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Generation and Utilization of NADPH in the Endoplasmic Reticulum: Impact on Metabolic and Hormonal Regulation

English title Generation and Utilization of NADPH in the Endoplasmic Reticulum: Impact on Metabolic and Hormonal Regulation
Applicant Odermatt Alexander
Number 140961
Funding scheme Project funding (Div. I-III)
Research institution Universität Basel
Institution of higher education University of Basel - BS
Main discipline Biochemistry
Start/End 01.05.2012 - 30.04.2015
Approved amount 451'266.00
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All Disciplines (2)

Discipline
Biochemistry
Endocrinology

Keywords (8)

inhibitor; redox; 11beta-hydroxysteroid dehydrogenase; endoplasmic reticulum; glucose-6-phosphate; pentose-phosphate pathway; NADPH; bile acid

Lay Summary (English)

Lead
Lay summary

The reduced pyridine nucleotide NADPH acts as cofactor for a multitude of reduction reactions and is essential for major physiological processes. NADPH cannot freely move across cellular membranes, and distinct pools exist in the cytoplasm, the mitochondria and the endoplasmic reticulum (ER). In contrast to the cytoplasm, much less is known on NADPH generation and utilization in the ER.

To date, only one enzyme has been identified in the ER that generates NADPH. It uses glucose-6-phosphate as substrate and its activity is dependent on the cellular energy state. The reactions within the ER that utilize NADPH are not well studied. To date only one enzyme within the ER is known to utilize NADPH. It interconverts inactive and active glucocorticoids and therefore represents a hormonal response mechanism to alterations in energy status of cells that express this enzyme.

There are, however, several important questions that need to be addressed: 1) what is the role of the NADPH generating enzyme in cells that do not express the glucocorticoid activating enzyme? 2) which other enzymes require NADPH in the ER for their function? 3) how can NADPH be generated in the absence of the glucose-6-phosphate dependent enzyme, are there other mechanisms to generate NADPH in the ER? 4) How do pyridine nucleotides reach the ER and how is the pool in the ER initially formed? 5) does the product of the NADPH generating enzyme in the ER react further, i.e. is there a pentose-phosphate cycle in the ER? 6) what are the consequences of impaired redox control and NADPH homeostasis in the ER for metabolic functions?

By employing biochemical and cell biological methods we attempt to identify additional enzymes within the ER that are dependent on NADPH. Upon identification, expression plasmids will be constructed, followed by expression of proteins in cells, functional characterization and investigation of their physiological roles. Moreover, we will characterize the role of the luminal glucocorticoid activating enzyme in the metabolism of other substrates, including bile acids, oxysterols and xenobiotics, with respect to NADPH dependence.  

The proposed research should significantly enhance the current knowledge on NADPH generation and utilization in the ER. The expected findings are relevant regarding the understanding of the coupling between cellular energy state, hormonal regulation, ER redox regulation, and oxidative stress-induced damage. Disturbed functions of the enzymes investigated are likely to be associated with metabolic diseases.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
11β-Hydroxysteroid dehydrogenase 1: Regeneration of active glucocorticoids is only part of the story.
Odermatt Alex, Klusonova Petra (2015), 11β-Hydroxysteroid dehydrogenase 1: Regeneration of active glucocorticoids is only part of the story., in The Journal of steroid biochemistry and molecular biology, 151, 85-92.
Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.
Tsachaki Maria, Birk Julia, Egert Aurélie, Odermatt Alex (2015), Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions., in Biochimica et biophysica acta, 1853(7), 1672-82.
11β-Hydroxysteroid dehydrogenase-1 is involved in bile acid homeostasis by modulating fatty acid transport protein-5 in the liver of mice.
Penno Carlos A, Morgan Stuart A, Rose Adam J, Herzig Stephan, Lavery Gareth G, Odermatt Alex (2014), 11β-Hydroxysteroid dehydrogenase-1 is involved in bile acid homeostasis by modulating fatty acid transport protein-5 in the liver of mice., in Molecular metabolism, 3(5), 554-564.
Acute effects of 3,4-methylenedioxymethamphetamine and methylphenidate on circulating steroid levels in healthy subjects.
Seibert Julia, Hysek Cédric M, Penno Carlos A, Schmid Yasmin, Kratschmar Denise V, Liechti Matthias E, Odermatt Alex (2014), Acute effects of 3,4-methylenedioxymethamphetamine and methylphenidate on circulating steroid levels in healthy subjects., in Neuroendocrinology, 100(1), 17-25.
Fructose promotes the differentiation of 3T3-L1 adipocytes and accelerates lipid metabolism.
Legeza Balázs, Balázs Zoltán, Odermatt Alex (2014), Fructose promotes the differentiation of 3T3-L1 adipocytes and accelerates lipid metabolism., in FEBS letters, 588(3), 490-496.
Ligand-based pharmacophore modeling and virtual screening for the discovery of novel 17β-hydroxysteroid dehydrogenase 2 inhibitors.
Vuorinen Anna, Engeli Roger, Meyer Arne, Bachmann Fabio, Griesser Ulrich J, Schuster Daniela, Odermatt Alex (2014), Ligand-based pharmacophore modeling and virtual screening for the discovery of novel 17β-hydroxysteroid dehydrogenase 2 inhibitors., in Journal of medicinal chemistry, 57(14), 5995-6007.
Cysteine-10 on 17 β -Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals.
Nashev Lyubomir G, Atanasov Atanas G, Baker Michael E, Odermatt Alex (2013), Cysteine-10 on 17 β -Hydroxysteroid Dehydrogenase 1 Has Stabilizing Interactions in the Cofactor Binding Region and Renders Sensitivity to Sulfhydryl Modifying Chemicals., in International journal of cell biology, 2013, 769536-769536.
Impaired oxidoreduction by 11β-hydroxysteroid dehydrogenase 1 results in the accumulation of 7-oxolithocholic acid.
Penno Carlos A, Morgan Stuart A, Vuorinen Anna, Schuster Daniela, Lavery Gareth G, Odermatt Alex (2013), Impaired oxidoreduction by 11β-hydroxysteroid dehydrogenase 1 results in the accumulation of 7-oxolithocholic acid., in Journal of lipid research, 54(10), 2874-2883.
Synthesis and biological analysis of benzazol-2-yl piperazine sulfonamides as 11β-hydroxysteroid dehydrogenase 1 inhibitors.
Hofer Sandra, Kratschmar Denise V, Schernthanner Brigitte, Vuorinen Anna, Schuster Daniela, Odermatt Alex, Easmon Johnny (2013), Synthesis and biological analysis of benzazol-2-yl piperazine sulfonamides as 11β-hydroxysteroid dehydrogenase 1 inhibitors., in Bioorganic & medicinal chemistry letters, 23(19), 5397-5400.
Synthesis of sterically encumbered 11β-aminoprogesterone derivatives and evaluation as 11β-hydroxysteroid dehydrogenase inhibitors and mineralocorticoid receptor antagonists.
Pandya Keyur, Dietrich David, Seibert Julia, Vederas John C, Odermatt Alex (2013), Synthesis of sterically encumbered 11β-aminoprogesterone derivatives and evaluation as 11β-hydroxysteroid dehydrogenase inhibitors and mineralocorticoid receptor antagonists., in Bioorganic & medicinal chemistry, 21(21), 6274-6281.
The microsomal enzyme 17β-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase.
Legeza Balázs, Balázs Zoltán, Nashev Lyubomir G, Odermatt Alex (2013), The microsomal enzyme 17β-hydroxysteroid dehydrogenase 3 faces the cytoplasm and uses NADPH generated by glucose-6-phosphate dehydrogenase., in Endocrinology, 154(1), 205-13.
Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells.
Chantong Boonrat, Kratschmar Denise V, Nashev Lyubomir G, Balazs Zoltan, Odermatt Alex (2012), Mineralocorticoid and glucocorticoid receptors differentially regulate NF-kappaB activity and pro-inflammatory cytokine production in murine BV-2 microglial cells., in Journal of neuroinflammation, 9, 260-260.

Collaboration

Group / person Country
Types of collaboration
Stephan Krähenbühl/Universität Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Gareth Lavery Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Reto Portmann Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Luigi Terracciano Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Michael Baker/UCSD United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Serge Rudaz, Martial Saugy Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Daniela Schuster/University of Innsbruck Austria (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Karl Fent Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Jörg Huwyler/FHNW Muttenz Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Stephan Herzig, Universität Heidelberg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Armin Wolf, Francois Pognan/iTox Novartis Switzerland (Europe)
- Industry/business/other use-inspired collaboration

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
SCAHT Research Meeting Individual talk Endocrine disruptors and steroid modulation 25.11.2014 Bern, Switzerland Odermatt Alexander;
Fortbildungszyklus Endokrinologie – Diabetologie Individual talk Enzymatic control of local glucocorticoid availability: thoughts on its role in inflammation and metabolic disease 18.11.2014 Bern, Switzerland Odermatt Alexander;
International Symposium Natural Products and Drug Discovery – Future Perspectives Poster Pharmacophore-based virtual screening unveils unexpected off-target effects of natural compounds 14.11.2014 Vienna, Austria Odermatt Alexander;
50th Congress of the European-Societies-of-Toxicology, Edinburgh, 2014 Poster Chemical tuning enhances both potency towards Nrf2 and in vitro therapeutic index of triterpenoids 07.09.2014 Edinburgh, Great Britain and Northern Ireland Odermatt Alexander;
Centre of Cardiovascular Sciences Seminar Individual talk Substrate promiscuity of 11β-hydroxysteroid dehydrogenase 1: there is more than glucocorticoid activation 28.04.2014 Edinburgh, Great Britain and Northern Ireland Odermatt Alexander;
49th Congress of the European-Societies-of-Toxicology, Interlaken 2013 Poster The organotin tributyltin and TNF-alpha induce 17 beta-hydroxysteroid dehydrogenase type 3 expression 01.09.2013 Interlaken, Switzerland Fürstenberger Cornelia; Odermatt Alexander;
49th Congress of the European-Societies-of-Toxicology, Interlaken 2013 Poster Validation of high throughput 2D hepatocyte, micro-patterned co-cultured hepatocyte, and 3D hepatocyte spheroid models for prediction of rodent and human liver toxicity 01.09.2013 Interlaken, Switzerland Odermatt Alexander;
49th Congress of the European-Societies-of-Toxicology, Interlaken 2013 Poster Construction of a mouse MA-10 Leydig cell line stably expressing a HSD17B3 luciferase reporter 01.09.2013 Interlaken, Switzerland Fürstenberger Cornelia; Odermatt Alexander;
49th Congress of the European-Societies-of-Toxicology, Interlaken 2013 Poster Pharmacophore-based virtual screening in the search for endocrine disrupting chemicals-Successful case studies 01.09.2013 Interlaken, Switzerland Odermatt Alexander;
49th Congress of the European-Societies-of-Toxicology, Interlaken 2013 Poster Effects of MDMA and methylphenidate on steroid levels in healthy humans 01.09.2013 Interlaken, Switzerland Seibert Julia; Odermatt Alexander;
Science Seminars Individual talk Intracrine Regulation of Steroid Hormone Action 07.05.2013 Konstanz, Germany Odermatt Alexander;
2nd Congress on Steroid Research Chicago 2013 Poster Quantification of Multiple Bile Acids Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry: Impact of Uninephrectomy on Circulating Bile Acids in Rats 10.03.2013 Chicago, United States of America Odermatt Alexander;
2nd Congress on Steroid Research Chicago 2013 Poster Cytoplasmic Orientation of the Microsomal Enzyme 17β-hydroxysteroid Dehydrogenase Type 3 10.03.2013 Chicago, United States of America Odermatt Alexander;
2nd Congress on Steroid Research Chicago 2013 Poster Mineralocorticoid and Glucocorticoid Receptors Modulate NF-κB Activity and Neuroinflammation in Murine BV-2 Microglial Cells 10.03.2013 Chicago, United States of America Odermatt Alexander;
2nd Congress on Steroid Research Chicago 2013 Poster Virtual Screening as a Strategy for the Identification of Xenobiotics Disrupting Corticosteroid Action 10.03.2013 Chicago, United States of America Odermatt Alexander;
Endocrinology Seminar Series University of Birmingham Individual talk 11β-Hydroxysteroid Dehydrogenase 1 is a multi-functional carbonyl reductase: from cortisone to fungicides 07.12.2012 Birmingham, Great Britain and Northern Ireland Odermatt Alexander;
18th North American Regional ISSX Meeting, Dallas 2012 Talk given at a conference Impact of 11β-hydroxysteroid dehydrogenase 1 on the metabolism of xenobiotics 14.10.2012 Dallas, United States of America Odermatt Alexander;
48th Congress of the European-Societies-of-Toxicology, Stockholm 2012 Poster 11 beta-HSD2 inhibition by the pesticide thiram: The role of cysteine residues 17.06.2012 Stockholm, Sweden Odermatt Alexander;
48th Congress of the European-Societies-of-Toxicology Stockholm 2012 Poster Fluoxymesterone inhibits glucocorticoid inactivation 17.06.2012 Stockholm, Sweden Odermatt Alexander; Fürstenberger Cornelia;


Self-organised

Title Date Place
Seminars in Drug Discovery and Development, 11beta-HSD1: translational and therapeutic aspects 21.05.2014 Basel, Switzerland
Seminars in Drug Discovery and Development, THE KEAP1-NRF2 CELL DEFENCE PATHWAY: PHYSIOLOGICAL, PHARMACOLOGICAL & TOXICOLOGICAL RELEVANCE TO HUMAN HEALTH 02.04.2014 Basel, Switzerland
Seminara in Drug Discovery and Development, B-1 Cell Lymphoma and Leukemia in Mice Lacking the Steroid and Xenobiotic Receptor SXR (NR1I2) 19.03.2014 Basel, Switzerland
Seminars in Drug Discovery and Development, Silencing Myc Transcription with Promoter-Targeting Small Interfering RNA Inhibits Prostate Cancer Stem Cell Maintenance and Tumorigenicity 27.11.2013 Basel, Switzerland
49th Congress of the European Societies of Toxicology, Chair of Session Computational toxicology & Endocrine disruption 02.09.2013 Interlaken, Switzerland
Seminars in Drug Discovery and Development, Old drugs, new tricks: Regulation of cellular homeostasis by the HIV protease inhibitors 10.04.2013 Basel, Switzerland
Translational Medicine Seminar 20.03.2013 Basel, Switzerland
Congress on Steroid Research, Chair of Plenary Session 4: Neurosteroids 10.03.2013 Chicago, United States of America
Seminars in Drug Discovery and Development, Uric acid: a risk factor for renal and cardiovascular disease 07.11.2012 Basel, Switzerland

Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Info Tag Pharmazeutische Wissenschaften German-speaking Switzerland 2014

Awards

Title Year
Annual Research Meeting, Xenobiotic Risk Research, Best Students Presentation by Carlos Penno 2013

Associated projects

Number Title Start Funding scheme
127330 The role of the endoplasmic reticulum in the metabolism of xenobiotics 01.10.2009 ProDoc
159454 Impact of the NADPH pool in the endoplasmic reticulum on metabolic and hormonal regulation 01.05.2015 Project funding (Div. I-III)
124912 The Regulation of Glucocorticoid and Mineralocorticoid Hormone Action by 11beta-Hydroxysteroid Dehydrogenases 01.05.2009 Project funding (Div. I-III)
150760 Studies in the Molecular Sciences Enabled by High Sensitivity NMR 01.12.2013 R'EQUIP

Abstract

Purpose and aim of the projectThe reduced pyridine nucleotide NADPH acts as cofactor for a multitude of essential reduction reactions. The enzymes involved in NADPH synthesis and regeneration, and the metabolic reactions utilizing NADPH in the cytoplasm, have been extensively investigated. In contrast, much less is known on NADPH generation and utilization in the endoplasmic reticulum (ER). The recent recognition of the role of hexose-6-phosphate dehydrogenase (H6PDH) in generating luminal NADPH provided an explanation for the maintenance of the NADP+/NADPH pool in the ER. Biochemical evidence further indicated the existence of all enzymes of the pentose-phosphate pathway in the ER. However, so far only H6PDH has been characterized at the molecular level, and further research is required to elucidate this pathway in the ER. Recent cell-based experiments and studies with transgenic mice lacking H6PDH demonstrated the importance of luminal NADPH for glucocorticoid activation. Moreover, H6PDH-deficient mice suffered from myopathy and activation of the unfolded-protein response (UPR), whereby glucocorticoid-independent impairment of ER redox regulation was suggested as underlying mechanism. H6PDH activity depends on glucose-6-phosphate (G6P) availability. G6P can be provided from the cytoplasm by the G6P-transporter (G6PT) or, alternatively, by a luminal isomerase converting fructose-6-phosphate (F6P) to G6P that we characterized recently. Nevertheless, the gene encoding this enzyme remains to be identified. We have previously shown that H6PDH determines the reaction direction of 11beta-hydroxysteroid dehydrogenase 1 (11b-HSD1) to catalyze the reduction of inactive 11-keto- to active 11b-hydroxyglucocorticoids. Also, we found that 11b-HSD1 metabolizes several other substrates, including oxysterols, bile acids and carbonyl containing xenobiotics. The physiological relevance of these additional functions is poorly understood and requires further investigation.So far, 11b-HSD1 is the only NADPH-dependent enzyme in the ER that has been thoroughly characterized. Evidence from H6PDH-deficient transgenic mice and cell-based experiments emphasizes the existence of other NADPH-dependent luminal enzymes. Therefore, we aim to further characterize the mechanisms of NADPH generation in the ER and to search for enzymes that utilize NADPH in this compartment. We propose to:I.elucidate the mechanisms involved in the regulation of NADPH generation in the ER by:a) an attempt to identify the gene for the luminal hexose-6-phosphate isomerase;b) characterization of the luminal 6-phosphogluconate dehydrogenase (6PGDH) activity;c) trying to identify enzymes other than H6PDH that generate NADPH in the ER; andII.characterize NADPH-dependent reactions in the ER by:a) determination of the impact of 11b-HSD1 on bile acid metabolism and profiles;b) determination of 17b-HSD3 membrane topology and functional impact of H6PDH;c) performing a search for substrate(s) of 11b-HSD3 and elucidating its function; andd) trying to identify additional ER luminal reductases that utilize NADPH.For the identification of luminal enzymes we will use rat liver microsomes and apply an activity-guided purification strategy. A combination of fractionation, affinity purification, gel-electrophoretic separation and identification by mass spectrometry will be applied. Upon identification, expression plasmids will be constructed, followed by functional characterization. The proposed research should significantly enhance the current knowledge on NADPH generation and utilization in the ER. The expected findings are relevant regarding the understanding of the coupling between cellular energy state, hormonal regulation, ER redox regulation, and oxidative stress-induced damage. Disturbed functions of the enzymes investigated are likely to be associated with metabolic disturbances.
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