SFEBES2022 Poster Presentations Adrenal and Cardiovascular (66 abstracts)
Depleting NAD+ pools specifically in the endoplasmic reticulum lumen impairs 11β-hydroxysteroid dehydrogenase activity
Ali Kabli 1 , Silke Heising 1 , Samuel Heaselgrave 1 , Yasir Elhassan 1 , Rowan Hardy 1 , Oyvind Stromland 2 , Mathias Ziegler 2 , Stuart Morgan 1 , David Hodson 1,3 & Gareth Lavery 1,4
1University of Birmingham, Birmingham, United Kingdom; 2University of Bergen, Bergen, Norway; 3University of Oxford, Oxford, United Kingdom; 4University of Nottingham, Nottingham, United Kingdom
Introduction: The endoplasmic reticulum (ER) lumen enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) obtains NADPH from hexose-6-phosphate dehydrogenase to reduce cortisone to the active glucocorticoid cortisol. Cells depleted in NAD+ (parent molecule of NADPH) have impaired 11β -HSD1 activity, which can be rapidly rescued with supplementation of the NAD+ precursor nicotinamide riboside. This suggests the existence of an ER-specific pathway to NAD(P)(H). Here we begin to use the poly-ADP-ribose assisted protein localization assay to study ER-specific NAD+ metabolism using 11β-HSD1 as a readout.
Methods: HepG2 human liver cells and C2C12 mouse muscle cells were transfected with ER targeted constructs containing the catalytic unit of poly-ADP-ribose polymerase 1 (ER-PARP1-EGFP which utilises NAD+ to produce a polymer of poly-ADP-ribose (PAR-ylation) in a NAD+ concentration dependent manner), and control ER-EGFP only (all experiments n=3, in triplicate). 48 hrs after transfection cells were analysed by fluorescence microscopy for target localisation, PAR-ylation levels assessed by western blotting, and 11β-HSD1 activity measured by assaying cortisone to cortisol conversion.
Results: Fluorescence microscopy for eGFP confirmed successful targeting of PARP1 to the ER with minimal effect on cell viability. Western blotting confirmed elevated ER PAR-ylation which was not seen in EGFP only controls, suggesting increased rates of NAD+ depletion. In HepG2 and C2C12 cells 11β-HSD1 activity was lower by 12±2% and 10±2% respectively in ER-PARP1-EGFP cells compared to ER-EGFP control cells which was not a result of altered expression of the 11β-HSD1 enzyme.
Conclusion: ER NAD(P)(H) availability and maintenance impacts upon redox sensitive enzymes is poorly understood. Here we show preliminary evidence that depleting ER NAD+ through targeted PARP1 activity can impair NADPH dependent 11β-HSD1 activity. Further experiments are fully evaluating the potential of a dynamic NAD(P)(H) pathway in the ER compartment.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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