Molecular and Cellular Endocrinology 301 (2009) 137–141 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce 11-Hydroxysteroid dehydrogenase 1 reductase activity is dependent on a high ratio of NADPH/NADP + and is stimulated by extracellular glucose Anna A. Dzyakanchuk a , Zoltán Balázs a , Lyubomir G. Nashev a , Kurt E. Amrein b , Alex Odermatt a,∗ a Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland b F. Hoffmann-La Roche Ltd., Vascular and Metabolic Diseases, Basel, Switzerland article info Article history: Received 27 June 2008 Received in revised form 11 August 2008 Accepted 12 August 2008 Keywords: 11-Hydroxysteroid dehydrogenase Glucocorticoid Glucose Glucose-6-phosphate Hexose-6-phosphate dehydrogenase NADPH abstract To assess the impact of the NADPH/NADP + ratio and the influence of extracellular glucose on 11- hydroxysteroid dehydrogenase 1 (11-HSD1) activity, we applied microsomal preparations and intact HEK-293 cells expressing 11-HSD1 in the presence or absence of hexose-6-phosphate dehydroge- nase (H6PDH). A NADPH/NADP + ratio of ten or higher was required for efficient microsomal 11-HSD1 reductase activity. Measurements in intact cells suggested that the ER-luminal NADPH concentration is highly sensitive to fluctuating extracellular glucose levels. Lowering glucose in the culture medium dose-dependently decreased 11-HSD1 reductase activity and diminished the cortisol/cortisone ratio measured after 24h of incubation. Coexpression with H6PDH potentiated 11-HSD1 reductase activity at high glucose. This effect was significantly decreased at low glucose, with concomitantly increased 11-HSD1 dehydrogenase activity. In contrast, 11-HSD1 reductase activity in H4IIE liver cells and in 3T3-L1 adipocytes was less sensitive to changes in the medium. 11-HSD1 dehydrogenase activity was observed in H4IIE cells only at subphysiological glucose levels, indicating a highly efficient supply of sub- strate for H6PDH and NADPH generation in the ER-lumen. Our results suggest significant cell type-specific differences in ER-luminal NADPH generation that might allow a fine-tuned regulation of glucocorticoid action. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Tissue-specific intracellular activation of glucocorticoids is catalyzed by 11-hydroxysteroid dehydrogenase 1 (11-HSD1), converting cortisone to cortisol (or 11-dehydrocorticosterone to corticosterone in rodents). 11-HSD1 is highly expressed in liver and white adipose tissue but also in skeletal muscles, certain regions of the brain and in macrophages. 11-HSD1 is anchored in the endoplasmic reticulum (ER) membrane, with its catalytic site facing the lumen (Frick et al., 2004; Odermatt et al., 1999). Recently, it has been suggested that H6PDH-mediated NADPH generation in the ER-lumen (which is characterized by an oxidative environment with an estimated ratio of oxidized to reduced glutathione of 1:1-3 (Braakman et al., 1992), is a key regulatory factor of tissue-specific glucocorticoid activation Abbreviations: 11-HSD, 11-hydroxysteroid dehydrogenase; G6P, glucose- 6-phophate; G6PT, endoplasmic reticulum glucose-6-phophate transporter; ER, endoplasmic reticulum; H6PDH, hexose-6-phosphate dehydrogenase; TLC, thin layer chromatography. ∗ Corresponding author. Tel.: +41 61 267 1530; fax: +41 61 267 1515. E-mail address: alex.odermatt@unibas.ch (A. Odermatt). (Atanasov et al., 2004; Banhegyi et al., 2004; Bujalska et al., 2005). H6PDH has been shown to physically interact with 11-HSD1 in the ER-lumen, allowing efficient supply of cosubstrate NADPH to 11-HSD1 (Atanasov et al., 2008). A known substrate for H6PDH is glucose-6-phosphate (G6P), transported from the cytoplasm into the ER-lumen via the membrane-bound G6P tansporter (G6PT). The latter protein is thought to colocalize with G6Pase-, the enzyme catalyzing the terminal step of gluconeogenesis and glycogenoly- sis and generating glucose from G6P (Chou et al., 2002). Thus, both glucose catabolism as well as anabolism seem to be tightly linked to intracellular glucocorticoid activation through the availability of G6P. Indeed, glucocorticoids induce a state of insulin resistance, leading to reduced suppression of glucose production and periph- eral glucose uptake (Rizza et al., 1982). In vivo, glucocorticoids can cause both impaired insulin-dependent glucose uptake in the periphery and enhanced gluconeogenesis in the liver (Andrews and Walker, 1999; Rizza et al., 1982; Rooney et al., 1993). Transgenic mice lacking a functional 11-HSD1 are resistant to high fat diet-induced obesity (Kotelevtsev et al., 1997). In con- trast, animals specifically overexpressing 11-HSD1 in adipose tissue develop visceral obesity, insulin resistance and hyperten- sion (Masuzaki et al., 2001), and hepatic 11-HSD1 overexpression results in fatty liver, insulin-resistance and hypertension (Paterson 0303-7207/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2008.08.009