Presence of a Functional Receptor for GLP-1 in Osteoblastic Cells, Independent of the cAMP-Linked GLP-1 Receptor BERNARDO NUCHE-BERENGUER, 1 SERGIO PORTAL-NU ´ N ˜ EZ, 2 PAOLA MORENO, 1 NIEVES GONZA ´ LEZ, 1 ALICIA ACITORES, 1 ANA LO ´ PEZ-HERRADO ´ N, 2 PEDRO ESBRIT, 2 ISABEL VALVERDE, 1 AND MARI ´ A L. VILLANUEVA-PEN ˜ ACARRILLO 1 * 1 Department of Metabolism, Nutrition & Hormones, Fundacio´n Jime´nez Dı ´az, Madrid, Spain 2 Bone & Mineral Metabolism Laboratory, Fundacio´n Jime´nez Dı ´az, Madrid, Spain Glucagon-like peptide 1 (GLP-1) controls glucose metabolism in extrapancreatic tissues through receptors other than the pancreatic cAMP-linked GLP-1 receptor; also, GLP-1 induces an insulin- and PTH-independent bone anabolic action in insulin-resistant and type-2 diabetic rats. Here we searched for the presence and characteristics of GLP-1 receptors in osteoblastic MC3T3-E1 cells. [ 125 I]-GLP-1 specific binding to MC3T3-E1 cells was time- and temperature-dependent, reaching maximal value at 30 min at 258C; in these conditions, [ 125 I]-GLP-1 binding was dissociable, and displaced by GLP-1, partially by GLP-2, but not by exendin-4 (Ex-4), exendin-9 (Ex-9), glucagon or insulin; Scatchard analysis of the unlabeled GLP-1 data showed high and low affinity binding sites; cross-linking of GLP-1 binding revealed an estimated 70 kDa band, almost undetectable in the presence of 10 6 M GLP-1. GLP-1, Ex-9, insulin or glucagon failed to modify cellular cAMP content, while GLP-2 and Ex-4 increased it. However, GLP-1 induced an immediate hydrolysis of glycosylphosphatidylinositols (GPIs) generating short-lived inositolphosphoglycans (IPGs), and an increase in phosphatidylinositol-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) activities; Ex-4 also affected GPIs, but its action was delayed with respect to that of GLP-1. This incretin was found to decrease Runx2 but increased osteocalcin gene expression, without affecting that of osteoprotegerin or the canonical Wnt pathway activity in MC3T3-E1 cells which do not express the pancreatic GLP-1 receptor. Our data demonstrate for the first time that GLP-1 can directly and functionally interact with osteoblastic cells, possibly through a GPI/IPG-coupled receptor. J. Cell. Physiol. 225: 585–592, 2010. ß 2010 Wiley-Liss, Inc. Parenteral feeding is associated with reduced bone mass, suggesting that hormones released after nutrients absorption — as it is the case of incretins — could have a role on bone turnover (Henriksen et al., 2003; Clowes et al., 2005). In fact, it has been demonstrated not only the presence of glucose-dependent insulinotropic (GIP peptide) receptor, coupled to the adenylate cyclase-cAMP system, in bone and osteoblastic cell lines (Bollag et al., 2000), but also that mice overexpressing GIP have an increased bone mass (Xie et al., 2007). With respect to the other well known incretin, glucagon-like peptide 1 (GLP-1)—a glucose-dependent insulinotropic hormone that has insulin-independent antidiabetic properties (Creutzfeldt, 2001)—it has recently been demonstrated its insulin-independent bone anabolic action—by continuous infusion of the peptide for 3 days—in both insulin-resistant and type 2 diabetic rat models, in which GLP-1 exerts a normalizing effect on their impaired bone structure (Nuche-Berenguer et al., 2009). Yet, the mechanisms by which GLP-1 is acting on bone turnover are unknown. A previous study showed that genetic disruption of the pancreatic GLP-1 receptor in mice results in an increased bone resorption associated with decreased thyroid calcitonin expression (Yamada et al., 2008); however, the possibility that GLP-1 might act directly on bone cells through this receptor or other uncharacterized receptors is not conclusive from that data. GLP-1 has direct actions upon glucose and lipid metabolism in extrapancreatic tissues such as liver, skeletal muscle and fat (Valverde et al., 1994; Villanueva-Pen ˜acarrillo et al., 1994; Sancho et al., 2005), which are exerted through specific receptors (Valverde et al., 1993; Delgado et al., 1995; Villanueva-Pen ˜acarrillo et al., 1995). However, the GLP-1 receptor, at least in liver and muscle, seems to be different in structure and/or function to that reported in the pancreas (Thorens, 1992), as GLP-1 does not promote cAMP formation in these tissues or cells (Delgado et al., 1995; Villanueva- Pen ˜acarrillo et al., 1995; Yang et al., 1998; Nishizawa et al., 2000); instead, it induces inositolphosphoglycan (IPG) generation (Ma ´rquez et al., 1998; Luque et al., 2002). Moreover, in rats, GLP-1, by prolonged treatment, exerts a control on GLUT-2 and GLUT-4 expression in normal as well as in type 2 and type 1 diabetic states (Villanueva-Pen ˜ acarrillo et al., 2001). No previous attempts have currently been made to characterize the presence of putative GLP-1 receptors or their signaling in bone cells. In the present study, we have searched for the presence and characteristics of GLP-1 receptors in a well characterized osteoblastic cell line. In an attempt to evaluate the function of these receptors, we also assessed in these cells the possible interaction of GLP-1 with osteoblastic genes which are involved Contract grant sponsor: Instituto de Salud Carlos III and Ministerio de Ciencia e Innovacio ´n; Contract grant numbers: PI 060076, PI 080922, RETICEF (RD06/ 0013/1002), CIBERDEM (CP08/00158) *Correspondence to: Marı ´a L. Villanueva-Pen ˜acarrillo, Department of Metabolism, Nutrition & Hormones, IIS-Fundacio ´ n Jime ´nez Dı ´az, Avda. Reyes Cato ´ licos 2, 28040 Madrid, Spain. E-mail: mlvillanueva@fjd.es Received 9 April 2010; Accepted 7 May 2010 Published online in Wiley Online Library (wileyonlinelibrary.com.), 19 May 2010. DOI: 10.1002/jcp.22243 ORIGINAL ARTICLE 585 Journal of Journal of Cellular Physiology Cellular Physiology ß 2010 WILEY-LISS, INC.