Constant expression of hexose-6-phosphate dehydrogenase during differentiation of human adipose-derived mesenchymal stem cells Silvia Senesi 1 *, Paola Marcolongo 1 *, Ivana Manini 2 , Rosella Fulceri 1 , Vincenzo Sorrentino 2 , Miklo ´ s Csala 3,4 , Ga ´bor Ba ´ nhegyi 1,3,4 and Angelo Benedetti 1 1 Department of Pathophysiology, Experimental Medicine and Public Health, University of Siena, Via A. Moro no. 1, 53100 Siena, Italy 2 Molecular Medicine Section, Department of Neuroscience and Center for Stem Cell Research, University of Siena, Siena, Italy 3 Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary 4 Pathobiochemistry Research Group of the Hungarian Academy of Sciences, Budapest, Hungary (Correspondence should be addressed to A Benedetti; Email: benedetti@unisi.it) *S Senesi and P Marcolongo contributed equally to this work Abstract The reductase activity of 11b-hydroxysteroid dehydrogenase type 1 (HSD11B1) plays an important role in the growth and differentiation of adipose tissue via the prereceptorial activation of glucocorticoids. This enzyme colocalizes with hexose-6- phosphate dehydrogenase (H6PD) at the luminal surface of the endoplasmic reticulum membrane, and the latter enzyme provides NADPH to the former, which can thus act as an 11b-reductase. It was suggested that, during adipogenesis, the increased expression of H6PD causes a dehydrogenase-to-reductase switch in the activity of HSD11B1. However, only the expression of the HSD11B1 has been extensively studied, and little is known about the expression of H6PD. Here, we investigated the expression and the activity of H6PD in the course of the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) and murine 3T3-L1 cells. It was found that H6PD is already present in adipose-derived stem cells and in 3T3-L1 fibroblasts even before the induction of adipogenesis. Moreover, mRNA and protein levels, as well as the microsomal H6PD activities remained unchanged during the differentiation. At the same time a great induction of HSD11B1 was observed in both cell types. The observed constant expression of H6PD suggests that HSD11B1 acts as a reductase throughout the adipogenesis process in human ADMSCs and murine 3T3-L1 cells. Journal of Molecular Endocrinology (2008) 41, 125–133 Introduction Hexose-6-phosphate dehydrogenase (H6PD) catalyzes the first two reactions of the pentose phosphate pathway in the endoplasmic reticulum (ER), thereby generating reduced NAD (NADPH) within the luminal compart- ment (Buetler & Morrison 1967, Takahashi & Hori 1978, Ozols 1993, Collard et al. 1999, Mason et al. 1999, Clarke & Mason 2003). It uses a separate pool of glucose-6- phosphate and NADP C distinct from the cytosolic ones (Ba ´nhegyi et al. 2004, Czegle et al. 2005). It has been proposed that the luminal [NADPH]/[NADP C ] ratio determines the direction of the activity of the ER luminal enzyme 11b-hydroxysteroid dehydrogenase type 1 (HSD11B1; Atanasov et al. 2004, Hewitt et al. 2004, Odermatt et al. 2006). Indeed, HSD11B1 in vitro catalyzes the reversible conversion between (inactive) 11-ketoglucocorticoids and (active) 11-hydroxygluco- corticoids, although in intact cells acts predominately as a reductase consuming NADPH and activating glucocorticoids. The local activation of glucocorticoids could have various pathophysiological roles, such as stimulation of adipogenesis (Seckl & Walker 2004, Tomlinson et al. 2004, Draper & Stewart 2005). An enhanced lipogen- esis, for example, is considered to be key event in abdominal obesity and metabolic syndrome (Tomlinson & Stewart 2002, Seckl et al. 2004, Andrew et al. 2005). An important role in adipogenesis has been attributed to H6PD (Atanasov et al. 2004, Hewitt et al. 2004, Odermatt et al. 2006). It was demonstrated that mutations in genes of both HSD11B1 and H6PD in a triallelic digenic model of inheritance result in the loss of HSD11B1 reductase activity (Draper et al. 2003). During the differentiation of human omental adipose stromal cells, a switch was observed in HSD11B1 activity from dehydrogenase to reductase direction (Bujalska et al. 2002). Later an increased H6PD expression was reported during adipocyte differentiation and it was associated with the previously described switch in HSD11B1 activity (Atanasov et al. 2004). A transient mild increase in 125 Journal of Molecular Endocrinology (2008) 41, 125–133 DOI: 10.1677/JME-08-0028 0952–5041/08/041–125 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org