBePo-cohorts for association with diabetes mellitus type 2 and body weight. The Luciferase-assay revealed a cell-specific regulation of 11beta-HSD1 and a repressor, which was active in all 3 cell models. Accordingly, a cell-specific regulation was observed in transactivation-assays with C/EBP-proteins -alpha, -beta and -delta. The 11beta-HSD1 enzyme expression and activity was specifically modified by the here investigated endogenous and exogenous factors, which was demonstrated in-vitro but also in-vivo in various experimental settings. The characterisation of the MeSyBePo-cohorte revealed no association between genotype and clinical phenotype, although a trend for an increased body weight and diabetes mellitus type 2 was detected. This work demonstrated a cell-specific regulation of the 11beta-HSD1 promoter. Furthermore, a binding site for TATA-binding proteins was detected in HepG2 and undifferentiated 3T3-L1 cells. A pivotal role in regulation of 11beta-HSD1 promoter activity was demonstrated for the C/EBP-proteins, especially in liver cells. The in-vivo-Studies revealed a regulation of enzyme expression and activity by endogenous, exogenous and pharmacological substances, which was confirmed and analyzed in more detail in cell culture experiments.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1) is an enzyme that predominantly converts inactive glucocorticoids (cortisone in human and most mammals, 11dehydro-corticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively). Thus 11β-HSD1 amplifies intracellular levels of glucocorticoids. Studies in globally 11β-HSD1 deficient mice have revealed changes in glucocorticoid-regulated physiological and pathological processes, including metabolism, aging, arthritis and angiogenesis. The function of macrophages, which play an important role in inflammation, is also altered. For example, 11β-HSD1 deficiency in macrophages causes a delay in their acquisition of phagocytic capacity. To dissect the role of macrophage 11β-HSD1 in angiogenesis, arthritis and obesity, both in vitro macrophage stimulation and in vivo functional assays in macrophage-specific 11β-HSD1 knockout mice, were conducted. Thioglycollate-elicited peritoneal macrophages from globally 11β-HSD1 deficient and control C57BL/6 mice were used for in vitro studies. In M1/M2 macrophage polarisation experiments, 11β-HSD1 deficient macrophages showed increased expression of mRNAs encoding pro-inflammatory factors upon lipopolysaccharide and interferon-ϒ treatment and decreased expression of pro-resolution genes with interleukin-4 stimulation. However, at cytokine or protein levels, there was little difference between the genotypes except for decrease IL12 p40 levels in 11β-HSD1 deficient macrophages. Hypoxic stress failed to show differences between genotypes in hypoxia-regulated gene expression. These data do not support a strong role for macrophage 11β-HSD1 in inflammation regulation, nor in response to hypoxia, at least when measured in vitro. The discrepancy between transcriptional and translational responses is currently unexplained, but may reflect altered posttranscriptional activity. To investigate the role of macrophage 11β-HSD1 in vivo, macrophage-specific Hsd11b1 knockout mice, LysM-Cre Hsd11b1 flox/flox (MKO) mice and Hsd11b1flox/flox littermate controls were generated. In MKO mice, 11β-HSD1 protein levels and enzyme activity were reduced by >80% in resident peritoneal macrophages. However, 11β-HSD1 protein and enzyme activity levels were unchanged or only modestly reduced in thioglycocollate-elicited peritoneal neutrophils, monocytes/macrophages, or in bone marrow-derived macrophages, despite >80% decrease in Hsd11b1 mRNA levels in these cells. A relatively long half-life of 11β-HSD1 protein compared to that of circulating myeloid cells may underlie this mismatch between transcriptional and translational expression. Furthermore, following 12 days of inflammatory arthritis induced by K/BxN serum transfer, the reduction in 11β-HSD1 protein levels in circulating neutrophils of MKO mice is consistently around 50%, which corroborates the above explanation. MKO mice and littermate controls were subjected to inflammatory models which may involve resident macrophages. First, to address the role of 11β-HSD1 in macrophages in angiogenesis, sponge implants were inserted subcutaneously into the flanks of adult male mice and harvested after 21 days. Chalkley counting on hematoxylin and eosin stained sponge sections showed significantly increased angiogenesis in MKO mice (scores: 5.2±1.0 versus 4.3±0.7; p<0.05, n=9-11). Cdh5 expression (encoding VE-cadherin, a marker of endothelial cells) was higher in sponges from MKO mice (relative expression: 1.5±0.9 versus 0.8±0.6; p<0.05), as was Il1b (encoding IL-1 beta, a marker of inflammation, relative expression: 6.5±6.4 versus 1.5±0.9; p<0.05). Vegfa mRNA (encoding vascular endothelial growth factor alpha) was unchanged, with a trend for higher Angpt1 (encoding angiopoietin 1, p=0.09) expression levels in the MKO group. These results suggest that lack of 11β- HSD1 in resident macrophages increases their pro-angiogenic activity, independently of VEGF-. The K/BxN serum transfer model of arthritis was used to investigate the role of macrophage 11β-HSD1 in arthritis. Adult male MKO and control mice received a single i.p. injection of 125μl K/BxN serum per mouse, followed by 21 days of clinical scoring to assess joint inflammation. The onset of inflammation (d1-8) was similar between MKO and control mice, but MKO mice exhibited greater clinical inflammation scores in the resolution phase of arthritis (d13-21; area-under-the-curve: 86.6±14.7 versus 60.1±13.4; p<0.005), indistinguishable from globally 11β-HSD1- deficient mice. Hematoxylin and eosin staining revealed pronounced fibroplasia predominantly in the supporting mesenchyme associated with the tenosynovium, with new bone and blood vessel formation. These results suggest that macrophage 11β-HSD1 deficiency is fully accountable for the worse arthritis resolution phenotype in the globally 11β-HSD1 deficient mice, but not the earlier onset of inflammation with global 11β-HSD1 deficiency. Macrophage activation states are closely linked with adipose insulin sensitivity. Globally 11β-HSD1 deficient mice are protected from high fat diet induced insulin resistance and adipose tissue hypoxia and fibrosis. To study the effect of macrophage 11β-HSD1 deficiency on insulin sensitivity, adult male MKO and control mice were given a 14 week high fat diet, which typically causes insulin resistance in control but not globally 11β-HSD1 KO mice. The level of fibrosis in subcutaneous adipose tissues was reduced as indicated by quantification of picrosirius red staining of collagen, though GTT data so far does not support protection from insulin resistance in MKO mice. In summary, in vitro macrophage polarisation experiments do not support a strong role of 11β-HSD in M1/M2 macrophage polarisations or response to hypoxia. However, MKO mice reveal, for the first time, an important in vivo role of macrophage 11β-HSD1 to promote angiogenesis and facilitate resolution of K/BxN serum transfer induced arthritis. Modulation of fibrosis is context dependent. Reduced adipose fibrosis may be one of the mechanisms that improve insulin sensitivity. Meanwhile, these findings suggest caution regarding the potential side effects of 11β-HSD1 inhibitors in treating metabolic disease in patients with inflammation-related co-morbidities, such as rheumatoid arthritis.

Obesity is associated with an increased risk of diabetes type 2, dyslipidaemia and atherosclerosis. These cardiovascular and metabolic abnormalities are exacerbated by dietary fats such as cholesterol and its metabolites. High adipose tissue glucocorticoid levels, generated by the intracellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) are also implicated in the pathogenesis of obesity, metabolic syndrome and atherosclerosis. Transgenic mice over-expressing 11β-HSD1 selectively in adipose tissue develop the metabolic syndrome whereas 11β-HSD1-/- mice have a ‘cardioprotective’ phenotype, deriving in part from improved adipose tissue function. Consistent with this, prototypical therapeutic 11β-HSD1 inhibitors ameliorate metabolic disturbances associated with obesity. 11β-HSD1 also inter-converts the atherogenic oxysterols 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7β-HC). Work presented in the first part of the thesis defines the impact of these alternative substrates on the metabolism of glucocorticoids in adipocyte cell lines (3T3-L1 and 3T3-F442A). 11β-HSD1 catalyses the reduction of 7KC in mature adipocytes leading to accumulation of 7β-HC. Oxysterol and glucocorticoid conversion by 11β-HSD1 was competitive and occurred within a physiologically-relevant IC50 range of 450nM for 7KC inhibition of glucocorticoid metabolism. Working as an inhibitor of 11β-HSD1 activity, 7KC decreased the regeneration of active glucocorticoid and limited the process of preadipocyte differentiation. 7-oxysterols did not display intrinsic activation of the glucocorticoid receptor (GR). However, when co-incubated with glucocorticoid, 7KC repressed, and 7β-HC enhanced GR transcriptional activity. The effect of 7-oxysterols resulted from the modulation of 11β-HSD1 reaction direction, at least in transfected HEK293 cells, and could be abrogated by over-expression of hexose 6-phosphate dehydrogenase, which supplies NADPH to drive the reductase activity of 11β-HSD1. 11β-HSD1 inhibition protects from atherosclerosis, yet it is unknown whether it is an effect of alterations in the metabolism of 7-oxysterols. 7KC and 7β-HC did not activate the potential cognate receptor LXRα and FXR/RXR in transactivation assays. No differential regulation of key gene targets of LXRα, FXR and RORα in the liver and fat depots of high fat fed 11β-HSD1-/- and wild type mice was observed. To further determine the molecular basis for the metabolically beneficial phenotype of 11β-HSD1-/- mice I analysed global gene expression in subcutaneous and mesenteric adipose tissues of high fat-fed (4 weeks) 11β-HSD1-/- and congenic C57BL/6J mice by microarrays, followed by pathway analysis, gene clustering and realtime-PCR validation of transcripts with >1.5-fold difference between genotypes. 11β-HSD1-/- mice gained less weight and distributed adipose tissue to subcutaneous rather than visceral depots. Broadly, high fat-fed 11β-HSD1-/- mice showed up-regulation of transcripts in subcutaneous fat (70% of 1622 differentially-expressed transcripts), but down-regulation in mesenteric adipose tissue (73% of 849 transcripts). Genes up-regulated in 11β-HSD1-/- subcutaneous adipose were associated with β-adrenergic signaling, glucose metabolism, lipid oxidation, oxidative phosphorylation, MAPK, Wnt/β-catenin, EGF, and PI3K/AKT insulin signaling pathways. Increased subcutaneous fat insulin signaling was confirmed by increased IRS-1 and Akt phosphorylation in vivo. Down-regulated genes in 11β-HSD1-/- mesenteric fat were associated with immune cells, NK-kappaB, Jak/Stat, SAPK/JNK, chemokine, toll-like-receptor and Wnt signaling pathways suggesting reduced immune cell infiltration in mesenteric adipose in high fat-fed 11β-HSD1-/- mice. 11β-HSD1 deficiency protects against metabolic disease by increasing peripheral fat insulin sensitivity and through a novel mechanism involving reduction in visceral fat immune/inflammatory cell function. Data presented in this thesis contribute to the understanding of the role of 11β-HSD1 in adipose tissues in obesity and, potentially, atherosclerosis.

Glucocorticoids profoundly influence the immune system and pharmacological doses exert potent anti-inflammatory actions. During inflammation, glucocorticoids limit oedema and influence cell trafficking, differentiation programmes and gene transcription in glucocorticoid-sensitive leukocytes. Within cells, glucocorticoid action is modulated by a pre-receptor mechanism; glucocorticoid metabolism by the enzyme 11β- hydroxysteroid dehydrogenase (11β-HSD). Two 11β-HSD isozymes exist: 11β-HSD1, which catalyses amplification of glucocorticoid levels in intact cells by oxo-reduction of intrinsically inert cortisone (11-dehydrocorticosterone in rodents) into active cortisol (corticosterone in rodents) and 11β-HSD2, which performs the opposite reaction. Thus, amplification of intracellular glucocorticoid levels by 11β-HSD1 may represent an endogenous anti-inflammatory mechanism. This hypothesis has been tested in Hsd11b1-/- mice (homozygous for a targeted disruption in the Hsd11b1 gene, encoding 11β-HSD1), using carageenan-induced pleurisy and experimental model of arthritis induced by injection of arthritogenic antibodies. In both models, Hsd11b1-/- mice showed more severe acute inflammation than control mice. During carrageenan-induced pleurisy, Hsd11b1-/- mice recruited more inflammatory cells to the pleural cavity than congenic controls, with a greater proportion of viable cells, at the onset and peak of pleurisy, suggesting a worse inflammatory response. Histological examination suggested impaired resolution of inflammation in Hsd11b1-/- mice with persistence of inflammation in the visceral pleura, activation of lymphoid aggregates, and uniquely in Hsd11b1-/- mice, formation of fibrous adhesions between lung lobes 48h after initiation of pleurisy. During experimental arthritis induced by injection of serum from arthritic K/BxN mice, clinical signs of inflammation occurred earlier in Hsd11b1-/- mice and were slower to resolve than in control mice. Histological assessment of the acute phase (2d) of arthritis showed no difference in joint pathology between genotypes, despite greater oedema and higher clinical scores in the Hsd11b1-/- mice. However, when the inflammation had resolved (21d following injection of serum), compared to control mice, Hsd11b1-/- mice showed more severe exostosis, intense periarticular inflammation, more collagen deposition and uniquely, ganglion cyst formation. At 21d, whereas basal (morning) plasma corticosterone levels were normal in control mice, they remained elevated in Hsd11b1-/- mice, suggesting ongoing inflammation and persistent activation of the hypothalamic-pituitary-adrenal axis. Mast cells are critical in the initiation of an inflammatory response and are essential in this model of arthritis. Mast cells expressed 11β-HSD1 (but not 11β-HSD2) mRNA and activity. Although mast cell number did not differ in joints or peritoneum of Hsd11b1-/- mice, 11-HSD1-deficient mast cells had a lower threshold for degranulation induced by K/BxN arthritogenic serum. As well as implicating a role for mast cell 11β-HSD1 in limiting initial inflammation in arthritis, these findings also have implications for infection, allergy and tolerance. Collectively, these data suggest that 11β-HSD1 deficiency worsens acute inflammation and results in slower resolution. Therefore, amplification of intracellular glucocorticoids levels, by 11β-HSD1, may represent an important mechanism to limit the acute inflammatory response and programme its subsequent resolution. Increasing leukocyte 11β-HSD1 or local delivery of substrate affords a novel approach for anti-inflammatory therapy.

Chronically elevated brain glucocorticoid (GC) levels impair cognition. Age-related cognitive deficits or "sickness" behaviour is often associated with neuroinflammation. In rodents, raised GC levels prior to lipopolysaccharide (LPS) administration potentiate neuroinflammation although GC suppresses neuroinflammation if administered after LPS. 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1) reductase activity can increase intracellular GC levels, including in the brain, without alteration in circulating levels. Deficiency/pharmacological inhibition of 11β-HSD1 is protective against age related cognitive impairment in both rodent and humans. However, the underlying mechanism remains unclear. 11β-HSD1 reductase activity is coupled to hexose-6-phosphate dehydrogenase activity, itself dependent on cellular energy status. Processes affected by deficiency/inhibition of 11β- HSD1 (e.g. acute inflammation, angiogenesis) are associated with increased glycolysis. Additionally, compared to C57BL/6J controls, adipose tissue of 11β-HSD1 deficient mice shows increased expression of glycolytic and oxidative metabolism genes in a rodent model of obesity, characterised by low-grade chronic inflammation. I hypothesised that 11β-HSD1 has a role in regulation of cellular energetics basally and following inflammation. 11β-HSD1 expression in the brain will be up-regulated during systemic inflammation. Following inflammation, 11β-HSD1 deficiency will attenuate the pro-inflammatory response and subsequently alter energy substrate uptake and/or utilisation in the key areas of brain (i.e. hypothalamus and the hippocampus) that sense and respond to inflammation and energy balance. To test my hypothesis, global 11β-HSD1 KO mice, primary macrophages in vitro and murine models of inflammations were utilised. 11β-HSD1 mRNA and protein expression were confirmed in the hypothalamus and the hippocampus of C57BL/6J mice. In the absence of inflammation, expression of inflammatory markers is low or negligible in the brains of Hsd11b1-/- mice similar to C57BL/6J controls. However, compared to C57BL/6J, Hsd11b1-/- mice show altered mRNA levels of metabolic transporters and enzymes in the hypothalamus and the hippocampus. Overall, the mRNA profiling suggests reduced dependence on glucose in the brains of Hsd11b1-/- mice, either through increased lactate availability (in the whole brain and hippocampus) or through increased glycolysis and mitochondrial number/function (in the hypothalamus). Primary macrophages were utilised to investigate the role of 11β-HSD1 in cellular energetics in vitro. In these cell based assays, glycolysis was found to be the predominant glucose metabolising pathway in C57BL/6J primary macrophages, consistent with the literature. Preliminary data suggested reduced glycolytic activity in Hsd11b1-/- compared to C57BL/6J primary macrophages. However, initial attempts to utilise these cell based assays on primary microglia were unsuccessful. Moreover, Hsd11b1 mRNAs in the brain (down-regulation with inflammation, discussed later) was found to be differentially regulated in comparison to Hsd11b1 mRNA levels in the macrophages (up-regulation with inflammation) hence further investigation was not pursued. To identify a model of peripheral inflammation where 11β-HSD1 is regulated in the brain in vivo, Staph. aureus induced acute lung inflammation and the K/BxN serum transfer induced model of arthritis were utilised. Increased expression of inflammatory markers in the brain was associated with reduced Hsd11b1 mRNA levels in the hippocampus of control mice in these models. Comparison of Hsd11b1-/- and C57BL/6J mice showed increased levels of mRNAs encoding metabolic transporters in the hypothalamus and the hippocampus of Hsd11b1-/- mice following inflammation in the K/BxN serum transfer model of arthritis suggesting increased energy substrate availability. Additionally, increased levels of mRNA encoding metabolic enzymes suggested increased glycolytic capacity and mitochondrial oxidative phosphorylation activity in the hippocampus but not the hypothalamus of Hsd11b1- /-, compared to C57BL/6J mice, following K/BxN serum induced arthritis. Overall, these data suggest that the reduction in expression of 11β-HSD1 could be a potential mechanism to increase energy substrate availability, glycolytic capacity and mitochondrial activity in the hippocampus to provide metabolic support for neuronal metabolism and function following peripheral inflammation. The role of 11β-HSD1 in the pro-inflammatory response and cellular energetics in the hippocampus was further investigated in a well characterised sterile peritonitis model of systemic inflammation in which a low to moderate dose of LPS was used. Mice were administered LPS or vehicle (0.9% saline) by a single i.p. injection and culled 3h, 6h or 9h post injection. Inflammation resulted in significant reduction in burrowing activity both in Hsd11b1-/- and C57BL/6J mice suggesting sickness behaviour.. The number of circulating immune cells, as a measure of peripheral inflammation, did not differ between genotypes. Similarly, plasma corticosterone levels were elevated following inflammation but no genotype difference was observed. However, levels of plasma 11-dehydrocorticosterone, the inert substrate for 11β- HSD1, were significantly elevated in the Hsd11b1-/-, compared to C57BL/6J mice, following inflammation. Levels of mRNA encoding inflammatory markers were lower in the hippocampus of Hsd11b1-/-, compared to C57BL/6J mice, following inflammation. Also, Hsd11b1 mRNA levels were reduced in the hippocampus of C57BL/6J mice following inflammation, consistent with the finding above. Principal component analysis on levels of mRNA encoding metabolite transporters and enzymes revealed a distinct metabolic response in the hippocampus of Hsd11b1-/-, compared to C57BL/6J mice, 6h post LPS. At the same time point in the hippocampus, levels of mRNAs encoding metabolite transporters and enzymes suggested an attenuated switch to aerobic glycolysis with maintenance of mitochondrial function/activity. Quantification of hippocampal energy metabolites using targeted metabolomics in the Hsd11b1-/- compared to C57BL/6J mice 6h post LPS showed correspondence with the mRNA results. Overall, these results suggest that reduced expression of 11β-HSD1 could be a potential mechanism to reduce the pro-inflammatory response and provide better metabolic support for neuronal function and metabolism in the hippocampus, following systemic inflammation. In summary, the current work provides evidence for neuroprotection with 11β-HSD1 deficiency, following systemic inflammation. The suggestive neuroprotection is at least in part mediated via an attenuated pro-inflammatory responses and increased energy substrate uptake and/or utilisation providing better metabolic support for neuronal function following inflammation. It argues for the development of tissue specific small molecule inhibitors of 11β-HSD1 that can cross the blood brain barrier as therapeutic agents against the adverse cognitive effects of systemic inflammation and/or inflammaging.

Diabetes Mellitus is characterized by high blood sugar and is caused by resistance to (type 2) or insufficiency of (type 1) the pancreatic β-cell hormone insulin. Most commonly, type 2 diabetes is associated with obesity whereas type 1 diabetes is largely a result of immune-mediated destruction of the β-cell. One rare but significant cause of type 2 diabetes is excess blood glucocorticoid levels (Cushing’s syndrome). High circulating glucocorticoids potently induce metabolic disorders including peripheral insulin resistance in key metabolic tissues (muscle, liver and fat) as well as directly suppressing β-cell function and can precipitate type 2 diabetes. However, in common forms of metabolic syndrome (visceral obesity, type 2 diabetes, increased cardiovascular disease risk) it appears that amplification of local tissue glucocorticoid action by increased levels of the intracellular enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), particularly in adipose tissue, is a key driver of the adverse metabolic phenotype rather than altered circulating glucocorticoid levels. 11β-HSD1 is also elevated in pancreatic islets from obese rodents. This thesis aimed to determine the role of 11β-HSD1 in pancreatic islets (β-cells) under normal conditions and its potential pathogenic role in the development of diabetes. We first determined that 11β-HSD1 acted primarily as a reductase (amplifying glucocorticoid action) in pancreatic islets. We then determined that islet 11β-HSD1 transcription is under the control of the promoters that express in other tissues like liver. Islet 11β-HSD1 is significantly regulated by factors relevant to the diabetic state; high glucose and insulin suppressed whereas fatty acids and TNFα increased 11β-HSD1 activity. To test whether the high islet 11β-HSD1 found in obese rodents was directly diabetogenic, we generated transgenic mice specifically overexpressing β-cell 11β-HSD1 under the mouse insulin promoter (MIP-HSD1 mice) in a mouse strain prone to develop β-cell failure when subjected to diabetic challenge (eg. chronic high fat feeding). Unexpectedly, MIP-HSD1tg/+ mice (expressing ~2 fold elevated 11β-HSD1 activity) exhibited markedly improved β-cell insulin secretory responses, whereas MIP-HSD1tg/tg mice had partially impaired β-cell insulin secretory function in vivo and in vitro. Moreover, MIP-HSD1tg/+ mice completely resisted the mild hyperglycaemia induced by multiple-low doses of the β-cell toxin streptozotocin (40mg/kg i.p. for 5 days) and partially resisted the profound hyperglycaemia induced by a single high dose of streptozotocin (180mg/kg). Notably, MIP-HSD1tg/+ mice exhibited lower macrophage infiltration (MAC-2) and higher T-regulatory cell (Foxp3) infiltration after these challenges with evidence of increased insulin-positive cells and maintenance of normal levels of proliferation-competent β-cells. Overall, MIP-HSD1tg/tg exhibited a partial protection from the streptozotocin challenge. Modestly increased 11β-HSD1 expression in β-cells unexpectedly supports compensatory insulin hypersecretion preventing type 2 diabetes and protects β-cells from inflammatory mediated damage in the setting of type 1 diabetes. Above a protective threshold, elevated β-cell 11β-HSD1 may result in β-cell dysfunction and diabetes. These findings have important implications for the currently advocated therapeutic strategies to inhibit 11β-HSD1 in the context of obesity and diabetes.

7-Oxysterols constitute the major component (40%) of oxidized low-density lipoprotein (oxLDL). They arise in the body via auto-oxidation of cholesterol and are known to induce endothelial dysfunction, oxidative stress and apoptosis in the vascular wall, prior to development of atherosclerosis. A novel pathway has been described for hepatic inter-conversion of 7-ketocholesterol (7-KC) and 7β -hydroxycholesterol (7β OHC) by the enzyme 11β-hydroxysteroid dehydrogenase type-1 (11β HSD1), better known for metabolizing glucocorticoids. Inhibition of 11βHSD1 is atheroprotective and the potential underlying mechanism for this may involve altered metabolism and actions of glucocorticoids. However, alterations in the metabolism of 7-oxysterols may also play an important role in this atheroprotective effect. The work described here addresses the hypotheses that (i) 7-oxysterols are substrates for murine 11βHSD1; (ii) inhibition of 11β HSD1 may abolish cellular metabolism of 7-oxysterols; (iii) this route of metabolism may modulate the actions of 7-oxysterols and glucocorticoids on murine vascular physiology. Murine 11β HSD1 inter-converted 7-oxysterols (Km=327.6±98ìM, Vmax=0.01±0.001pmol/ìg/min) but the regulation of reaction direction is different from that for glucocorticoids. Predominant dehydrogenation of 7β OHC to 7-KC was quantified in several models (recombinant protein, cultured cells stably transfected with 11β HSD1), in which predominant reduction of glucocorticoids was measured. Furthermore, in murine hepatic microsomes, dehydrogenation of 7β OHC occurred exclusively. In aortic rings in culture, however, both reduction and dehydrogenation of 7-oxysterols were evident. 7-Oxysterols and glucocorticoid substrates competed for metabolism by 11β HSD1, with 7β OHC inhibiting dehydrogenation of glucocorticoids (Ki=908±53nM). The circulating concentrations of 7-oxysterols in the plasma of C57Bl6 and 11β HSD1-/- mice were in the ìM range (0.02 – 0.13ìM). The disruption of 11β HSD1 has resulted in increased ratios of 7-KC and 7β OHC over total plasma cholesterol levels (*p<0.05). This finding suggested that 11β HSD1 is involved in metabolizing and determining the plasma levels of 7-KC and 7β OHC. To assess the consequences of these alterations for vascular function, studies were undertaken in aortic rings. Prolonged incubation with 7-oxysterols (20-25 ìM) showed a tendency to attenuate noradrenaline-mediated contractions of C57Bl6 aortae, but had no effect on contractions in response to 5-hydroxytryptamine or KCl. Similarly, endothelium-dependent and -independent relaxations of murine aortae were unaltered after exposure to 7-oxysterols. Thus in the mouse, 11β HSD1 may influence the balance of circulating and cellular 7-oxysterols which may have consequential effects on glucocorticoid action. Although this work suggests that concentrations present in murine tissues are unlikely to cause vascular dysfunction, they may influence further cellular events as yet undescribed. Under pathological conditions where high concentrations of 7-oxysterols occur, 11β HSD1 may influence the extracellular-transport and delivery of 7-KC and 7β OHC to the plaque. This work therefore proposes that inhibition of metabolism of 7-oxysterols by 11β HSD1 inhibitors, may contribute to the atheroprotective effects of these drugs.

Glucocorticoids (GC) have a negative effect on age-associated cognitive decline and the GC metabolising enzyme 11β-hydroxysteroid dehydrogenase Type 1 (11β- HSD1) plays a key role in these effects. Increased glucocorticoids exert detrimental effects on the volume and function of brain regions such as the prefrontal cortex (PFC) and hippocampus that are necessary for cognitive functions such as memory and working memory. Previous research has identified changes in cell populations, metabolite levels and structure within the brain as well as altered levels of inflammation with age, and studies have suggested these biomarkers to be associated with cognitive impairments. Aged mice with a deletion in 11ß-HSD1 (11β-HSD1-/- mice), resulting in lower levels of glucocorticoids within the brain, exhibit attenuated cognitive decline in hippocampal dependent spatial learning and memory with age. However, the mechanisms through which 11β-HSD1 contribute to age-associated cognitive decline remain unknown. However, previous genetic models of 11β-HSD1- /- mice have demonstrated residual 11β-HSD1 activity in the brain which may still exert some effects on cognitive processes. Furthermore, the effect of 11ß-HSD1 on working memory – a more cognitively demanding process essential for everyday decision making - has yet to be determined. This thesis tests the hypothesis that glucocorticoid action mediates age-associated cognitive impairment in spatial learning and memory and spatial working memory through alterations in cell activity, brain metabolite levels and neuroinflammatory processes. Therefore, we aimed to investigate if complete lifelong 11β-HSD1 deficiency would protect against age-associated working memory deficits as well as spatial learning and memory deficits, and its effect on associated neural markers. In particular, we determined changes in hippocampal metabolite levels, cell activity and inflammation as a function of ageing in a longitudinal manner. At 6, 12, 18 and 22 months, male 11β- HSD1-/- and C57BL/6J control mice were cognitively assessed in the Morris Water Maze (MWM) and Radial Arm Water Maze (RAWM) – tests for spatial reference memory and spatial working memory respectively. Magnetic resonance spectroscopy (1H-MRS) was performed to examine the hippocampal metabolite profile in the same mice at 6, 18 and 22 months. Following their final scan, mice were culled and brains dissected for analysis. Results revealed unaltered spatial learning with age in C57BL/6J and 11β-HSD1-/- mice and pointed to a development of alternative strategies for task completion as a result of repeated testing. Spatial memory was more susceptible to age-associated effects with impairments in wild-type mice but not 11β-HSD1-/- mice, though not completely immune from the effects of repeated testing. These impairments were correlated with glutamate/glutamine levels and glial fibrillary acidic protein (GFAP), whilst GFAP was further correlated with 11β-HSD1 protein expression. Working memory was impaired with age in both 11β-HSD1-/- and wild-type mice, suggesting 11β-HSD1 deletion may be detrimental to cognitive processes in the prefrontal cortex. In conclusion, impaired memory with age may be attributed to increased glial reactivity and altered glutamate/glutamine cycling in the hippocampus, and lifelong removal of 11β-HSD1 may alter these processes. However, lifelong removal of 11β-HSD1 may not be as beneficial to working memory processes suggesting that 11β-HSD1 and glucocorticoid action play a key role in working memory processes.

Glucocorticoids (GC) are regulators of permissive and adaptive physiology. GC excess can lead to metabolic complications including type 2 diabetes and metabolic syndrome. Levels are regulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which reactivates GC. 11β-HSD1 activity is deregulated in a range metabolic disorders in which GC levels are normal. I hypothesise that 11β-HSD1 is a critical regulator of adipose tissue sensitivity to GC excess, and that through 11β-HSD1 depletion adipose tissue will be desensitised to GCs and resist metabolic deregulation. Using 11β-HSD1 KO mice in a model of GC excess we demonstrate that 11β-HSD1 mediates the adverse metabolic effects of GC excess on a global scale. I further investigated brown adipose tissue (BAT) with GC excess. I demonstrate that 11β-HSD1 regulates BAT activity and mitochondrial function, possibly suppressing BATs thermogenic potential. I extended my studies to examine the potential for white adipose tissue (WAT) to assume markers of thermogneic and mitochondrial function in the context of 11βHSD1 and GC excess. The data suggest 11β-HSD1 may suppress the potential of WAT to assume a ‘BAT-like’ profile. These data show 11β-HSD1 loss of function confers a protective phenotype with GC excess and demonstrates it’s role in mediating the metabolic phenotype associated with GCs. These data support the idea that GCs can influence BAT and WAT thermogenic potential and may increase knowledge of metabolic dysregulation in humans suffering form GC excess. This therefore highlights 11β-HSD1 as an exciting potential target for the treatment for the metabolic disease associated with GC excess.

Chronic glucocorticoid (GC) excess (Cushing’s syndrome, pharmacotherapy) causes metabolic and cardiovascular disease. This might be predicted from the known metabolic (dyslipidaemia, insulin resistance/hyperglycaemia) and hypertensive effects of chronically elevated GC levels. Intracellular GC levels within target tissues are controlled by 11β-hydroxysteroid dehydrogenases. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by Hsd11b1) is an enzyme that, in intact cells and in vivo, converts inert GCs (cortisone in humans, and 11- dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively). Consequently, 11β-HSD1 amplifies intracellular GC levels. Additionally, 11β-HSD1 is also involved in the metabolism of 7-oxysterols; it catalyses the reduction of 7-ketocholesterol (7-KC) to 7β-hydroxycholesterol (7β- HC). 7-KC may inhibit cholesterol biosynthesis through its ability to inhibit cleavage/processing of sterol regulatory element binding protein-2 (SREBP-2), the key regulator of cholesterol synthesis. Alteration of cholesterol homeostasis is a major risk factor for cardiovascular disease. Improvement of metabolic syndrome and attenuation of atherosclerosis are observed in susceptible rodent models with 11β- HSD1 deficiency or inhibition. Conversely, pilot data showed decreased levels of 7- KC as well as increased levels of cleaved SREBP-2 protein (the transcriptionally active form) in liver of mice with hepatic 11β-HSD1 overexpression (LOE mice), suggesting increased cholesterol biosynthesis. It was hypothesised that hepatic 11β- HSD1 promotes cholesterol biosynthesis through hepatic induction of SREBP-2 target genes in the cholesterol biosynthetic pathway. The hypothesis was tested in adult, male LOE and wild-type C57BL/6 mice. In mice fed a standard chow diet, hepatic levels of mRNA encoding hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase and HMG-CoA synthase, SREBP-2 targets in the cholesterol biosynthetic pathway, did not differ between genotypes. Compared to chow, a cholesterol-rich ‘Western’ diet (WD) decreased hepatic levels of mRNA encoding SREBP-2, HMG-CoA reductase and HMG-CoA synthase in wild-type as well as in LOE mice. These data imply that LOE mice show a normal physiological response with respect to cholesterol synthesis when challenged with cholesterol-rich diet, and, contrary to the hypothesis, liver 11β-HSD1 does not increase cholesterol biosynthesis via elevated expression of mRNAs encoding hepatic cholesterol biosynthetic enzymes. The liver X receptors (LXR) are well-known as sensors of oxysterols and regulators of genes involved in processes that decrease total body cholesterol levels i.e. reverse cholesterol transport and cholesterol excretion into bile. Cholesterol is the precursor to oxysterol LXR ligands. It was predicted that liver overexpression of 11β-HSD1 leads to activation of LXRα (the isoform with dominant roles in reverse cholesterol transport and whole-body cholesterol homeostasis) and its downstream targets involved in cholesterol efflux and excretion, in response to increased intracellular cholesterol levels. Indeed, levels of Lxrα mRNA were increased in livers of WD-fed LOE mice compared to wild-type mice on the same diet. There was no evidence for increased cholesterol clearance through bile acid synthesis in LOE mice as indicated by unchanged levels of hepatic Cyp7a1 mRNA between LOE and wild-type mice. However, consistent with being direct targets of LXRα, increased Abcg5 and Abcg8 mRNA levels were observed in livers of WD-fed LOE mice compared to WD-fed wild-type mice. These results corroborate findings in chow-fed LOE mice. Moreover, these data suggest that LOE mice ‘sense’ intracellular cholesterol excess and respond to it by increasing cholesterol efflux into the biliary lumen for excretion, thereby supporting a role for hepatic 11β-HSD1 in promoting biliary cholesterol secretion. To assess the effect(s) of hepatic 11β-HSD1 deficiency on cholesterol homeostasis as well as evaluate the importance of liver 11β-HSD1 in metabolic syndrome, liver-specific 11β-HSD1 knockout (LKO) mice were generated by crossing “floxed” Hsd11b1 mice with Alb-Cre transgenic mice in which Cre expression is restricted to hepatocytes. In liver of LKO mice, 11β-HSD1 mRNA, protein and enzyme activity were reduced by >80%, with no differences in 11β-HSD1 protein levels in kidney, adipose tissue or muscle between LKO and floxed Hsd11b1 littermate controls. These results indicate liver-specificity of Hsd11b1 knockdown in these mice. Body weight and weights of liver, adipose tissue, adrenal, muscle, kidney and brain were unaltered by liver-specific 11β-HSD1 deficiency on a standard chow diet. These mice were subject to a 14-week high fat (HF) diet, which typically causes metabolic syndrome in control but not globally 11β-HSD1 deficient mice. In HF-fed LKO mice, weights of the subcutaneous and epididymal fat depots were decreased compared to HF-fed control mice, resulting in an overall decrease in total white adipose tissue weight. Although no differences were observed in subcutaneous adipocyte hypertrophy between HF-fed LKO and control mice in a small number of samples tested, the above finding suggests that liver 11β-HSD1 influences adiposity and that liver-specific deficiency of 11β-HSD1 may reduce diet-induced adiposity. In terms of cholesterol homeostasis, no differences were observed in hepatic levels of mRNAs encoding cholesterol biosynthetic enzymes as well as those encoding enzymes/transporters for cholesterol catabolism/excretion between LKO and control mice, on either chow or HF diet. In summary, these data do not support a role for hepatic 11β-HSD1 in cholesterol synthesis. However, my evidence suggests that increased hepatic 11β-HSD1 promotes hepatobiliary cholesterol secretion. Finally, knockdown of liver 11β-HSD1, combined with HF feeding, reduces adiposity, suggesting that hepatic 11β-HSD1 may play a key role in adipose tissue lipogenesis/lipolysis and/or lipid storage, and that liver-specific 11β-HSD1 inhibition (or deficiency) may be advantageous in diet-induced obesity. Data presented in this thesis contribute to the understanding of the role of hepatic 11β-HSD1 in cholesterol homeostasis and metabolic syndrome.

Alterations in glucocorticoid (GC) biosynthesis and metabolism are associated with a variety of pathophysiological disorders including cholestasis, diabetes and other metabolic disorders. Bile acids (BA) are also important modulators of metabolic functions and regulate cholesterol, triglyceride and glucose homeostasis as well as being critical for dietary fat digestion, enterohepatic function, and postprandial thermogenesis. In intact cells and in vivo, the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme converts inactive GC precursors (cortisone in humans, and 11-dehydrocorticosterone in mice and rats) into their active forms (cortisol and corticosterone, respectively) thereby amplifying local intracellular GC levels. Interconversion by 11β-HSD1 of other sterols has also been described. These include conversions of 7keto-cholesterol to 7β-hydroxycholesterol, 7-oxodehydroepiandrosterone (7-oxo-DHEA) to 7α-hydroxy- and 7β-hydroxy DHEA, 7- oxo-lithocholic acid (LCA, a bile acid; BA) to chenodeoxycholic acid (CDCA, a 7α- hydroxylated BA) and ursodeoxycholic acid (UDCA, a 7β-hydroxylated BA) in human liver microsomes. In the liver, BA inhibit 11β-HSD1 but whether 11β-HSD1 regulates BA homeostasis is unclear. Evidence of molecular regulation of the enterohepatic recycling of bile acids by liver glucocorticoid receptor (GR) in mice does suggest a role for 11β-HSD1. It was therefore hypothesised that disruption of 11β-HSD1 expression in mice would impair BA recycling and might affect the relative concentrations of BA within the enterohepatic circuit. The primary objective of the current work was to investigate the impact of altered 11β-HSD1 on BA homeostasis. This was achieved using genetically modified mouse models with altered 11β-HSD1 expression, either globally or restricted to hepatocytes. BA are stored in the gall bladder and are released postprandially, to aid digestion. It was hypothesised that 11β-HSD1 deficiency might the affect the process of postprandial gall bladder emptying/refilling. Mice with global 11β-HSD1 knockout (Hsd11b1-/-) and age-matched control mice (C57Bl/6) were either fasted for 4h and culled or fasted for 4h and re-fed for another 4h before culling. Their response to fasting and re-feeding was assessed with specific focus on organs associated with BA recycling in the enterohepatic circuit (liver, gall bladder, serum and small intestine). Gall bladders of fasted Hsd11b1-/- and C57Bl/6 mice had similar volumes of bile but in fasted Hsd11b1-/- mice, BA concentrations were higher in serum and liver. As expected, re-feeding caused gall bladder emptying in C57Bl/6 mice with consequent increased serum and liver bile acid concentrations. In Hsd11b1-/- mice, the gall bladder did not empty and serum and liver BA concentrations were similar to the fasted state. To explore possible reasons for this, levels of mRNA encoding proteins known to be involved in hepatic BA transport were quantified using real-time q-PCR. Levels of mRNA encoding NTCP/ SCL10A1/ SCL10A1, the transporter responsible for most hepatocyte BA uptake, were increased in livers of fasted Hsd11b1-/- mice whereas levels of Slc51b mRNA, encoding the OST- transporter that facilitates BA removal from liver to the systemic circulation, and levels of Mrp2 and Atp8b1/FIC1 mRNAs (both encoding proteins which transport BA from liver into gall bladder) were decreased. This suggests that in fasted Hsd11b1-/- mice, BA transporter expression is altered to increase BA influx into hepatocytes and decrease efflux, to compensate for reduced levels of liver BA. These data together imply that bile acid recycling is controlled by 11β-HSD1 activity which regulates gall bladder emptying, hepatic BA concentration and BA transporter activity to ensure continuity of BA recycling within the enterohepatic circuit compartments. These changes may also affect digestion of lipids and fat-soluble micronutrients. Because 11β-HSD1 can directly metabolise secondary BA, it was predicted that 11β-HSD1 deficiency would lead to changes in the BA profile. Profiling of BA in the gall bladder was performed using mass spectrophotometry. In Hsd11b1-/- mice, 7α-hydroxylated BA predominated (cholic acid [CA]>α-muricholic acid [α- MCA]>CDCA>others), in contrast to C57Bl/6 mice in which 7β-hydroxylated BA predominated (ω-MCA>β-MCA>UDCA>others). The ratio of 7α:7β acids was therefore >100-fold greater in Hsd11b1-/- mice. This suggests that 11β-HSD1 either directly or indirectly controls the epimerisation of 7α- to 7β- hydroxylated BAs. Measurement of mRNAs encoding proteins important for hepatic BA biosynthesis in livers of fasted Hsd11b1-/- mice showed decreased expression of Scarb1/SR-B1, Cyp39a1 and Cyp27a1 (though with no change in levels of CDCA, the product of CYP27A1, in liver or bile fluid), compared to fasted control mice. Hepatic levels of Gpbar1/TGR5/GPBAR1 and Cyp3a11 mRNAs, encoding proteins important in BA detoxification, were increased and decreased, respectively. This suggests that Gpbar1/TGR5/GPBAR1, encoding G-protein coupled bile acid receptor (also called TGR5/GPBAR1) and an FXR target, could be induced to detoxify 7α-hydroxylated BA whereas expression of Cyp3a11, which catalyses the conversion of LCA to hyodeoxycholic acid (HDCA) is decreased; bile fluid of Hsd11b1-/- mice contained lower levels of LCA and little to no HDCA, though LCA and HDCA levels in liver were unaltered. Currently, the functional differences between 7α- and 7β- hydroxylated BA are not clear. However, these findings could have significant implications for bile acid-mediated transcription which, in turn, might affect lipid and sterol metabolism. Also, alterations in BA composition may have other physiological consequences via other pathways. Because cholesterol is the precursor of BA synthesis, it was hypothesised that western diet (WD) (containing cholesterol) would exacerbate and/or alter the phenotype of Hsd11b1-/- mice. Gall bladder weights of fasted Hsd11b1-/- and control C57Bl/6 mice did not change with western diet compared to chow diet. In control C57Bl/6 mice, the total BA concentration in the gall bladder increased in response to WD in comparison to chow diet. In contrast, Hsd11b1-/- mice showed no change in total BA concentration when fed on WD in comparison to chow. These data indicate that 11β-HSD1 is required by mice for the normal increase in total BA concentration in bile in response to dietary cholesterol. BA profiling of bile from control mice fed on WD showed no difference in the relative amounts of 7β-hydroxylated BA and 7α-hydroxylated BA to littermates fed on chow diet with the exception of β–MCA which increased, and α–MCA which decreased. Like chow-fed Hsd11b1-/- mice, BA profiling of bile from WD-fed Hsd11b1-/- mice showed a significant decrease in relative levels of 7β-hydroxylated BA (UDCA < β-MCA < others) and an increase in percentage of 7α-hydroxylated BAs (CA>α-MCA>CDCA>others) compared to C57Bl/6 controls. These data show that Hsd11b1-/- mice fail to show the normal increase in 7β-hydroxylated BA and decrease in 7α-hydroxylated BA observed in control mice in response to a cholesterol containing diet, suggesting 11β-HSD1 deficiency blunts the influence of cholesterol on BA composition. Measurement of hepatic mRNAs encoding BA transporters suggest that hepatocyte uptake of BA is decreased in C57Bl/6 on WD compared to those mice on chow diet, whereas this was not the case in Hsd11b1-/- mice where hepatic expression did not change with diet. Thus, Hsd11b1-/- mice failed to increase expression of Ntcp/ Scl10a1/ Scl10a1 appropriately, suggesting impaired hepatic BA uptake, while Slc51b (encoding OST-β) expression was increased, compared to control mice, possibly to reduce hepatic BA concentration by transporting BA out of hepatocytes into the systemic circulation. Therefore, Hsd11b1-/- mice may adapt to a cholesterol-induced increase in hepatic BA by blunting hepatic BA uptake via NTCP/ SCL10A1/ SCL10A1 and increasing hepatic efflux via OST-β. The effects of 11β-HSD1 deficiency upon BA recycling and BA profile and concentration within the enterohepatic circuit, could reflect 11β-HSD1 action within the liver or could be due to actions in other tissues.
To investigate the role of hepatic 11β-HSD1 specifically, 11β-HSD1 liver-specific knockout (Hsd11b1LKO), 11β- HSD1 liver-specific over-expressors (Hsd11b1LOE) and control mice with exon 3 of the Hsd11b1 gene “floxed” (Hsd11b1F) were studied. Findings from this study indicate a role for 11β-HSD1 in adaption to dietary cholesterol and suggest that hepatic 11β-HSD1 (as opposed to 11β-HSD1 in extra-hepatic tissues) is the main factor regulating BA metabolism. Also, work from this thesis demonstrates 11β-HSD1 is an important regulator of gall bladder emptying and filling, an important component of enterohepatic bile acid recycling. Based on these findings it is anticipated that therapeutic use of 11β-HSD1 inhibitors will result in BA imbalances within the enterohepatic circuit and therefore BA homeostasis. Care must therefore be observed when implementing therapeutic use of 11β-HSD1 inhibitors, with particular focus on patients with cholestasis, Addison’s disease and critically ill patients who already have known BA imbalances in their enterohepatic system.

Atherosclerosis and post-angioplasty neointimal proliferation, which are leading causes of cardiovascular morbidity and mortality, develop as a result of chronic or acute vascular injury producing inflammatory and proliferative responses in the vessel wall. Glucocorticoids, the stress hormones produced by the adrenal cortex, have anti-inflammatory and anti-proliferative characteristics and can also influence systemic cardiovascular risk factors. The systemic levels of these hormones are controlled by the hypothalamic pituitary adrenal axis. However, there is also a tissue-specific pre-receptor regulation of these hormones by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD); type 1 regenerates active glucocorticoids within the cells and type 2 inactivates glucocorticoids. Whilst it has been shown that the inhibition of 11β-HSD1 has favourable effect on cardiovascular risk factors and the inhibition of 11β-HSD2 results in hypertension; the effect of these enzymes on vascular lesion development is not known. The work described in this thesis tested the hypothesis that 11β-HSD1 inhibition reduces vascular lesion development due to improvement in cardiovascular risk factors, whereas 11β-HSD2 inhibition leads to adverse vascular remodelling. Apolipoprotein-E deficient (ApoE-/-) mice fed on western diet were used to study atherosclerosis, whereas neointimal proliferation was investigated using a well-established mouse model of wire-angioplasty. Vascular lesions were assessed using novel imaging and standard histological techniques. 11β-HSD1 inhibition reduced the size of atherosclerotic lesions and improved markers of plaque stability with a reduction in lipid content and increase in collagen content of the plaques. This was associated with a reduction in weight gain and blood pressure but without any effect on lipid profile. 11β-HSD1 inhibition did not produce any significant effect on neointimal proliferation in C57Bl/6J mice. However in ApoE-/- mice, 11β-HSD1 inhibition reduced neointimal proliferation with corresponding increase in size of patent lumen and with an associated reduction in macrophage content of neointimal lesions. 11β-HSD2 deletion produced an outward remodelling in un-injured vessels but there was no effect on neointimal proliferation after wire-angioplasty. Administration of a selective mineralocorticoid antagonist, eplerenone, reduced neointimal lesions significantly but to a similar degree in both C57Bl/6J and 11β-HSD2-/- mice, associated with a significant reduction in macrophage content of lesions but without any effect on blood pressure. Data in this thesis highlight the potential therapeutic application of 11β-HSD1 inhibition in reducing the size and vulnerability of atherosclerotic plaques and also reduction in neointimal proliferation (and hence post-angioplasty restenosis) in high risk patients with „metabolic syndrome‟ phenotype. The results also indicate that 11β-HSD2 has a limited, if any, role to play in the development of neointimal lesions.

The world is facing an unprecedented situation: soon the number of people over 60 years old will exceed the number of children, and more people at extreme old age than ever before. This is due to medical advances that have increased life expectancy and with it, the number of elderly. The latest data published by the WHO predicts that by 2050 the world’s population aged 60 years and older is expected to nearly double from 12% to 22% achieving a total 2 billion, up from 900 million in 2015. A longer life brings with it opportunities, however, there is little evidence to suggest that older people today are experiencing their later years in better health than their parents. While rates of severe disability have declined over the past 30 years, there has been no significant change in mild to moderate disability over the same period. If this added years are dominated by declines in physical and mental capacity, the implications for older people and for society will be more negative. Therefore, in the last years research has focused on the biology of ageing with the purpose of achieving better understanding of its mechanisms for preventing the onset and progression of age-related conditions. Besides, modern society is experiencing an increasingly common stressful lifestyle together to increased rates of metabolic stress caused, in part, by high-fat diet consumption. Several pieces of evidence state that environmental factors are essential in determining the development of different diseases as well as compromising healthy ageing. With upcoming age, the capability to fight against harmful stimuli decreases and the organism becomes more vulnerable to infections and disease. In agreement, stressful experiences have been identified as an important risk factor for cognitive impairment. Therefore, it is important to study the molecular mechanisms underpinning the effects of chronic stress on cognition and its relationship with ageing in order to unveil what challenges we might have to cope with as a society in the not-so-far future. In parallel with ageing, stress and neurodegenerative diseases, such as Alzheimer’s disease (AD), there is impaired glucocorticoid (GC) signalling. Disturbances in the GC-mediated stress response and individual’s adaptive abilities appear to increase the vulnerability of elderly to age-related pathologies. In consequence, the present doctoral dissertation has been focused on the study of the mechanisms involved in age-related neurodegeneration modified by GC excess attenuation through the inhibition of the enzyme 11β-HSD1 in an animal model of accelerated ageing, as well as, their response to chronic mild stress exposure. Last but not least, the present doctoral thesis has been devoted to evaluate the GC-mediated stress response to chronic moderate stressful situations and to metabolic stress underlying neurodegeneration and the potential role of 11β-HSD1 inhibition to restore those detrimental effects induced by stress. In summary, results obtained pointed out a protective role of the 11β-HSD1 inhibitor tested as improved cognitive and behavioural abilities of aged mice, as well as restored the deleterious effects induced by stressful conditions applied. Additionally, some of the molecular pathways related to ageing and neurodegeneration – particularly, AD neurodegeneration – were altered as a consequence of stress, but most of them were re-established after 11β-HSD1 inhibition, such as proteostasis, oxidative stress, neuroinflammation and epigenetics, among others. Overall, GC excess attenuation may become a potential therapeutic strategy for age-related cognitive decline.

Non-alcoholic fatty liver disease (NAFLD) is a worldwide health problem which includes steatosis (triglyceride accumulation alone), non-alcoholic steatohepatitis (NASH, with liver inflammation), fibrosis, cirrhosis and hepatocellular carcinoma. Liver fibrosis, which is a reversible response, is the final phase of most chronic liver disease and is characterized by accumulation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). Glucocorticoids (GCs) regulate many aspects of metabolism involved in NAFLD. Also, GCs limit HSC activation in vitro. Tissue GC levels are regulated by 11β- hydroxysteroid dehydrogenase-1 (11β-HSD1) which converts inactive 11- dehydrocorticosterone (DHC) into active corticosterone. Previous studies demonstrate that 11β-HSD1 deficiency improves fatty liver in obesity models, but the role of 11β-HSD1 in mechanisms involved in the progression and/or resolution of hepatic injury is largely unknown. I hypothesized that 11β-HSD1 modulates fibrotic and inflammatory responses during hepatic injury and/or the resolution phase. First I sought to address if the levels of 11β-HSD1 during different models of liver injury are dysregulated. In mice, 11β-HSD1 was down-regulated in choline deficient diet (CDD) induced steatosis, methionine and choline deficient diet (MCDD) induced NASH, carbon tetrachloride (CCL4) induced liver fibrosis and thioacetamide (TAA) induced liver fibrosis. In CCL4 injured livers, the down regulation of 11β- HSD1 was observed around the scar area. To test if 11β-HSD1 plays a key role in modulating liver inflammation and fibrosis responses in NAFLD and liver fibrosis I used initially11β-HSD1 knockout (KO) mice. 11β-HSD1 KO showed higher HSC activation only in the High fat feeding model but not in CDD and MCDD models. In the CCL4 injury model, despite reduced hepatocellular injury, 11β-HSD1 KO mice showed enhanced collagen deposition during peak injury and increased fibrotic gene expression during the early resolution phase although unaltered inflammatory markers during both peak injury and resolution. To further dissect cell-specificity on the effect of 11β-HSD1, I repeated the CCL4-injury model using the hepatocyte-specific 11β-HSD1 KO (Alb-HSD1). Alb-HSD1 mice did not show increased susceptibility to fibrosis compared to control littermates suggesting that the 11β- HSD1 possibly modulates fibrotic response by affecting HSC function. To mechanistically address how GCs inhibit HSC activation in vitro I studied the effects of 11β-HSD1 on HSC in vitro. 11β-HSD1 expression was down-regulated during ‘spontaneous’ HSC activation, and 11β-HSD1 deficiency enhanced susceptibility to activation. The GC (11-DHC)’s inhibitory effect on HSC activation was reversed by 11β-HSD1 inhibition. Finally, to address the clinical relevance of 11β-HSD1 in hepatic injury and/or resolution a selective 11β-HSD1 inhibitor, UE2316, was used. UE2316 induced a pro-fibrotic phenotype in ob/ob mice and CCL4-treated C57BL/6 mice, but had no effect when administered only during injury resolution. In conclusion, 11β-HSD1 deficiency causes increased activation of HSCs following diet and chemical injury and promotes liver fibrosis. Effects of 11β-HSD1 inhibitors, which are a potential treatment for metabolic syndrome, are perhaps offset by adverse outcomes in liver.

The enzyme 11β-Hydroxysteroid Dehydrogenase 1 (11β-HSD1) catalyses the intracellular biosynthesis of the active glucocorticoid cortisol. Tissue specific dysregulation of the enzyme has been implicated in the development of metabolic syndrome and other associated diseases. Experiments with transgenic mice and prototype inhibitors show that inhibition of 11β-HSD1 in visceral adipose tissue and liver leads to a resistance of diet-induced hyperglycemia and a favourable lipid and lipoprotein profile as compared to controls. 11β-HSD1 inhibition has thus been proposed as an effective strategy to decrease intracellular glucocorticoid levels without affecting circulating glucocorticoid levels that are essential for stress responses. The clinical development of selective and potent drugs has therefore become a priority. In this research, a process of virtual screening employing the novel algorithm UFSRAT (Ultra Fast Shape Recognition with Atom Types) was used to discover compounds which had specific physicochemical and spatial atomic parameters deemed essential for inhibition of 11β-HSD1. The top scoring compounds were assayed for inhibitory activity against recombinant human and mouse enzyme, using a fluorescence spectroscopy approach. In addition, HEK-293 cell based assays with either human, mouse or rat enzymes were carried out using a scintillation proximity assay (SPA). The most potent compound competitively inhibited human 11β-HSD1 with a Kiapp value of 51 nM. Recombinant mouse and human enzyme were expressed, purified and characterised and used in a series of ligand binding assays. Further to this, an X-ray crystal structure of mouse 11β-HSD1 in complex with a tight binding inhibitor – carbenoxolone was solved.

Das Enzym 11beta-HSD1 stellt im intrazellulären Glucocorticoidstoffwechsel eine wichtige Prärezeptorkontrolle dar. Es reguliert die intrazelluläre Cortisolkonzentration durch die enzymatische Umwandlung des aus dem Blutkreislauf aufgenommenen und hormonell inaktiven Cortisons zum aktiven Cortisol. Die Bedeutung einer erhöhten 11beta-HSD1 Expression und Aktivität bei der Entstehung von Übergewicht und Insulinresistenz wurde bisher vorwiegend in Leber und Fettgewebe untersucht und nachgewiesen. Wenig erforscht sind die Funktionen der 11beta-HSD1 im Muskelgewebe. In dieser Arbeit wurde die Funktion und Regulation der 11beta-HSD1 im Skelettmuskel mithilfe der murinen Skelettmuskelzelllinie C2C12 und primärer humaner Myoblasten untersucht. Es konnte demonstriert werden, dass die 11beta-HSD1 in Abhängigkeit des Differenzierungsgrades exprimiert wird und als Oxo-Reduktase aktiv ist, sowie selbst einen Regulator der Differenzierung darstellt. Es zeigte sich ein Feed-Forward-Mechanismus des Cortisons, das die 11beta-HSD1 in den Skelettmuskelzellen akut und chronisch induzierte, sowie eine gleichzeitige Veränderung der GRalpha- und MRalpha-Expressionen gegenregulatorisch zur 11beta-HSD1. Die Daten aus der Mauszelllinie konnten zum größten Teil in primären humanen Myoblasten bestätigt werden. Zudem konnten mehrere Transkriptionsfaktoren wie CREB, Myogenin und MEF-2c identifiziert werden, die in den verschiedenen Phasen der Differenzierung unterschiedliche Relevanz für die Regulation der 11beta-HSD1 Promotoraktivität hatten. Des Weiteren wurden die Proteolyserate und die Expression der E3-Ubiquitin-Ligasen Atrogin-1 und MuRF-1 11beta-HSD1-abhängig durch Cortison induziert. Trotz alledem führte eine Langzeit-Stimulation mit Cortison zu einer 11beta-HSD1-abhängigen Induktion der Differenzierung mit einer Veränderung der Muskelfasertypen in Richtung langsam-zuckender Muskelfasern, was eine Bedeutung für das klinische Bild der glucocorticoid-induzierten Muskelatrophie haben kann.
The enzyme 11beta-HSD1 functions as an important pre-receptor control of intracellular glucocorticoid action regulating the intracellular cortisol concentration by enzymatic conversion of the hormonal inactive cortisone up-taken from blood circulation to the active cortisol. A pivotal role of an increased 11beta-HSD1 expression and activity for the development of overweight and insulin resistance has been analysed and demonstrated particularly in liver and adipose tissue. However, the functions of 11beta-HSD1 in skeletal muscle tissue are rarely investigated. For analysis of function and regulation of the 11beta-HSD1 in skeletal muscle the murine skeletal muscle cell line C2C12 as well as primary human myoblasts from healthy volunteers were used. 11beta-HSD1 was shown to be expressed and functionally active as oxo-reductase in human and murine skeletal muscle cells dependent on the differentiation but as well to function as a regulator of differentiation itself. The stimulation experiments revealed a feed-forward-mechanism of cortisone that induced 11beta-HSD1 acutely and chronically. Concurrently, GRalpha and MRalpha were expressed contra-regulatory to 11beta-HSD1. For the most part these data were confirmed in human primary myoblasts. Several transcription factors as CREB, Myogenin and MEF-2c were identified having different relevance for regulation of 11beta-HSD1 promoter activity during the different phases of differentiation. Furthermore, treatment with cortisone increased protein degradation and expression of the two E3-ubiquitin-ligases Atrogin-1 and MuRF-1 in an 11beta-HSD1-dependent way. Nonetheless, a long-term stimulation by cortisone revealed an 11beta-HSD1-dependent induction of differentiation accompanied by modification of muscle fiber type composition towards slow-twitch muscle fibers that may play a role for the clinical picture of glucocorticoid-induced muscle atrophy.

Glucocorticoids, key hormonal regulators of the stress response, powerfully influence inflammation and metabolism. Reducing excessive glucocorticoid exposure is beneficial in treating metabolic and cognitive disorders, but manipulating systemic endogenous glucocorticoids risks compromising their beneficial effects. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates glucocorticoids in target tissues and thus inhibition of this enzyme presents a clinical opportunity to reduce tissue-specific glucocorticoid action. Active glucocorticoids also exert potent angiostatic effects by binding the glucocorticoid receptor (GR), and 11β-HSD1 inhibitors have proven beneficial in models of myocardial infarction by promoting angiogenesis. The possibility that 11β-HSD1 inhibitors may increase pathological angiogenesis, such as that seen in solid tumours, remains unaddressed. This project tested the hypothesis that 11β-HSD1 inhibition promotes tumour growth as a result of increased angiogenesis, using murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC). Murine SCC or PDAC cells were injected (1x106 cells/flank) into WT female mice fed either standard diet, or diet containing the 11β-HSD1 inhibitor UE2316 (175 mg/kg, N=6/group), or into 11β-HSD1 knockout (Del1) mice fed standard diet. Developing tumours were measured by callipers over several weeks, before animals were culled and tissues collected. SCC tumours grew more rapidly in UE2316-treated mice to reach a significantly (P < 0.01) larger final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). PDA tumours were unaffected by 11β-HSD1 inhibition or deletion. Immunofluorescent co-staining of tumour sections for CD31/α-smooth muscle actin revealed no differences in vessel density, and RT-qPCR showed no difference in angiogenic factor expression, after 11β-HSD1 inhibition/deletion in either tumour type. GR and 11β-HSD1 RNA expression were greater in SCC vs PDAC tumours (P < 0.001), as was 11β-HSD1 activity (P < 0.0001). In studies using the aortic ring assay of ex vivo angiogenesis, 11β-HSD1 deletion, but not inhibition with UE2316, was shown to prevent glucocorticoid-mediated angiostasis. The growth/viability of tumour cell lines was not affected by UE2316 or corticosterone, as assessed by live cell imaging using the Incucyte imaging system. RNA-sequencing of SCC tumours revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated tumours, and that extracellular matrix regulation was also altered by UE2316. Imaging of tumour sections using Second Harmonic Generation microscopy confirmed that UE2316 altered Type I collagen deposition in SCC (P < 0.001) but not PDAC. 11β-HSD1 inhibition can increase tumour growth, possibly via suppression of inflammatory/immune cell signalling and alteration of the extracellular matrix, and tumours with higher GR and 11β-HSD1 content, such as SCC, may be more at risk. Interestingly this investigation found no evidence of increased angiogenesis in vivo or ex vivo after UE2316 treatment, suggesting that 11β-HSD1 inhibition does not promote angiogenesis in all ischaemic environments. Future work must focus on the effects of 11β-HSD1 inhibition on the immune and extracellular matrix component of the tumour microenvironment. While promotion of pathological angiogenesis does not appear to pose a major threat, 11β-HSD1 inhibitors may still interact with the immune and inflammatory environment in tumours to the detriment of health.

Orientador: Agnaldo Lopes da Silva Filho
Resumo: Introdução: As proteínas de choque térmico (“Heat Shock Proteins”) são produzidas em resposta ao estresse patofisiológico nas células animais e não só fazem parte de várias etapas da carcinogênese, atuando principalmente como agentes antiapoptóticos, como também estão implicadas em mecanismos de resistência à quimioterapia em vários tipos de tumores. Objetivo: O presente estudo visa comparar a expressão dos genes TRAP1, HSPB1, HSPD1, HSPA1L e HSPA1A nas amostras de CEO (no tumor primário ou na metástase) com a expressão dos mesmos em amostras de tumores ovarianos benignos e tecido ovariano normal e correlacionar a expressão gênica com o prognóstico das pacientes e com a resistência ao tratamento com platina. Métodos: Foram avaliadas amostras de 51 pacientes operadas no Hospital Vera Cruz, entre os anos de 2008 a 2011, divididas em quatro grupos: CEO primário (n = 14), CEO metastático (n = 11), cistoadenoma seroso ovariano (n = 07) e ovário normal (n = 19). Utilizou-se a técnica de qRT-PCR para determinar o perfil de expressão dos genes. Resultados: As pacientes incluídas neste estudo apresentavam idade média de 56,75 anos. Não houve diferença significativa (valor-P> 0,050) na comparação entre a expressão dos genes e os grupos estudados. Os genes HSPA1A, HSPA1L e TRAP1 foram subexpressos e se diferiram significativamente dos genes em indivíduos com ovário normal. A expressão dos genes analisados não correlacionou se com as variáveis quantitativas, como idade, menarca, e tempo ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Heat Shock Proteins are produced in response to pathophysiological stress and take part in several stages of carcinogenesis, acting primarily as anti-apoptotic agents. They are also implicated in resistance to chemotherapy in several types of tumors. Herein we correlated the expression of genes encoding these proteins and the clinical and pathological aspects of patients with ovarian cancer (OC). METHODS: 51 patients included in the study were divided into four groups: those with primary EOC (n = 14), metastatic EOC (n = 11), ovarian serous cystadenoma (n = 7), with no evidence of ovarian malignancy or control (n = 11). The 57 tumor samples obtained were submitted to RNA extraction and reverse transcription. qRT-PCR was performed to compare the expression of TRAP1, HSPB1, HSPD1, HSPA1A and HSPA1L in primary and HSP60, HSP70, HSPA1L genes did not differ among the groups (p-value> 0.050) .HSPA1A, HSPA1L and TRAP1 we underexpressed in the primary and metastatic EOC groups with HSPA1L showing the lowest expression with compare with normal ovary tissue. TRAP1 expression was higher in tumors at stage I/II than at stages III/IV. Grade II subjects showed higher HSPB1 expression. There was no correlation between HSPs expression and age, menarche, parity, period after menopause initiation and CA-125. HSPA1A gene was negatively correlated with the risk of dying of OC. There was no differences between HSP expression gene evaluated and overall and disease-free survival. In conclusion, we ... (Complete abstract click electronic access below)
Mestre

The tetrahydroisoquinoline (THIQ) core structure is explored as a steroidomimetic nucleus with attractive pharmaceutical properties. A library was synthesised employing Pomeranz-Fritsch, Pictet-Spengler, Bischler-Napieralski strategies yielding 77 final targets, substituted at every position, for biological evaluation. Complementary strategies overcame synthetic difficulties, sometimes yielding two products in a single cyclisation. Three compounds were initially tested against a panel of 19 nuclear receptors (NRs) and exhibited broad substitution-dependent activity. 2-(4-Chlorophenyl)-1-isopropyl-1,2,3,4-tetrahydroisoquinolin-6-ol fully inhibited every NR at 100 µM, confirming the THIQ as a lead for optimisation. Compounds were evaluated for cytotoxicity against 60 cell lines by the NCI (USA), exhibiting moderate to insignificant cytotoxicity. Three compounds showed ca. 30-90% of average growth inhibition and were selected for a five dose test. Off-target evaluation highlighted compounds with activity against glucagon-like peptide 1 secretion, calcitonin gene-related peptide receptor antagonism and with >100% inhibition against the metabotropic glutamate receptor 2. Estrogen receptor-related receptor α (ERRα), a constitutively active orphan NR, is a hormone-dependent cancer target and diethylstilboestrol (DES), a known inverse agonist, possesses similarities to THIQs. THIQs tested against ERRα revealed no general SAR rules, but showed a lower degree of efficacy in a commercial TR-FRET assay, with 1-benzyl-2-(4-chlorophenyl)-4-methyl-1,2,3,4-tetrahydroisoquinolin-6-ol showing 79% efficacy at 100 µM as an inverse agonist, being more active than DES (64% at 100 µM). Inhibition of steroidogenic enzymes like 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is an emerging approach for the treatment of HDBC, compared to other current clinical strategies. THIQs evaluated against 17β-HSD1 showed good activity in both whole cell and cell lysate assays, with the best inhibitor, 2-(4-chlorophenyl)-4-isopropyl-1,2,3,4-tetrahydroisoquinolin-6-ol, possessing an IC50 value of 336 nM. The value of THIQ as a drug-like steroidomimetic scaffold is thus established and this work reveals straightforward strategies to optimise potency and selectivity for a range of potential targets by structural and stereochemical iteration.

Numerous epidemiological studies show a strong association between low birth weight and later life hypertension and metabolic disease. Excessive in utero exposure to glucocorticoids (‘stress hormones’) has been hypothesized to be important in such developmental ‘programming’, acting via crucial physiological, gene expression or structural changes in the developing fetus. Normally, the fetus is protected from the high levels of maternal glucocorticoids by an enzymic placental barrier, 11 betahydroxysteroid dehydrogenase type 2 (11β-HSD2). In the placenta, 11β-HSD2 efficiently converts active maternal glucocorticoids (cortisol in humans; corticosterone in rodents) to physiologically inactive 11-keto forms. In previous studies in rats, maternal administration of dexamethasone, a synthetic glucocorticoid which is minimally metabolized by 11β-HSD2, or carbenoxolone, a potent inhibitor of 11 β-hydroxysteroid dehydrogenase, increased glucocorticoid load to the fetus. This resulted in lower offspring birthweight and later life hypertension and hyperglycemia — important components of the metabolic syndrome. These programming effects were seen when dexamethasone was administered selectively during the third week of gestation. We have used this well-validated model of programming to dissect the molecular mechanisms that mediate the programming of hypertension. In accord with previous observations, administration of dexamethasone (100μg/kg/day) to pregnant rats during the last week of pregnancy significantly reduced offspring birthweight by 10%. Moreover, the 9 month-old adult offspring had systolic hypertension (9% rise) accompanied by significant hypokalemia (10% fall K+). The coexistence of hypertension and hypokalemia suggested that prenatal overexposure to dexamethasone might increase mineralocorticoid activity in the kidney. Intriguingly, although offspring of dexamethasone-treated dams had 46% lower plasma renin concentrations (consistent with intravascular fluid volume expansion), 24-hour total urinary aldosterone levels were significantly reduced compared to controls (reduction of 56%). Maternal dexamethasone treatment was associated with a permanent decrease in 11β- HSD2 mRNA and activity in the kidney of the offspring (45% and 36% respectively). 11β-HSD2 plays an important role in regulation of renal sodium reabsorption (and thereby blood pressure) by acting as a pre-receptor barrier to MR access, preventing glucocorticoids from activating MR in the distal nephron. Thus, the decrease in renal 11β-HSD2 activity would allow greater endogenous glucocorticoids to activate MR, likely accounting for the low-renin, low-aldosterone hypokalemic hypertensive phenotype observed in these offspring. Other components of mineralocorticoid or glucocorticoid signaling pathways, including mineralocorticoid receptor (MR), glucocorticoid receptor (GR) and 11-beta hydroxysteroid dehydrogenase type 1 (11β-HSD1) were not altered in the offspring kidney by prenatal glucocorticoid exposure. Dexamethasone-programmed offspring also showed exaggerated mineralocorticoid activity with increased kalliuresis in response to exogenously administered corticosterone, suggesting that the decrease in renal 11β-HSD2 is functionally important. In this respect, our rat model resembles the syndrome of apparent mineralocorticoid excess where reduced 11β-HSD2 allows illicit activation of MR by glucocorticoids, resulting in excessive sodium reabsorption, hypertension and hypokalemia. We also studied the effects of maternal dexamethasone on offspring erythropoietin expression in the kidney. This followed from previous observations that identified the hepatocyte nuclear factor 4 alpha (HNF4α) as a key gene up-regulated in dexamethasone-programmed offspring liver, where it might be involved in mediating hyperglycemia. HNF4α is also expressed in the kidney. The role of HNF4α in the kidney is not fully understood, but has been implicated in regulation of erythropoietin synthesis. As in the liver, prenatal exposure to dexamethasone caused a significant increase (64% increase) in renal HNF4α expression. The increase in renal HNF4α mRNA was observed early (in one week old offspring) and persisted into adulthood. This was associated with significantly elevated levels of erythropoietin in circulation (110% increase). Moreover, animals that were exposed to prenatal dexamethasone had significantly increased red blood cell mass (7% increase), presumably as a result of upregulation of erythropoietin.

Pancreatic β-cells sense the concentration of glucose in the systemic circulation through metabolism of the sugar molecule. Failure to correlate the blood sugar concentration to an appropriate metabolic signal disrupts the function of the β-cell as a controller of glucose homeostasis and may contribute to the development of type 2 diabetes mellitus. Release of insulin is pulsatile and this thesis presents data that support that metabolism drives such pulsatile release. It is also found that increase in insulin release in response to elevation of the glucose concentration is only seen when the rise in glucose induces a prompt and sustained increase in mitochondrial metabolism. Such activation of mitochondrial metabolism depended on the metabolic state of the β-cell prior to the glucose challenge. In this context, prolonged periods of elevated levels of fatty acids are harmful to the pancreatic β-cell. To study the protein expression changes induced by fatty acids a protocol for islet protein profiling and identification of differently expressed proteins were developed. By using this protocol it was discovered that oleate decreased the cellular level of the chaperone peptidyl-prolyl isomerase B. The protocol was also used to study protein expression in islets obtained from mice fed a high-fat and/or a high-sucrose diet. Excess of glucocorticoids in the systemic circulation also cause a diabetic phenotype. Tissue response to glucocorticoids is regulated by the intracellular concentration of the active form of glucocorticoids, which is formed from the inactive form by the enzyme 11β-hydroxysteroid dehydrogenase type 1. It was found that pancreatic islets produce 11β-HSD1 protein in relation to substrate availability and that the amount of islet 11β-HSD1 protein was negatively correlated with insulin secretion.

One of the major paradigms in structural biology, according to which a rigid well-folded 3D structure is required for protein function, has clearly changed over the last decades. Recent studies show that some proteins despite the absence of a stable secondary or tertiary structure, play important roles in a number of biological processes, such as differentiation, transcription regulation, DNA condensation, mRNA processing, and apoptosis. Such proteins are known as intrinsically disordered proteins (IDPs) and are characterized by high flexibility and plasticity, that facilitate their interactions with a broad range of binding partners, such as proteins, membranes, nucleic acids and other molecules of biological relevance. (Uversky & Dunker, 2010). Intrinsically disordered proteins are also prone to misfolding and tend to evade normal clearance pathways. In turn, the combination of misfolding and lack of clearing mechanisms can result in aberrant processes, often associated with the onset of pathologies. In these cases, it is common to observe progressive protein aggregation into intracellular and/or extracellular deposits. The consequence is a diverse group of neurodegenerative disorders, each of which entails the aggregation of particular proteins in characteristic patterns and locations (Jucker & Walker, 2013). Under some particular conditions (e.g. environmental change or mutation) even native folded proteins might lose theirs stable, biochemically functional forms, and for this reason they can be investigated by the same methodologies used for IDPs. Examples of such proteins are beta amyloid peptide (Aβ) (intrinsically disordered in the monomeric form) associated to the Alzheimer’s disease (AD), and superoxide dismutase (SOD1) (which is intrinsically disordered in the reduced apo form) related to amyotrophic lateral sclerosis (ALS). These proteins are the main topics of this thesis. In this work we will present detailed structural characterization of Aβ prefibrillar and fibrillar assemblies by Solid State Nuclear Magnetic Resonance (SSNMR) that is crucial for understanding Aβ precise mechanism aggregation pathways and identifying toxic Aβ species involved in Alzheimer’s disease. This thesis will demonstrate also attempt of reverting side effects caused by cisplatin, that is effectie drug strongly inhibiting process of SOD1 oligomerization. That could open a way toward a novel therapeutic strategies in amyotrophic lateral sclerosis (ALS), since the clinical use of cisplatin is still limited cause of the development of neurotoxicity and others undesirable effects.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Lenka Popovská Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human 17β-HSD1 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1; SDR28C1; EC 1.1.1.62) belongs to the SDR superfamily and catalyzes the NAD(P)(H) dependent oxidoreduction of hydroxyl/keto groups at position C17 of androgens and estrogens and in this manner regulate intracellular availability of steroid hormone ligands to their nuclear receptors. This pathway constitutes a pre-receptor control mechanism. Estradiol is the most potent female sex steroid and the only one responsible for estrogen action in women. The ovary is the primary source of estradiol present in the circulation of premenopausal women, but circulating estrone and androgens originating from the adrenal gland are also converted to estradiol in peripheral tissues such as adipose tissue. After menopause, estrogen biosynthesis in peripheral tissues has a major role in estrogen action. For production of recombinant protein 17β-HSD1 bacterial strain E. coli DH10B with inserted vector pOTB7, which contained coding sequence of the respective enzyme, was used. The sequence of the enzyme was isolated from...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Candidate: Tereza Červinková Supervisor: PharmDr. Lukáš Červený, Ph.D. Title: Beneficial Effects of 11β-HSD1 Inhibition on Cognitive Performance in a Mouse Model of Alzheimer's Disease The increased life expectancy goes hand in hand with ageing-related cognitive impairments. Alzheimer's disease (AD) is the most common type of dementia being an irreversible and progressive brain disorder with loss of cognitive functions. Recent studies suggest that excess of glucocorticoid (GC) action exerts deleterious effects on the hippocampus and causes impaired spatialmemory. In addition, it has been demonstrated that aged mice with cognitive deficits show increased gene expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in the hippocampus and parietal cortex. The Senescence-Accelerated Mouse Prone 8 (SAMP8) strain is a spontaneous animal model of accelerated ageing. Many studies indicate that SAMP8 harbour the behavioural and histopathological signatures of AD. In the present study, we evaluated the neuroprotective effects of 11β-HSD1 inhibition by a potent pyrrolidine-based compound RL-118 and/or effects of diet on cognitive performance in different groups of SAMP8 by conducting behavioural and...

Obesity is one of the leading cause metabolic disorders in the western-industrialised countries. Metabolic disorders such as obesity, insulin resistance and non-alcoholic fatty liver disease is caused by the high-fat-diet intake in those population. The excess fat accumulation in the body causes alterations in various metabolic pathways, one of which is the conversion active glucocorticoids to inactive glucocorticoids. Although there are several studies that convey the effect of heavy alcohol consumption is linked with the pathogenesis of metabolic disorders and alcoholic fatty liver disease, the effect of light-to-moderate alcohol consumption is largely unknown. A number of epidemiological studies has demonstrated that regular light-moderate alcohol consumption lowers the risk of life threatening diseases such as cardiovascular disease and non-alcoholic fatty liver disease (NAFLD). Thus, in this study we investigated the effect of chronic light-to-moderate alcohol consumption in high-fat diet-fed rats on the hepatic regulation of 11β-HSD1 protein expression and its enzyme activity together with measurements of serum and hepatic corticosterone levels. The results demonstrated that light-to-moderate alcohol consumption down-regulated the expression of 11β-HSD1 protein in the liver together with decreased enzyme activity. In parallel, light-to-moderate alcohol also lowered the serum and liver corticosterone levels in rats fed with high fat diet. In conclusion, the present study demonstrated that chronic light-moderate alcohol consumption down-regulated 11β-HSD1 protein expression and decreased enzymatic activity in the liver of HFD-fed rats, which was associated with a reduction in circulating and liver corticosterone levels. Thus, the present study provides mechanistic evidence for the earlier epidemiological studies that indicate light-to-moderate alcohol consumption lowers the risk of development of metabolic syndrome leading to NAFLD, at least in part, via targeting 11β-HSD1.

Research Doctorate - Doctor of Philosophy (PhD)
The remarkable cellular communication events that characterise the highly species specific interactions observed during the ontogeny of mammalian fertilization, represent some of the most intriguing in all of biology. Given the 60 years or so of research conducted to elucidate the precise mechanisms that underpin these interactions, it is surprising that they still remain largely unknown. This can be mostly attributed to the unique luminal environment in which the sperm reside following insemination and the direct effects that these fluids have on their functionality. Although immense controversy surrounds the precise ligand responsible for the spermatozoas binding to the oocyte’s zona pellucida, considerable contention is also afforded to the mechanism by which they bind. A number of landmark papers have recently emerged to suggest that these initial binding events may be facilitated by the formation and presentation of multimeric zona pellucida receptor complexes on the sperm surface during their terminal maturation, rather than the widely held paradigm that the zona pellucida receptor is a single molecular entity. During these studies the use of blue native polyacrylamide gel electrophoresis, for the first time in mammalian sperm, has provided direct evidence that a number of multimeric zona receptor complexes indeed reside on the apical plasma membrane of capacitated sperm and that two of these complexes have the ability to interact with the zona pellucida. Proteomic analysis of these two complexes has indicated that molecular chaperones (CCT/TRiC complex and HSPD1) are responsible for the formation of each complex, and individually, these complexes contain a number of receptor proteins (ZPBP2, ZP3R and ADAMTS10) that potentially provide the zona pellucida affinity. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.

Stress is a widespread phenomenon in the western society of these days. It is a risky factor for health and well-being of the majority of people. Based on these facts, it is the main subject for the field of "stress physiology" research, which aims to study processes occurring during stress response and tries to elucidate mechanisms leading to stress-induced health impairment. The first aim of this thesis was to describe effects of psycho-social stress on organism. The second aim was to find out if can stress applied in juvenile age affect the stress response in adulthood. If so, how is the role of glucocorticoid-metabolism enzyme 11β-HSD1 in this influence? To answer these questions, two different animal models inducing stress response in the laboratory rat were used. The first one is the model of mild social stress based on the resident-intruder paradigm. Our results show efficancy of this model. Fisher 344 male rats treated under this model for seven consecutive days show highly elevated plasma corticosterone concentrations and elevated expression of the glucocorticoid receptor gene in the pituitary. Behavioral analysis demonstrates a decreased social behavioral profile of the intruders, suggesting submisive social position of these animals in the resident-intruder paradigm. The second model used is...

O presente relatório de estágio encontra-se subdividido em três capítulos. O primeiro capítulo diz respeito à revisão sistemática sobre os inibidores da 11ß-hidroxiesteróide desidrogenase tipo 1 (11ß-HSD1) no tratamento da Diabetes Mellitus Tipo 2. A diabetes é uma patologia cada vez mais frequente na sociedade, tornando-se numa das principais causas de morte em todo o mundo. Por este motivo, novos alvos terapêuticos têm vindo a ser estudados. A 11ß-HSD1 é uma enzima que se expressa primariamente no fígado e tecido adiposo e é responsável pela redução da cortisona à sua forma ativa cortisol que, consequentemente, pode levar a alterações metabólicas, como a resistência à insulina e hiperglicemia. Desta forma, a inibição da 11ß-HSD1 pode constituir uma nova abordagem terapêutica para a Diabetes Mellitus Tipo 2. Com este trabalho pretende-se rever sistematicamente a evidência científica disponível acerca deste tópico. Para tal, foi realizada uma pesquisa em três bases de dados e através dos quais foram incluídos 15 artigos neste estudo. Apesar dos altos níveis inibitórios que esta classe apresenta no fígado e tecido adiposo, os estudos até agora publicados não têm demonstrado resultados comercialmente atrativos em populações diabéticas. Além disso, os efeitos dos inibidores da 11ß-HSD1 a longo prazo carecem de ensaios clínicos. Porém, esta enzima continua a ser um alvo promissor para o desenvolvimento de fármacos neste âmbito, especialmente devido à sua eficácia no controlo dos vários fatores que constituem a síndrome metabólica e ao seu potencial para múltiplas indicações em doentes com diabetes, incluindo a cicatrização de feridas e a perda de peso. O segundo capítulo relata a experiência adquirida durante o estágio curricular em farmácia hospitalar no Centro Hospitalar Universitário da Cova da Beira (CHUCB). Aqui são descritas as diferentes áreas que tive a oportunidade de acompanhar, bem como as diversas funções e atividades que o farmacêutico aí desempenha. O terceiro capítulo é referente ao estágio em farmácia comunitária, realizado na Farmácia Matias Pereira. Aqui, é apresentado globalmente todo o funcionamento da farmácia comunitária e a legislação que regula o setor, assim como as tarefas realizadas durante o estágio.
The present dissertation is divided in three chapters. The first one refers to the systematic review about the 11ß-HSD1 inhibitors in type 2 diabetes mellitus treatment. Diabetes is a pathology more and more frequent in society, becoming one of the main causes of death worldwide. For this reason, new therapeutic targets have been studied. 11ß-HSD1 is an enzyme that is expressed primarily in liver and adipose tissue and is responsible for reducing cortisone to its active form cortisol which, consequently, can lead to metabolic changes such as insulin resistance and hyperglycaemia. In this way, the inhibition of 11ß-HSD1 may offer a new therapeutic approach for type 2 diabetes mellitus. This work intends to systematically review the available scientific evidence on this topic. To this end, a search was conducted in three databases and through which 15 articles were included in this study. Despite the high inhibitory levels that these drugs have in liver and adipose tissue, the studies published so far have not shown commercially attractive results in diabetic populations. In addition, the long-term effects of 11ß-HSD1 inhibitors require long-term clinical trials. However, this enzyme remains a promising target for drug development, especially due to its effectiveness in controlling the various factors that constitute the metabolic syndrome and its potential for multiple indications in patients with diabetes, including wound healing and weight loss. The second chapter reports on the experience acquired during the curricular internship in hospital pharmacy at CHUCB. Here are described the different areas that I had the opportunity to follow, as well as the various functions and activities that the pharmacist performs there. The third chapter refers to the internship in community pharmacy, held at Farmácia Matias Pereira. Here, the entire operation of the pharmacy and the legislation that regulates the sector are presented globally, as well as the tasks performed during the internship.