Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 7 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Titel på diss.-titelbl.: Islet glucose metabolism and insulin secretion in two animal models of glucose intolerance. Härtill 5 uppsatser.

Glucocorticoid excess as a result of Cushing’s syndrome or pharmacological treatment can result in the development of obesity and type 2 diabetes mellitus (T2DM). The reactivation of cortisone to cortisol is catalysed by 11βHSD1 which is expressed widely but notably in adipose tissue and liver. Studies have shown dysregulation of cortisol in these tissues with obesity potentially promoting the development of T2DM. Inhibition of 11βHSD1 has been attempted as a novel treatment for T2DM with observed improvement in glycaemic control, body weight and blood pressure. The efficacy of such agents has been disappointing with few reaching phase 2 trials. With recent evidence of bidirectional activity of 11βHSD1 in vivo it becomes apparent that dysregulation may occur at an intracellular rather than tissue level. In this thesis I address several key outstanding questions concerning the physiology and regulation of 11βHSD1 including: 1. Whether combined therapy with metformin alters 11βHSD1 activity and obscures the efficacy of 11βHSD1inhibitors; 2. Whether the contribution of 11βHSD1 to local cortisol concentrations has been under-estimated by considering total rather than free cortisol turnover; and 3. Whether recycling between cortisol and cortisone in adipose tissue and skeletal muscle in obesity is a neglected feature of 11βHSD1 biochemistry and function. Eight obese healthy men with and without type 2 diabetes were recruited to a randomised placebo controlled cross over trial. They received 4 weeks treatment with metformin and placebo. Participants with T2DM additionally received gliclazide as a further control. Using the deuterated tracer D4-cortisol 11βHSD1 activity was measured. Metformin treatment increased whole body 11βHSD1 in both groups postulated as a result of improved insulin sensitivity. 11βHSD1 is located within cells and so contributes to free tissue cortisol concentrations but perhaps less so to total (protein-bound) cortisol in plasma. It has been shown that 11βHSD1 contributes almost half of total circulating cortisol concentrations at rest. This measurement relied upon blood sampling during steady state deuterated cortisol (D4-cortisol) infusion with measurements of total (free plus protein bound) cortisol which may have underestimated true 11βHSD1 activity. This was therefore investigated by comparing 11βHSD1 activity as calculated using total compared with free cortisol tracer enrichments. Equilibrium dialysis was performed separating free from bound portions in plasma samples taken from healthy volunteers who received D4-cortisol infusion. Analysis revealed similar measurements of 11βHSD1 activity using free compared with total cortisol implicating rapid turnover of glucocorticoids between the free and bound pools. On first discovery 11βHSD1 was seen to be a dehydrogenase enzyme in vitro. Later work recognised reductase activity in vivo and up until recently 11βHSD1 has been viewed as a predominantly reductase enzyme. As with other enzymes in the same family, the ability to catalyse both reductase and dehydrogenase depends upon the availability of substrate and co substrate. Whether dysregulation of 11βHSD1 in the settings of obesity and T2DM is the result of alteration in directionality at a cellular level is not known. Firstly bidirectional activity of 11βHSD1 was confirmed in vitro using HEK-293 cells stably transfected with 11βHSD1. The influence of obesity and acute perturbation with hyperinsulinaemia was subsequently investigated in vivo in a random order cross over single blinded case control study involving ten normal weight and ten obese healthy male volunteers. D4-cortisol and deuterated cortisone (D2-cortisone) were infused for the measurement of reductase and dehydrogenase activity of 11βHSD1 respectively with measurements taken across forearm muscle and abdominal subcutaneous adipose tissue. Across whole body, lean and obese individuals displayed similar 11β-reductase and 11β-dehydrogenase activity. Across tissue, 11β-reductase and 11β-dehydrogenase activity was different from zero across adipose tissue in obese individuals and across skeletal muscle in lean individuals providing further evidence of tissue specific differences in 11βHSD1 with obesity. With the addition of hyperinsulinaemia, reductase and dehydrogenase activity was somewhat increased in lean individuals although there was no statistically significant difference between lean and obese individuals. Across tissue there was a trend for obese individuals to display increased 11β-reductase activity across adipose tissue with hyperinsulinaemia. Comparing the rates of reductase and dehydrogenase activity revealed predominantly reductase activity across tissue in obese and dehydrogenase activity in lean individuals. The development of direction specific inhibitors targeting reductase activity by 11βHSD1 may prove efficacious for the treatment of obesity. In conclusion, 11βHSD1 acts as a bidirectional enzyme in vitro and in vivo. Overall directionality of enzyme activity is altered in a tissue specific manner in the setting of obesity. We have shown that this intracellular regulation of cortisol is reflected equally in the metabolically active free pool and total plasma pool. The efficacy of 11βHSD1 inhibitors as novel agents for the treatment of T2DM and coexisting obesity is not diminished by co-prescription with metformin but may prove more efficacious through the development of reductase specific inhibitors.

Glucocorticoids (Gcs) are a commonly used drug to target the glucocorticoid receptor (GR). The GR has a myriad of cellular and physiological effects, however, Gcs are clinically used for the treatment of inflammatory conditions due to the potent anti-inflammatory actions of GR. The anti-inflammatory effects come with serious side effects e.g. metabolic disease. I examine the role of lipid rafts in modulating the anti-inflammatory actions of Gcs, and the role of circadian rhythms in the control of Gc side effects. I tested the role of caveolin-1 (Cav1), a constituent of membrane lipid rafts, and its role in Gc suppression of inflammation. Gene expression analysis of mouse lung tissue showed that genetic depletion of Cav1 (CAV1KO) results in increased transactivation of Gc target genes. The increased Gc action, however, does not result in an increased effect on suppression of inflammation in a model of innate immunity: aerosolised lipopolysaccharide (LPS) induced lung inflammation or in a model of adaptive immunity: Ovalbumin. CAV1KO mice were protected from LPS induced inflammation, despite increased cytokine production. This suggests a differential response to LPS in lung parenchyma and alveolar macrophages dependent on Cav1. CAV1KO results in a pro-inflammatory phenotype in macrophages, and the opposite in parenchymal tissue. These data suggest that while Cav1 is an upstream regulator of Gc response, it does not have a strong enough effect to alter the ability of GR to repress inflammation in vivo. Gc treatment results in a strong metabolic phenotype, with aberrant energy metabolism, insulin resistance and hepatosteaotosis, I investigated how this side effect interacts with circadian rhythms, another key determinant of energy metabolism. Using transcriptomics of whole lung and liver taken during the day or the night, I demonstrate that the metabolic actions of Gc in the liver can be temporally separated, whilst maintaining consistent anti-inflammatory actions in both liver and lung. This temporal gene regulation by Gc is controlled by REV-ERB, a rhythmically expressed, orphan nuclear receptor, part of the core clock machinery, via a direct interaction with GR at key regulatory DNA loci. Genetic deletion of REV-ERB protects mice from the hepatosteotosis associated with Gc treatment. Taken together, these data suggest that Gcs are regulated upstream of the receptor by the core consitutent of membrane lipid rafts; Cav1, which modulates the Gc response in vivo. Also, that the GR action can be controlled by dosing at different times of day, separating the detrimental metabolic effects of Gcs from the beneficial anti-inflammatory effects. This is enabled through a direct interaction between GR and REV-ERB at key gene regulatory sites.

Reduced availability of high energy-content prey (nutritional stress) is a predominant hypothesis to explain the decline of Steller sea lion (Eumetopias jubatus) populations in western Alaska from the late 1970' s to the late 1990' s. Animals may physiologically respond to consuming insufficient prey by increasing stress levels and decreasing metabolic rates. It may thus be possible to identify nutritional stress by measuring concentrations of glucocorticoids (stress) and thyroid hormones (metabolism) shed in feces. However, techniques to measure thyroid hormone concentrations from Steller sea lion feces have not been developed. We quantified variation of triiodothyronine (T3) and thyroxine (T4) concentrations in Steller sea lion feces following injections of thyrotropin (TSH) into four captive animals. Glucocorticoids (GC) were also assayed to examine any relationship to stimulated thyroid hormone secretion. We found that fecal T3 peaked 48 h post-injection and increased 25-57% in three sea lions (all animals, p=0.03). Pre-injection GC increases indicated stress from isolation for baseline fecal collections, but post-injection increases could not be confirmed as a response to TSH injections or as a product of the study design. The results demonstrated that pre- and post-injection changes in fecal GC and T3 concentrations were consistent with predictions of an increased stress response and metabolic rate within the animals. We then measured T3 and GC concentrations in 834 Steller sea lion fecal samples collected in 2005 and 2006 from 15 sites (haulouts and rookeries) between British Columbia and the Central Aleutian Islands. Overall, GC concentrations did not differ between haulout populations (western 2006 pre-pupping and eastern 2005 post-pupping). Fecal hard-part analyses revealed a lower energy-content diet in the western population, suggesting that diet quality is a relevant hypothesis to explain slightly higher GC concentrations found in the western population, specifically the Aleutian Islands region. However, nutritional stress could not be substantiated through T3 concentrations. The rookeries possessed the highest energy-content diets, but also exhibited a nutritional stress response with a significantly higher GC and lower T3 concentration than either haulout population (possibly related to lactation or decreased foraging opportunities), but T3 comparisons performed at scales of site and region were inconclusive. / Science, Faculty of / Zoology, Department of / Graduate

Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.

Glucocorticoids (GCs) have an important role in anti-inflammation, apoptosis and immunomodulatory activity. GCs exert their effect by binding to their receptor, glucocorticoid receptor (GR), which subsequently triggers receptor dimerisation, nuclear translocation and eventually causes impact on transcriptional activity. Such regulatory mechanism is complex as it is not only controlled at the transcription level, but also at the post translational level with other contributing factors such as protein stability and cofactor recruitment. Glucocorticoids are commonly used as part of the chemotherapeutical protocols for lymphoid malignancies and have been successfully implicated in treating childhood acute lymphoblastic leukaemia (ALL). Nevertheless, resistance and side effects such as muscle atrophy and osteoporosis still occur frequently.With the advance in high-throughput technology, vast amount of data on various scales, including genomics, proteomics, and metabolomics make the molecular study of cancer more complicated. The rise of systems biology helps the scientist to address this problem with the use of computation. Although the concept and the approach may vary depending on the research fields, the ultimate goal remains the same which is to create a comprehensive understanding of biological processes and to forecast outcome.The goal of this body of work is to better understand glucocorticoid induced apoptosis in acute lymphoblastic leukaemia by adopting a systems biology approach. As the Bcl-2 family, particularly Bim is known to be a key determinant of GC-induced apoptosis, we investigated the molecular mechanism of GC induction of Bim. By adopting ordinary differential equation modelling approach, we were able to make prediction and investigate details of Bim regulation by GCs. Further to this, we carried out an integrated microarray analysis in various ALL to study GC resistance and identified crucial candidate gene c-Jun as a regulator of Bim and Erg as a determinant for GC resistance. These results allowed us to refine our models and enabled more answers to be addressed. In conclusion, our findings not only suggest potential regulatory mechanisms for determining GC sensitivity, they also aid us to find potential biomarkers for determining GC resistance. More importantly, this study represents a successful example for utilising systems biology to study the genetic complexity in cancer.

Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

In primary rat hepatocyte cultures, growth hormone was shown to depress tyrosine aminotransferase levels induced with hydrocortisone. Both induction by glucocorticoid and repression by growth hormone could be demonstrated in cultures several days old.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21027号 / 医科博第88号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 杉田 昌彦, 教授 濵﨑 洋子, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM