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Articles de revues sur le sujet "HSOD1"
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Bouldin, Samantha D., Maxwell A. Darch, P. John Hart et Caryn E. Outten. « Redox properties of the disulfide bond of human Cu,Zn superoxide dismutase and the effects of human glutaredoxin 1 ». Biochemical Journal 446, no 1 (27 juillet 2012) : 59–67. http://dx.doi.org/10.1042/bj20120075.
Texte intégralRésumé :
The intramolecular disulfide bond in hSOD1 [human SOD1 (Cu,Zn superoxide dismutase 1)] plays a key role in maintaining the protein's stability and quaternary structure. In mutant forms of SOD1 that cause familial ALS (amyotrophic lateral sclerosis), this disulfide bond is more susceptible to chemical reduction, which may lead to destabilization of the dimer and aggregation. During hSOD1 maturation, disulfide formation is catalysed by CCS1 (copper chaperone for SOD1). Previous studies in yeast demonstrate that the yeast GSH/Grx (glutaredoxin) redox system promotes reduction of the hSOD1 disulfide in the absence of CCS1. In the present study, we probe further the interaction between hSOD1, GSH and Grxs to provide mechanistic insight into the redox kinetics and thermodynamics of the hSOD1 disulfide. We demonstrate that hGrx1 (human Grx1) uses a monothiol mechanism to reduce the hSOD1 disulfide, and the GSH/hGrx1 system reduces ALS mutant SOD1 at a faster rate than WT (wild-type) hSOD1. However, redox potential measurements demonstrate that the thermodynamic stability of the disulfide is not consistently lower in ALS mutants compared with WT hSOD1. Furthermore, the presence of metal cofactors does not influence the disulfide redox potential. Overall, these studies suggest that differences in the GSH/hGrx1 reaction rate with WT compared with ALS mutant hSOD1 and not the inherent thermodynamic stability of the hSOD1 disulfide bond may contribute to the greater pathogenicity of ALS mutant hSOD1.
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Coelho, Fernando R., Asif Iqbal, Edlaine Linares, Daniel F. Silva, Filipe S. Lima, Iolanda M. Cuccovia et Ohara Augusto. « Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme ». Journal of Biological Chemistry 289, no 44 (18 septembre 2014) : 30690–701. http://dx.doi.org/10.1074/jbc.m114.586370.
Texte intégralRésumé :
The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp32 residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp32 residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1WT and hSOD1G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp32 residue in the process. The results showed that Trp32 residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp32 residue (bovine SOD1 and hSOD1W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp32 residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.
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Zhang, Kun, Yuejuan Zhang, Jing Zi, Xiaochang Xue et Yi Wan. « Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity inE. colivia Mutation of Free Cysteine Residues ». BioMed Research International 2017 (2017) : 1–12. http://dx.doi.org/10.1155/2017/4817376.
Texte intégralRésumé :
Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields inE. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 inE. coliwhereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it.Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.
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Álvarez-Zaldiernas, Cristina, Jun Lu, Yujuan Zheng, Hongqian Yang, Juan Blasi, Carles Solsona et Arne Holmgren. « Cellular Redox Systems Impact the Aggregation of Cu,Zn Superoxide Dismutase Linked to Familial Amyotrophic Lateral Sclerosis ». Journal of Biological Chemistry 291, no 33 (3 juin 2016) : 17197–208. http://dx.doi.org/10.1074/jbc.m115.708230.
Texte intégralRésumé :
Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys57 and Cys146, which have been linked to protein stability and folding via forming a disulfide bond, and Cys6 and Cys111 as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS.
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FENG, LIN, Yan Dan Dan, Chen Ya Wen, Fletcher Emmanuella E, Shi Hai Feng, Han Bang Xing et Zhou Yang. « Cloning, purification and enzymatic characterization of recombinant human Superoxide dismutase 1 expressed in Escherichia coli ». Acta Biochimica Polonica 65, no 2 (8 juillet 2018) : 235–40. http://dx.doi.org/10.18388/abp.2017_2350.
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Superoxide dismutase 1 (SOD1) is a metalloenzyme that catalyzes disproportion-action superoxide into molecular oxygen and hydrogen peroxide. In this study, the human SOD1 (hSOD1) gene was cloned, expressed, and purified. The hSOD1 gene was amplified from a pool of Bxpc3 cell cDNAs by PCR and cloned into expression vector pET-28a (+). The recombinant soluble hSOD1 was expressed in E.coli BL21 (DE3) at 37°C and purified by Nickel column affinity chromatography. The soluble hSOD1 was produced with a yield of 5.9 ug/mL medium. Considering that metal ions have a certain influence on the structure and activity of protein, we researched the influences of different concentrations of Cu2+ and Zn2+ on hSOD1 activity at induction and the time of activity detection. The results implied Cu2+ and Zn2+ can’t enhance SOD1 expression, however can improve the catalytic activity at induction. Furthermore, most of bivalent cations have an improve effect on enzyme activity at the time of detection.
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Islam, Rafique, Emily L. Kumimoto, Hong Bao et Bing Zhang. « ALS-linked SOD1 in glial cells enhances ß-N-Methylamino L-Alanine (BMAA)-induced toxicity in Drosophila ». F1000Research 1 (9 novembre 2012) : 47. http://dx.doi.org/10.12688/f1000research.1-47.v1.
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Environmental factors have been implicated in the etiology of a number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, the role of environmental agents in ALS remains poorly understood. To this end, we used transgenic fruit flies (Drosophila melanogaster) to explore the interaction between mutant superoxide dismutase 1 (SOD1) and chemicals such as ß-N-methylamino L-alanine (BMAA), the herbicide agent paraquat, and superoxide species. We expressed ALS-linked human SOD1 (hSOD1A4V, and hSOD1G85R), hSOD1wt as well as the Drosophila native SOD1 (dSOD1) in motoneurons (MNs) or in glial cells alone and simultaneously in both types of cells. We then examined the effect of BMAA (3 mM), paraquat (20 mM), and hydrogen peroxide (H2O2, 1%) on the lifespan of SOD1-expressing flies. Our data show that glial expression of mutant and wild type hSOD1s reduces the ability of flies to climb. Further, we show that while all three chemicals significantly shorten the lifespan of flies, mutant SOD1 does not have a significant additional effect on the lifespan of flies fed on paraquat, but further shortens the lifespan of flies fed on H2O2. Finally, we show that BMAA shows a dramatic cell-type specific effect with mutant SOD1. Flies with expression of mutant hSOD1 in MNs survived longer on BMAA compared to control flies. In contrast, BMAA significantly shortened the lifespan of flies expressing mutant hSOD1 in glia. Consistent with a neuronal protection role, flies expressing these mutant hSOD1s in both MNs and glia also lived longer. Hence, our studies reveal a synergistic effect of mutant SOD1 with H2O2 and novel roles for mutant hSOD1s in neurons to reduce BMAA toxicity and in glia to enhance the toxicity of BMAA in flies.
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Martin, Lee J., Danya A. Adams, Mark V. Niedzwiecki et Margaret Wong. « Aberrant DNA and RNA Methylation Occur in Spinal Cord and Skeletal Muscle of Human SOD1 Mouse Models of ALS and in Human ALS : Targeting DNA Methylation Is Therapeutic ». Cells 11, no 21 (31 octobre 2022) : 3448. http://dx.doi.org/10.3390/cells11213448.
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Amyotrophic lateral sclerosis (ALS) is a fatal disease. Skeletal muscles and motor neurons (MNs) degenerate. ALS is a complex disease involving many genes in multiple tissues, the environment, cellular metabolism, and lifestyles. We hypothesized that epigenetic anomalies in DNA and RNA occur in ALS and examined this idea in: (1) mouse models of ALS, (2) human ALS, and (3) mouse ALS with therapeutic targeting of DNA methylation. Human superoxide dismutase-1 (hSOD1) transgenic (tg) mice were used. They expressed nonconditionally wildtype (WT) and the G93A and G37R mutant variants or skeletal muscle-restricted WT and G93A and G37R mutated forms. Age-matched non-tg mice were controls. hSOD1 mutant mice had increased DNA methyltransferase enzyme activity in spinal cord and skeletal muscle and increased 5-methylcytosine (5mC) levels. Genome-wide promoter CpG DNA methylation profiling in skeletal muscle of ALS mice identified hypermethylation notably in cytoskeletal genes. 5mC accumulated in spinal cord MNs and skeletal muscle satellite cells in mice. Significant increases in DNA methyltransferase-1 (DNMT1) and DNA methyltransferase-3A (DNMT3A) levels occurred in spinal cord nuclear and chromatin bound extracts of the different hSOD1 mouse lines. Mutant hSOD1 interacted with DNMT3A in skeletal muscle. 6-methyladenosine (6mA) RNA methylation was markedly increased or decreased in mouse spinal cord depending on hSOD1-G93A model, while fat mass and obesity associated protein was depleted and methyltransferase-like protein 3 was increased in spinal cord and skeletal muscle. Human ALS spinal cord had increased numbers of MNs and interneurons with nuclear 5mC, motor cortex had increased 5mC-positive neurons, while 6mA was severely depleted. Treatment of hSOD1-G93A mice with DNMT inhibitor improved motor function and extended lifespan by 25%. We conclude that DNA and RNA epigenetic anomalies are prominent in mouse and human ALS and are potentially targetable for disease-modifying therapeutics.
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Self, Wade K., Kathleen Schoch, James Bollinger, Tracy Cole, Holly Kordasiewicz, Randall Bateman et Timothy Miller. « 2342 Protein production as an early pharmacodynamics biomarker for RNA-targeting therapies ». Journal of Clinical and Translational Science 2, S1 (juin 2018) : 24. http://dx.doi.org/10.1017/cts.2018.110.
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OBJECTIVES/SPECIFIC AIMS: We aimed to develop an assay to measure new protein synthesis after Antisense Oligonucleotide treatment, which we hypothesized to be the earliest biochemical identification of RNA-targeting therapy efficacy. METHODS/STUDY POPULATION: We treated 2 transgenic animal models expressing proteins implicated in neurodegenerative disease: human tau protein (hTau) and human superoxide dismutase 1 (hSOD1), with ASO against these mRNA transcripts. Animals received isotope-labeled 13C6-Leucine via drinking water to label newly synthesized proteins. We assayed target protein synthesis and concentration after ASO treatment to determine the earliest identification of ASO target engagement. RESULTS/ANTICIPATED RESULTS: hTau ASO treatment in transgenic mice lowered hTau protein concentration 23 days post-treatment in cortex (95% CI: 0.05%–64.0% reduction). In the same tissue, we observed lowering of hTau protein synthesis as early as 13 days (95% CI: 29.4%–123%). In hSOD1 transgenic rats, we observed lowering of 13C6-leucine-labeled hSOD1 in the cerebrospinal fluid 30 days after ASO treatment compared with inactive ASO control (95% CI: 12.0%–48.4%). DISCUSSION/SIGNIFICANCE OF IMPACT: In progressive neurodegenerative diseases, it is crucial to develop measurements that identify treatment efficacy early to improve patient outcomes. These data support the use of stable isotope labeling of amino acids to measure new protein synthesis as an early pharmacodynamics measurement for therapies that target RNA and inhibit the translation of proteins.
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Brasil, Aline de Araújo, Mariana Dias Castela de Carvalho, Ellen Gerhardt, Daniela Dias Queiroz, Marcos Dias Pereira, Tiago Fleming Outeiro et Elis Cristina Araujo Eleutherio. « Characterization of the activity, aggregation, and toxicity of heterodimers of WT and ALS-associated mutant Sod1 ». Proceedings of the National Academy of Sciences 116, no 51 (3 décembre 2019) : 25991–6000. http://dx.doi.org/10.1073/pnas.1902483116.
Texte intégralRésumé :
Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and insod1Δyeast cells. Using Sod1-KD cells we found that the WT–A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies.
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Queiroz, Raphael F., Verônica Paviani, Fernando R. Coelho, Emerson F. Marques, Paolo Di Mascio et Ohara Augusto. « The carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity is inhibited by the radical scavenger tempol ». Biochemical Journal 455, no 1 (13 septembre 2013) : 37–46. http://dx.doi.org/10.1042/bj20130180.
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The nitroxide tempol inhibited the carbonylation and covalent dimerization of human superoxide dismutase 1 caused by its bicarbonate-dependent peroxidase activity. Tempol acted by scavenging the produced carbonate radical and by recombining with hSOD1-Trp32• radicals as indicated by MS/MS evidence.
Thèses sur le sujet "HSOD1"
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Thyssen, Stella [Verfasser], Jochen [Akademischer Betreuer] Weishaupt et Oliver [Akademischer Betreuer] Wirths. « Einfluss des Proteinaggregationshemmstoffs anle138b auf Beginn und Verlauf der Amyotrophen Lateralsklerose im transgenen hSOD1-Mausmodell / Stella Thyssen. Gutachter : Jochen Weishaupt ; Oliver Wirths. Betreuer : Jochen Weishaupt ». Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2014. http://d-nb.info/1053119283/34.
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White, Christopher Iain. « Cardiovascular 11β-HSD1 : its role in myocardial physiology and pathophysiology ». Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/23391.
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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.
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Leiva, Martínez Rosana. « Polycyclic group optimization in 11β-HSD1 inhibitors and their pharmacological evaluation ». Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/457770.
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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).
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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.
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Andres, Janin. « Untersuchungen über Regulationsmechanismen der 11beta-Hydroxysteroid Dehydrogenase Typ 1 ». Phd thesis, Universität Potsdam, 2008. http://opus.kobv.de/ubp/volltexte/2009/3303/.
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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.
5
Zhang, Zhenguang. « Role of macrophage 11β-HSD1 in inflammation mediated angiogenesis, arthritis and obesity ». Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9553.
Texte intégralRésumé :
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.
6
Wamil, Małgorzata. « Protective role of 11β-HSD1 inhibition in the metabolic syndrome and atherosclerosis ». Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/3891.
Texte intégralRésumé :
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.
7
Coutinho, Agnes Elizabeth. « Consequences of 11β-hydroxysteroid dehydrogenase deficiency during inflammatory responses ». Thesis, University of Edinburgh, 2009. http://hdl.handle.net/1842/4190.
Texte intégralRésumé :
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.
8
Verma, Manu. « Deficiency of 11β-HSD1 modulates energy homeostasis in the brain following systemic inflammation ». Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/33323.
Texte intégralRésumé :
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.
9
Liu, Xiaoxia. « Regulation and function of 11β-hydroxysteroid dehydrogenase (11β-HSD1) in pancreatic β-cells ». Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5582.
Texte intégralRésumé :
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.
10
Mitić, Tijana. « Role of murine 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) in the metabolism of 7-oxysterols ». Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4416.
Texte intégralRésumé :
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.
Chapitres de livres sur le sujet "HSOD1"
1
Colas, D., J. London, R. Cespuglio et N. Sarda. « Polysomnography in transgenic hSOD1 mice as Down syndrome model ». Dans Advances in Down Syndrome Research, 165–71. Vienna : Springer Vienna, 2003. http://dx.doi.org/10.1007/978-3-7091-6721-2_15.
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Scott*, James S., et Jasen Chooramun. « Chapter 5. 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Inhibitors in Development ». Dans Drug Discovery, 109–41. Cambridge : Royal Society of Chemistry, 2012. http://dx.doi.org/10.1039/9781849735322-00109.
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Paderes, Genevieve D., Klaus Dress, Buwen Huang, Jeff Elleraas, Paul A. Rejto et Tom Pauly. « Structure-Based and Property-Compliant Library Design of 11β-HSD1 Adamantyl Amide Inhibitors ». Dans Methods in Molecular Biology, 191–215. Totowa, NJ : Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-931-4_10.
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Reynolds, Rebecca M., et Scott P. Webster. « Translational Research in Stress Neuroendocrinology : 11β-Hydroxysteroid Dehydrogenase 1 (11β-HSD1), A Case Study ». Dans Neuroendocrinology of Stress, 327–50. Chichester, UK : John Wiley & Sons, Ltd, 2015. http://dx.doi.org/10.1002/9781118921692.ch14.
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Naredo-Gonzalez, G., MA Jansen, GD Merrifield, OB Sutcliffe, MK Hansen, R. Andrew et BR Walker. « Non-Invasive In-Vivo Monitoring of 11β-HSD1 Activity Using19F-Magnetic Resonance Spectroscopy. » Dans Posters I, P3–32—P3–32. Endocrine Society, 2010. http://dx.doi.org/10.1210/endo-meetings.2010.part3.p1.p3-32.
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Tiganescu, A., AE Mayes, R. Hardy, PM Stewart et EA Walker. « Is Increased 11β-HSD1 Expression a Key Factor Underpinning Intrinsic and Extrinsic Skin Aging?. » Dans Posters I, P3–31—P3–31. Endocrine Society, 2010. http://dx.doi.org/10.1210/endo-meetings.2010.part3.p1.p3-31.
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Kienitz, T., M. Michaelis, P. Hofmann, F. Gotz et M. Quinkler. « Regulation of 11beta-Hydroxysteroid Dehydrogenase Type 1 (11beta-HSD1) Expression in the Rat Heart by Androgens. » Dans Posters I, P3–34—P3–34. Endocrine Society, 2010. http://dx.doi.org/10.1210/endo-meetings.2010.part3.p1.p3-34.
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Latif, Syed A., Renshan Ge et David J. Morris. « Differential Inhibition of 11β-HSD1 Dehydrogenase by 5β-Ring-a-Reduced Metabolites of 11-Desoxy-Steroid Hormones ». Dans BASIC/TRANSLATIONAL - Steroidogenesis & ; Its Disorders, P2–592—P2–592. The Endocrine Society, 2011. http://dx.doi.org/10.1210/endo-meetings.2011.part3.p8.p2-592.
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Lavery, Gareth G., Laura L. Gathercole, Stuart A. Morgan, Agnieszka Zielinska, Khalid Saqib, Beverley Hughes, Elizabeth A. Walker et Paul M. Stewart. « Lack of a Beneficial Metabolic Profile in Liver-Specific 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Knockout Mice ». Dans BASIC/TRANSLATIONAL - Molecular Mechanisms Regulating Glucose Homeostasis & ; Insulin Action, OR23–4—OR23–4. The Endocrine Society, 2011. http://dx.doi.org/10.1210/endo-meetings.2011.part3.or4.or23-4.
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Esteves, CL, V. Kelly, T.-M. Man, JR Seckl et KE Chapman. « Dietary Regulation of 11β-HSD1 in Adipose Tissue Is Mediated by the C/EBPβ Isoforms LAP and LIP. » Dans The Endocrine Society's 92nd Annual Meeting, June 19–22, 2010 - San Diego, OR19–5—OR19–5. Endocrine Society, 2010. http://dx.doi.org/10.1210/endo-meetings.2010.part3.or1.or19-5.
Texte intégralActes de conférences sur le sujet "HSOD1"
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Finke, U., J. Schneider, E. Friderichs, L. Flohé et H. Giertz. « MYOCARDIAL SALVAGE BY COMBINED TREATMENT WITH RECOMBINANT SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR AND RECOMBINANT HUMAN SUPEROXIDE DISMUTASE IN A CANINE CORONARY THROMBOSIS MODEL* ». Dans XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643570.
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Recanalization of thrombotic coronary occlusion with fibrinolytic treatment is a promising approach to salvage jeopardized ischemic myocardium. However, the success of thrombolytic treatment of myocardial infarction may be curtailed by the risk inherent to reperfusion. Cell damage upon reoxygenation after an ischemic period is tentatively attributed to the formation of oxygen-derived free radicals. Improved myocardial salvage is therefore expected from coadministration of a free radical scavenger and fibrinolytic treatment. We tested this hypothesis in a canine model of left anterior circumflex coronary artery (LCX) thrombosis. Thrombolysis was achieved with the fibrin- selective single-chain urokinase-type plasminogen activator of recombinant origin (r-scu-PA). As enzymatic scavenger of oxygen radicals recombinant human superoxide dismu-tase (r-HSOD) was used. The three experimental groups were: group I (n=4) did not receive any treatment after LCX thrombosis; in group II (n=9) at 100 min after LCX thrombosis r-scu-PA (20 μg/kg/min i.v. for 30 min) was infused; dogs in group III (n=8) received concomitant treatment with r-scu-PA and r-HSOD (10 mg/kg i.v. for 60 min). Infarct size as percent of the risk zone was 38.2 ± 4.1 in group I, 25.3 ± 3.7 in group II (p ≤ 0.05 vs group I) and 14.9 ± 3.2 in group III (p ≤ 0.05 vs group II). Incidence of reperfusion arrhythmias and increase in plasma CK were significantly diminished by r-HSOD when compared to dogs receiving r-scu-PA only. There were no significant differences in hemodynamic parameters between the groups. In conclusion, in terms of infarct size reduction, suppression of reperfusion arrhythmias and attenuation of intracellular enzyme release the combined treatment with r-scu-PA and r-HSOD yielded a significant higher myocardial salvage versus thrombolytic treatment alone in a canine LCX thrombosis model.* This work is part of the doctoral dissertation of Miss U. Fincke
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Vitiello, M., V. Kunnathully, G. Orsolini, O. Viapiana, F. Poli, A. Mattè, L. De Franceschi, L. Idolazzi, D. Gatti et M. Rossini. « AB0045 Il-17 induced glucocorticoid insensitivity might be dependent on the reduced 11beta-hsd1 enzyme activity ». Dans Annual European Congress of Rheumatology, 14–17 June, 2017. BMJ Publishing Group Ltd and European League Against Rheumatism, 2017. http://dx.doi.org/10.1136/annrheumdis-2017-eular.2681.
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Grumbach, Y., A. Hu, JS Grunstein, G. Nino, L. Gober et MM Grunstein. « Regulation of 11ß-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Expression in Human Airway Smooth Muscle (HASM) Cells. » Dans American Thoracic Society 2009 International Conference, May 15-20, 2009 • San Diego, California. American Thoracic Society, 2009. http://dx.doi.org/10.1164/ajrccm-conference.2009.179.1_meetingabstracts.a6313.
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Nikmaturrohana, Qurin, Sri Rahayu Lestari et Betty Lukiati. « Potential of single bulb garlic (Allium sativum) active compound as an 11β-HSD1 inhibitor in obesity through in silico method ». Dans PROCEEDINGS OF THE 3RD INTERNATIONAL SEMINAR ON METALLURGY AND MATERIALS (ISMM2019) : Exploring New Innovation in Metallurgy and Materials. AIP Publishing, 2020. http://dx.doi.org/10.1063/5.0002532.
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