ODP443 Targeting NAD+ availability modulates 11β- Hydroxysteroid dehydrogenase 1 activity in mouse and human cell lines and primary cells

Four nicotinamide adenine dinucleotide coenzymes – NAD+, NADH, NADP+, and NADPH – are the central catalysts of metabolism. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is NADPH-dependent reductase that catalyses the reduction of the inactive glucocorticoid cortisone) to active cortisol (11-dehydrocorticosterone to corticosterone in rodents). 11β-HSD1 is in the endoplasmic reticulum (ER) lumen, and the level of NADPH in the ER regulates enzyme activity. Little is known about the biochemical mechanisms controlling cytosolic/ER NAD(P)(H) crosstalk. Here we examined how perturbed cellular NAD+ availability modulates 11β-HSD1 enzyme activity in the ER across a range of mouse and human cell types. 11β-HSD1 activity was measured in a series of transformed and primary mouse and human liver (HepG2), muscle, and Human Dermal Fibroblast cells (HDFn) depleted for NAD+ using FK866 to inhibit nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. To replete NAD+ we supplemented cells with 0.5mM of NAD+ precursors nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). FK866 (100nM, 48h) was effective at depleting NAD+ by approximately 90% in C2C12 myotubes and 50% in HepG2 liver cells. In each cell line, 11β-HSD1 reductase activity was decreased by FK866 compared to untreated controls (80% in C2C12 P<0. 0001, 55% in HepG2, P=0. 03 and 65% in HDFn P=0. 002). Restoring NAD+ level with NR (0.5mM, 6h) in C2C12 and (0.5mM, 24h) in HepG2 and HDFn rescued 11β-HSD1 enzyme activity compared with no precursor treatment (60% in C2C12 P=0. 0007, 52% in HepG2 P=0. 03 and 68% in HDFn P=0. 0006). Human dermal fibroblasts treated with TNFa (10nM, 24h) boosted 11β-HSD1 expression 4-fold, however this was not able to overcome the FK866 induced drop-in reductase activity. These data show that depletion of NAD+ and impaired 11β-HSD1 reductase activity in the ER is a generalised phenomenon across cell types. This suggests a crosstalk between cytosolic and ER NAD(P)(H) pools that can respond rapidly to changes in NAD+ precursor availability. This could provide new insight into the regulation of cellular glucocorticoid metabolism and ER NAD(P)(H) homeostasis. Presentation: No date and time listed