Die vorliegende Arbeit diskutiert methodische Aspekte und Ergebnisse eigener pharmakoepdemiologischer Untersuchungen zum Risiko von okulärer Hypertension, Glaukom und Katarakt unter verschiedenen Darreichungsformen von Glukokortikoiden. Prospektive Studien der frühen 60er Jahre haben gezeigt, daß die Verabreichung topischer Glukokortikoide am Auge bei ca. einem Drittel der Bevölkerung zu einem Augeninnendruckanstieg führt. Bei langdauernder Therapie kann sich ein Kortikosteroidglaukom entwickeln, das in seiner Symptomatik und den klinischen Befunden einem primären Offenwinkelglaukom entspricht. Für orale Glukokortikoide untersuchten wir das Risiko von okulärer Hypertension und Offenwinkelglaukom in einer großen Fall-Kontroll-Studie, die 9.793 augenärztliche Patienten mit neu diagnostizierter okulärer Hypertension und Offenwinkelglaukom einschloß und 38.325 augenärztliche Kontrollpatienten ohne diese Erkrankungen. Die Einnahme oraler Glukokortikoide war mit einem Risikoanstieg von über 40% verbunden. Es zeigte sich ein deutlicher Anstieg des Risikos mit zunehmender Glukokortikoid-Tagesdosis: Für Patienten, die mehr als 80 mg Hydrokortisonäquivalent pro Tag erhalten hatten, war das Risiko über 80% erhöht. Unsere Berechnungen zeigten, daß unter solch hohen Dosen 93 zusätzliche Fälle von okulärer Hypertension oder Offenwinkelglaukom pro 10.000 Patienten und Jahr auftreten können. In derselben Fall-Kontroll-Studie analysierten wir auch das Risiko für inhalative und nasale Glukokortikoide. Zwar ist für diese Glukokortikoidformen das Risiko systemischer Glukokortikoidnebenwirkungen durch die topische Applikation deutlich reduziert, doch legen verschiedene klinisch-pharmakologische Untersuchungen nahe, daß inhalative Glukokortikoide in hoher Dosierung systemische Effekte ausüben können. Verschiedene Einzelfallberichte ließen ein erhöhtes Risiko von okulärer Hypertension und Glaukom für inhalative und nasale Glukokortikoide möglich erscheinen. Unsere Fall-Kontroll-Studie zeigte, daß inhalative Glukokortikoide, wenn sie in hohen Tagesdosen kontinuierlich über 3 Monate verabreicht werden, das Risiko von okulärer Hypertension und Offenwinkelglaukom um über 40% erhöhen. Wir beobachteten kein erhöhtes Risiko für nasale Glukokortikoide. In einer weiteren Fall-Kontroll-Studie untersuchten wir das Kataraktrisiko für inhalative Glukokortikoide. Orale Glukokortikoide sind ein etablierter Risikofaktor für eine Katarakt. Für inhalative Glukokortikoide lagen widersprüchliche Studienergebnisse vor. Während mehrere kleine Studien an Kindern kein erhöhtes Risiko gezeigt hatten, war in einer großen populationsbasierten australischen Studie ein erhöhtes Kataraktrisiko unter inhalativen Glukokortikoiden beobachtet worden. Wir konnten das Ergebnis der australischen Studie in unserer Fall-Kontroll-Studie bestätigen, die 3.677 Fallpatienten und 21.868 Kontrollpatienten einschloß. Eine Verabreichung inhalativer Glukokortikoide über mehr als 3 Jahre führte zu einer Verdreifachung des Risikos einer Kataraktextraktion. Das Risiko war nur für hohe Dosen inhalativer Glukokortikoide statistisch signifikant erhöht, nicht jedoch für niedrige bis mittlere Tagesdosen. Zusammengefaßt zeigen die Ergebnisse unserer Studien, daß inhalative Glukokortikoide in hoher Dosierung trotz topischer Applikation zu systemischen Glukokortikoidkomplikationen am Auge führen können. Dies läßt es geboten erscheinen, bei Patienten, die inhalative Glukokortikoide in hoher Dosierung erhalten, augenärztliche Kontrolluntersuchungen durchführen zu lassen / This work presents methodological aspects and results of own pharmacoepidemiologic studies investigating the risk of ocular hypertension, glaucoma and cataract for different forms of glucocorticoids.. Prospective studies of the early 60ies have shown that administration of topical glucorticoids at the eye will lead to ocular hypertension in about one third of the population. If ophthalmic glucocorticoid treatment is prolonged, a corticosteroid glaucoma may develop which closely resembles primary open-angle glaucoma.. We investigated the risk of ocular hypertension or open-angle glaucoma for oral glucocorticoids in a large case-control-study which included 9,793 ophthalmology patients with newly diagnosed ocular hypertension or open-angle glaucoma and 38,325 ophthalmology patients without these diseases (controls). Intake of oral glucocorticoids led to an increase in risk by over 40%. The risk increased markedly with the daily dose of glucocorticoid. For patients who had received more than 80 mg hydrocortisone-equivalent per day, the risk was more than 80% elevated. Our calculations showed that for such high doses, 93 additional cases of ocular hypertension or glaucoma per 10,000 patients and year may be expected. In the same case-control study, we analysed the risk of ocular hypertension and open-angle glaucoma for inhaled and nasal glucocorticoids. These forms of glucocorticoids have been developed to reduce the risk of systemic glucocorticoid complications by topical administration. Some clinical pharmacology studies have shown that high doses of inhaled glucocorticoids may cause systemic effects. Some published case reports suggested an increased risk of ocular hypertension and glaucoma for inhaled and nasal glucocorticoids. Our case-control study showed that high dose, continuous administration of inhaled glucocorticoids for more than 3 months increases the risk of ocular hypertension or open angle glaucoma by more than 40%. We did not observe an increased risk for nasal glucocorticoids. In another case-control study, we investigated the risk of cataract for inhaled glucocorticoids. Oral glucocorticoids are an established risk factor for cataract. For inhaled glucocorticoids, there have been contradictory results from several studies. Whereas some small studies in children did not show an increased risk, a population-based larger study from Australia demonstrated an elevated risk. We confirmed this increase in risk in our case-control study which included 3,677 elderly cases and 21,868 elderly controls. We observed a more than 3-fold risk of cataract extraction in patients who had been treated with inhaled glucocorticoids for more than three years. The risk was significantly increased only for high daily doses of glucocorticoids, but not for low-to-medium doses. In summary, the results of our studies show that high doses of inhaled glucocorticoids despite their topical administration may lead to systemic complications of glucocorticoids at the eye. Therefore it is recommended to have patients who are prescribed high daily doses of inhaled glucocorticoids examined by an ophthalmologist.

T lymphocytes are an essential component of the adaptive immune response and are highly versatile in function. Each T cell has a unique T cell receptor that can recognize an antigenic peptide in the context of the major his to compatibility complex (MHC) encoded molecules, thus offering a high degree of specificity to the immune response. T cells play a central role in the development of an effective host immune response and the quantitative and qualitative regulation of the T cell response is critical. T cells develop in the thymus, an important primary immune organ, where immature thymocytes undergo differentiation and maturation. Through the process of thymic differentiation, immature cluster of differentiation (CD)4-CD8- thymocytes progress to a CD4+CD8+ stage and are subjected to positive and negative selection to give rise to MHC restricted, single positive CD4+ or CD8+ naive T cells that emigrate from the thymus and populate the peripheral lymphocyte pool. Thymic atrophy is well known to occur naturally during the process of aging with thymocyte depletion and reduced thymic output. Along with age associated changes leading to atrophy, the thymus is exquisitely sensitive to starvation and several stresses. In addition, thymic atrophy is a characteristic feature during several viral, bacterial and parasitic infections. Egress of immature thymocytes, loss of thymic populations due to sensitivity to glucocorticoids and cytokine modulation, etc. have been variously proposed to be involved in this process. However there is limited understanding on the numerous mechanisms involved and the crosstalk between these diverse pathways. In this study, a model for thymic atrophy during acute Salmonella enterica serovar Typhimurium (S. typhimurium) infection was developed. S. typhimurium is a Gram negative bacterium that resides and grows in intracellular compartments within host cells. It causes gastroenteritis in humans but leads to typhoid like disease in mice, similar to that caused by S. typhi in humans. Initially, it was established that acute infection of C57BL/6 mice with 108 CFU S. typhimurium, via the oral, i.e. the physiological, route of infection leads to extensive depletion (8-10 fold) of thymocytes in an infection-dependent manner. Infected mice had higher CFU burden in the Peyer’s patches, spleen, liver, and mesenteric lymph node (MLN) as compared to the thymus. The thymic atrophy was dependent upon the infection caused by live S. typhimurium since oral feeding of mice even with higher doses (1010 CFU) of heat-killed bacteria did not lead to thymic atrophy. The susceptible populations in the thymus were identified by staining for expression of CD4 and CD8 on cell surface using specific monoclonal antibodies tagged to fluorophores, e.g. Fluorescein isothiocyanate (FITC) and phycoerythrin (PE), respectively. The double labelled samples were analyzed by flow cytometry. Interestingly, significant death of CD4+CD8+, the major population of thymocytes, but not single positive thymocytes or peripheral lymphocytes (MLN and spleen cells), was observed at later stages during infection. To gain greater understanding of the processes involved, the mechanisms leading to thymic atrophy were investigated. To this purpose, small molecule inhibitors and mice lacking key molecules important for the immune response were utilized. Also, various assays to assess death of thymocytes, including analysis of death markers such as Annexin V based detection of membrane flipping and caspase activation were performed. I. The extrinsic death pathway involving Fas/FasL interactions is a major death pathway. Therefore, the expression and functional role of the components of the pathway in this model of thymocyte death was investigated. It was observed that thymocytes from infected mice expressed more Fas and Fas ligand (FasL) on their surface than cells from uninfected mice. To address the role of the death receptor, Fas, infection studies were performed with lpr mice that lack functional Fas expression. The depletion of CD4+CD8+ thymocytes in lpr mice was comparable to that in C57BL/6 mice indicating that it was independent of the Fas pathway. However, extensive loss of mitochondrial membrane potential was observed upon analysis with mitochondrial potential specific dyes MitoTracker Red and DiOC6. Most likely, the intrinsic death pathway involving mitochondrial depolarization is involved in this model of thymic atrophy. II. Since thymocytes are known to be sensitive to glucocorticoids both in vitro and in vivo, the involvement of the same in this model of thymic atrophy was assessed. The amounts of cortisol, a glucocorticoid, as detected by ELISA, were elevated during infection. To investigate the functional implication of the increase in cortisol, studies were performed using RU486, a glucocorticoid receptor antagonist. RU486 did not modulate cortisol amounts and treatment of mice with RU486 did not affect CFU burden or survival of mice. However there was a moderate rescue in the number of viable CD4+CD8+ thymocytes, with only a 3-4 fold drop as compared to the 8-10 fold drop in vehicle treated infected mice. III. As glucocorticoids appeared to play a partial role in this model, it was reasonable to assume that other pathways were also involved in the thymic atrophy. The quantitative and qualitative modulation of the cytokine milieu has a profound effect upon the thymus. In fact, inflammatory cytokines, Tnfα and Ifnγ, increased upon infection. In order to study the role of Ifnγ mediated inflammatory responses in this model, infection studies with Ifnγ-/- mice were performed. Ifnγ-/- mice had higher CFU and lower survival; however the drop in thymocyte numbers was 3-4 fold as compared to the 8-10 fold drop in the infected C57BL/6 mice, again indicating a partial involvement of the Ifnγ mediated pathways. In order to study the interactions, if any, between the two pathways mentioned above, corticosteroid signaling was blocked in the Ifnγ-/- mice with RU486. Upon infection, the number of CD4+CD8+ thymocytes was significantly higher in Ifnγ-/- mice treated with RU486 (~1.5 fold drop in viable thymocyte numbers) along with lower caspase 3 activity and mitochondrial damage. Importantly, cortisol amounts in infected Ifnγ-/- mice were comparable to those in infected C57BL/6 mice and the administration of RU486 did not modulate Tnfα and Ifnγ cytokine amounts in sera. Thus, the glucocorticoid and Ifnγ mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S. typhimurium nfection. IV. Although thymic atrophy is known to occur, a detailed characterization of cell surface changes in thymocyte populations has not been performed. To investigate this aspect, thymocytes and MLN cells from uninfected and infected animals were stained for cell surface expression of CD3, CD4, CD5, CD8, CD24, CD25, CD44, CD69, MHC I and MHC II. This analysis was initially performed by studying the changes in expression of these molecules within the total thymocyte and MLN populations. Although there was no change in the expression of CD25 and MHC II in the total thymocyte population upon infection, CD24 expression reduced, whereas, the expression of CD3, CD5, CD44, CD69 and MHC I increased. Notably, changes in the frequency of expression of CD3, CD69 and MHC I were observed before the development of extensive thymic atrophy. The depletion of majority of the CD4+CD8+ thymocytes enriches the mature CD4+ or CD8+ thymocyte population This was corroborated with the observation that, upon in vitro stimulation with PMA and Ionomycin (pharmacological agents used to activate T cells) the residual thymocytes from infected mice produced more IL2 compared to thymocytes from uninfected mice. Subsequently, cells were stained with anti-CD4-FITC, anti-CD8-PE and a third biotinylated antibody, which was detected by a streptavidin-APC conjugate, against one of the remaining six markers. This three colour analysis made it possible to determine the changes in the expression of the third marker in each of the CD4-CD8-, CD4+CD8+, CD4+ and CD8+ populations upon infection. Distinct differences were observed in the phenotypes of uninfected and infected CD4+CD8+ thymocytes and the latter were CD3high, CD5high, CD24low, CD69high and MHC Ihigh indicating that the surviving population had a possibly more mature phenotype. Also, the changes in the phenotypes of the thymocyte populations were dependent upon the extent of thymic atrophy as indicated by time course and CFU studies with C57BL/6 and BALB/c mice respectively. Finally, the roles of glucocorticoids, Ifnγ and Nos2 in modulation of expression of these markers during infection were addressed. Interestingly, the expression of CD3, CD24 and MHC class I significantly correlated with increase in the number of surviving thymocytes upon inhibition of glucocorticoids signaling and in Ifnγ-/- mice. The implications of these changes in the thymocyte surface phenotype during thymic atrophy are discussed. V. Finally, the roles of downstream signalling molecules in S. typhimurium induced thymic atrophy were studied. Although the MAP kinase family members, Erk, Jnk and p38 have been implicated to play a role in the positive and/or negative selection of thymocytes during development, their role in infection induced thymocyte depletion has not been studied. Interestingly, the amounts of Jnk and pJnk, but not p38, increased in thymocytes upon infection. Importantly, pJnk amounts increased predominantly in CD3-/low thymocytes during infection. Furthermore, inhibition of Jnk signalling, using a specific inhibitor SP600125, lead to an increase in survival of CD4+CD8+ thymocytes during infection due to multiple reasons: lowering of cortisol, Tnfα and Ifnγ amounts, and better maintenance of thymic architecture. Thus, inhibition of Jnk mediated signaling protected CD4+CD8+ and CD3-/low thymocytes from death during S. typhimurium infection. Overall, the main conclusions of this study are as follows: First, extensive analysis of the surface phenotype of cells during thymic atrophy throws light on the sensitive and resistant thymocyte populations, thus offering a potential predictive marker profile. Second, glucocorticoids, Ifnγ and, importantly, Jnk mediated signaling play functional roles in the death of immature CD4+CD8+ thymocytes during S. typhimurium infection. The mechanistic details uncovered in this study may be important in designing effective strategies for reducing thymic atrophy during other infections. In fact, enhancement of thymic output may lead to greater numbers and diversity of thymic T cell emigrants in the periphery which is likely to enhance host responses during infections.

The fetus is highly responsive to the level of glucocorticoids in the gestational environment. Perturbing glucocorticoids during fetal development could yield long-term consequences. Extending prior research about effects of prenatally exposed synthetic glucocorticoids (sGC) on brain structural development during childhood, we investigated functional brain correlates of cognitive conflict monitoring in term-born adolescents, who were prenatally exposed to sGC. Relative to the comparison group, behavioral response consistency (indexed by lower reaction time variability) and a brain correlate of conflict monitoring (the N2 event-related potential) were reduced in the sGC exposed group. Relatedly, source localization analyses showed that activations in the fronto-parietal network, most notably in the cingulate cortex and precuneus, were also attenuated in these adolescents. These regions are known to subserve conflict detection and response inhibition as well as top-down regulation of stress responses. Moreover, source activation in the anterior cingulate cortex correlated negatively with reaction time variability, whereas activation in the precuneus correlated positively with salivary cortisol reactivity to social stress in the sGC exposed group. Taken together, findings of this study indicate that prenatal exposure to sGC yields lasting impacts on the development of fronto-parietal brain functions during adolescence, affecting multiple facets of adaptive cognitive and behavioral control.

The fetus is highly responsive to the level of glucocorticoids in the gestational environment. Perturbing glucocorticoids during fetal development could yield long-term consequences. Extending prior research about effects of prenatally exposed synthetic glucocorticoids (sGC) on brain structural development during childhood, we investigated functional brain correlates of cognitive conflict monitoring in term-born adolescents, who were prenatally exposed to sGC. Relative to the comparison group, behavioral response consistency (indexed by lower reaction time variability) and a brain correlate of conflict monitoring (the N2 event-related potential) were reduced in the sGC exposed group. Relatedly, source localization analyses showed that activations in the fronto-parietal network, most notably in the cingulate cortex and precuneus, were also attenuated in these adolescents. These regions are known to subserve conflict detection and response inhibition as well as top-down regulation of stress responses. Moreover, source activation in the anterior cingulate cortex correlated negatively with reaction time variability, whereas activation in the precuneus correlated positively with salivary cortisol reactivity to social stress in the sGC exposed group. Taken together, findings of this study indicate that prenatal exposure to sGC yields lasting impacts on the development of fronto-parietal brain functions during adolescence, affecting multiple facets of adaptive cognitive and behavioral control.

Master of Science / Department of Clinical Sciences / James W. Carpenter / In many species, temporary increases in glucocorticoids (GC) can be used to identify changes in adrenal activity in response to acute stressors. For this research, GC metabolites were identified in fecal extracts from various Atelopus species. The objectives were to identify possible correlates between GCs and health status, assess the impact of husbandry protocols on adrenal activity, and evaluate the sub-lethal effects of antifungal bacteria used for protection of frogs against the chytrid fungus (Batrachochytrium dendrobatidis; Bd). The first study examined whether fecal GC concentrations can be correlated with animal health and behavior changes in a captive setting. Atelopus zeteki with varying degrees of dermatitis were categorized based on the severity of their skin abnormalities and GC metabolite concentrations were analyzed to detect correlations between severity of disease and GC metabolite concentrations. Similarly, behaviors that may indicate elevated stress levels (e.g., time spent in hide) were analyzed to detect correlation between disease and behavior changes. There was no correlation between fecal GC metabolites and health status of the animal or between health status and amount of time spent in hide. The second study established ex situ colonies of two Panamanian frog species, Atelopus certus and Atelopus glyphus, to determine how male group size affects behavior and GC levels. When housed in groups of eight, animals initially had elevated GC concentrations and interacted aggressively, but these instances declined substantially in the first 2 weeks of being housed together. Thus, captive Atelopus populations can be housed in same-sex enclosures without causing sub-lethal stress on the individuals involved. The third study examined the ability of antifungal bacterium from Central America to propagate on Atelopus skin as a preventative treatment for Bd and the sub-lethal effects of each bacteria species on adrenal function based on GC analysis. Four species of bacteria (Pseudomonas sp., Pseudomonas putida, Chryseobacterium indolgenes, and Stenotrophomonas maltophili) were found to be successful Bd inhibitors in vitro. There were no detectable effects of bacterial exposure with GC metabolite concentrations over time for any of the treatments assessed.

Call number: LD2668 .T4 1985 D42 / Master of Science

The effect of synovial membrane products and mechanical pressure upon the metabolism of articular cartilage proteoglycans has been studied in vitro. The degradation of cartilage proteoglycans was studied in an organ culture system and measured as the release of [35S ] sulphate from prelabelled cartilage. The effect of synovial membrane products upon the synthesis of proteoglycans was studied in a chondrocyte monolayer system and the effect of mechanical pressure upon the synthesis of proteoglycans in an organ culture system. In both types of experiments [35S] sulphate was used as precursor. The findings may be summarized as follows 1 Conditioned synovial medium (control-SM) enhanced the degradation and reduced the synthesis of cartilage proteoglycans. In addition the degradation was further enhanced when the synovial tissue had been cultured in the presence of dextran sulphate. 2 Conditioned medium from synovial tissue cultured in the presence of indo-methacin (indo-SM), significantly reduced the synthesis of cartilage proteoglycans in chondrocyte cultures and reduced, although non-significantly, the degradation of proteoglycans in whole cartilage cultures. 3 Addition o f the prostaglandins E1 or E2 (PGE1 or PGE2 ) together with indo-SM to the cartilage cultures greatly enhanced cartilage degradation whereas the addition of PGE1 or PGE2 together with control-SM had no effect compared with that of control-SM alone. 4 Conditioned medium from synovial tissue cultured in the presence of low doses of glucocorticoids reduced cartilage degradation compared with control-SM. However, addition of control-SM together w ith low concentrations of glucocorticoids to the cartilage cultures significantly enhanced cartilage degradation. 5 Conditioned medium from synovial tissue cultured with actinomycin D or cycloheximide did not enhance cartilage degradation compared with cartilage cultured alone. 6 A continuous pressure of approximately 30 kgfcm-2 on cultures of cartilage reduced both the synthesis and the degradation o f cartilage proteoglycans. Although it is difficult to extrapolate from the in vitro to the in vivo situation, it is proposed that some factor(s) from the synovial membrane have the capacity to enhance the degradation and reduce the synthesis o f articular cartilage proteoglycans. From these experiments it cannot be completely excluded that treatm ent of arthritic joints with non-steroidal or streroidal anti-inflammatory drugs may result under certain conditions in enhanced joint damage. It is also suggested that under certain conditions the metabolism o f cartilage proteoglycans could be directly affected by mechanical stress. / <p>Diss. Umeå, Umeå universitet, 1982, härtill 6 uppsatser</p> / digitalisering@umu

Adipose tissue is not homogenous as individual fat depots display regional variation in their physiological properties. It follows that body fat distribution is increasingly being recognised as a major determinant of metabolic disease risk. At the cellular level, depot-specific properties are exhibited by adipocyte precursors during in vitro culture and persist for many generations, suggesting these cells retain an ‘intrinsic memory’ of their anatomical origin which is epigenetic in nature. A primary aim was to identify depot-specific genes whose expression may be regulated by DNA methylation in adipose precursors. Using two paired preadipocyte cell lines derived from human subcutaneous abdominal and gluteal adipose tissue - to represent upper and lower body fat with their opposing associations with cardiovascular disease and diabetes respectively - depot-specific gene expression and DNA methylation profiles were detected. Furthermore, the expression of certain genes in preadipocytes was found to change in response to treatment with the DNA demethylating agent 5-azacytidine, which suggests DNA methylation may regulate depot-specific gene expression. A secondary aim was to investigate whether glucocorticoids – which are important determinants of body fat distribution – exert their effects through DNA methylation. The synthetic glucocorticoid dexamethasone was found to modulate the expression of some of the differentially expressed genes in preadipocytes, with this effect possibly being mediated by DNA methylation. It has been postulated that depot-specific phenotypes in adipose tissue may arise from developmental differences. Several genes were found to be expressed in a depot-specific fashion during a differentiation time course, suggesting regional variation in adipogenesis may contribute to the generation of depot-specific phenotypes. Overall, the data presented suggests regional variation within subcutaneous white adipose tissue exists and supports the notion that DNA methylation patterns can, in part, determine adipose tissue heterogeneity.

Inflammation plays a key role in the underlying pathogenesis of obesity and its associated health risks, with increased markers of inflammation evident in both liver and adipose tissue. In parallel, there is dysregulation of glucocorticoid metabolism in obesity, with increased adipose levels of the glucocorticoid-regenerating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) and increased hepatic levels of 5α-reductase type 1 (5αR1), which catalyses the reduction of glucocorticoids. Both the mechanisms and consequences of this glucocorticoid metabolism dysregulation remain unclear, however, there is evidence that it may be related to inflammation. In vitro studies have demonstrated that pro-inflammatory markers upregulate 11βHSD1 expression in adipocytes, potentially explaining increased expression of this enzyme in obesity. Previous work has also demonstrated that the glucocorticoid metabolites produced by 5αR1 lack the metabolic effects of the parent glucocorticoid, but retain its anti-inflammatory properties, indicating that increased expression of hepatic 5αR1 may serve to dampen down inflammation in the liver. The hypotheses addressed in this thesis are that in obesity, inflammation regulates adipose glucocorticoid metabolism through 11βHSD1, and that hepatic glucocorticoid metabolism regulates the inflammatory state of the liver through 5αR1. The role of inflammation in the regulation of 11βHSD1 was assessed in vivo in mice treated with the anti-inflammatory compound sodium salicylate (salicylate). In diet-induced obese mice, salicylate downregulated 11βHSD1 expression and activity selectively in visceral adipose tissue, alongside improved glucose tolerance, reduced plasma non-esterified fatty acids, and changes in adipose lipid metabolism. 11βHSD1-deficient mice fed a high-fat diet were resistant to the insulin sensitising effects of salicylate treatment. These results indicate a novel role for 11βHSD1 down-regulation in mediating the insulin sensitising effect of anti-inflammatory treatment. The mechanisms underpinning the anti-inflammatory properties of 5α-reduced glucocorticoids were explored in vitro and in vivo. In lipopolysaccharide-stimulated murine macrophages, both 5α-reduced glucocorticoid metabolites tested, namely 5α-dihydrocorticosterone (5αDHB) and 5α-tetrahydrocorticosterone (5αTHB), suppressed tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) release, although to a lesser extent than corticosterone (B). Similar to B, both 5αDHB and 5α THB suppressed phosphorylation of intra-cellular inflammatory signalling mitogen-activated protein kinases (MAPK) proteins c-Jun N-terminal kinase (JNK) and p38, as well as increasing protein expression of MAPK phosphatase-1 (MKP-1). Treatment of phorbol ester-stimulated HEK293 kidney cells with these 5α-metabolites revealed that 5αDHB suppressed nuclear factor κB (NFκB) and activator protein-1 (AP-1) activation to a similar extent to that of B, whilst 5αTHB increased activation of these pro-inflammatory transcription factors, indicating cell-specific effects of 5αTHB. In conclusion, reduced intra-adipose glucocorticoid regeneration by 11βHSD1 mediates the insulin sensitising effects of salicylate, suggesting that altered glucocorticoid metabolism may reflect altered intra-adipose inflammation in obesity. Furthermore, these data support the concept that this enzyme provides a therapeutic target in obesity-related metabolic disorders. 5α-reduced metabolites of glucocorticoids have similar anti-inflammatory properties to the parent glucocorticoid, indicating that the elevated hepatic levels of 5α-reductase in obesity may be a protective mechanism to limit the adverse metabolic effects of glucocorticoids upon the liver, but maintain the beneficial anti-inflammatory properties. These 5α-reduced glucocorticoid metabolites may provide a potential therapeutic treatment as selective glucocorticoid receptor modulators for inflammatory conditions.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2005. / Endogenous glucocorticoids (GCs) modulate many physiological functions in the human body and synthetic GCs are the most effective therapy in the treatment of inflammation, autoimmune and endocrine disorders. However, the long-term usage of synthetic GCs is associated with severe side-effects. GCs mediate their effects through the ligand-dependent transcription factor, the glucocorticoid receptor (GR), either by causing an increase (transactivation) or a decrease (transrepression) in gene transcription. The bioactivity of a ligand in GR-mediated transcriptional regulation is established by a transcriptional doseresponse curve, where the potency (EC50 value) and the efficacy (maximal response) of the ligand are determined. A central question is how different GR ligands elicit their differential physiological responses for the same gene in the same cell. The main aim of this thesis is to investigate if the phosphorylation of GR at serine 211 (Ser211) correlates with the potency and/or efficacy of a particular ligand in transactivation and transrepression of gene expression.