Altered GLUT4 trafficking in adipocytes in the absence of the GTPase Arfrp1 Deike Hesse a,1 , Angela Hommel a,1 , Alexander Jaschke a , Markus Moser b , Ulrike Bernhardt a , Claudia Zahn a , Reinhart Kluge a , Petra Wittschen c , Achim D. Gruber c , Hadi Al-Hasani a , Hans-Georg Joost a , Annette Schürmann a, * a Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany b Department of Molecular Medicine, Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany c Department of Veterinary Pathology, Freie Universität Berlin, D-14163 Berlin, Germany article info Article history: Received 10 March 2010 Available online 15 March 2010 Keywords: ARF-proteins Glucose transporter GLUT4 Golgi apparatus Golgin proteins abstract The GTPase ADP-ribosylation factor related protein 1 (ARFRP1) controls the recruitment of proteins such as golgin-245 to the trans-Golgi. ARFRP1 is highly expressed in adipose tissues in which the insulin-sen- sitive glucose transporter GLUT4 is processed through the Golgi to a specialized endosomal compartment, the insulin-responsive storage compartment from which it is translocated to the plasma membrane in response to a stimulation of cells by insulin. In order to examine the role of ARFRP1 for GLUT4 targeting, subcellular distribution of GLUT4 was investigated in adipose tissue specific Arfrp1 knockout (Arfrp1 adÀ/À ) mice. Immunohistochemical and ultrastructural studies of brown adipocytes demonstrated an abnormal trans-Golgi in Arfrp1 adÀ/À adipocytes. In addition, in Arfrp1 adÀ/À adipocytes GLUT4 protein accumulated at the plasma membrane rather than being sequestered in an intracellular compartment. A similar missort- ing of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which was associated with significantly elevated uptake of deoxyglucose under basal conditions. Thus, Arfrp1 appears to be involved in sorting of GLUT4. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The GTPase ARFRP1 (ARF-related protein 1) [1] is a member of the family of ADP-ribosylation factors (ARFs) that operate as GTP-dependent molecular switches in the regulation of intracellu- lar protein traffic and in Golgi function [2,3]. ARFRP1 is ubiqui- tously expressed with higher levels in white and brown adipose tissue, liver, kidney, and intestine [1] and is associated with trans-Golgi membranes [4]. ARFRP1 recruits a second GTPase, ARL1, and its effector the GRIP protein golgin-245 to the trans-Gol- gi network [4–6]. Conventional Arfrp1 À/À embryos died during early gastrulation between day 6 and 6.5 [7] due to adhesion de- fects [8]. The adipocyte-specific deletion of Arfrp1 resulted in a lip- odystrophic phenotype due to a defective lipid droplet growth and an elevated lipolysis [9]. We found SNAP23 (synaptosomal-associ- ated protein of 23 kDa) associated with small lipid droplets of con- trol adipocytes as described in the literature [10]. In contrast, SNAP23 was predominantly located in the cytosol and at the cell surface of Arfrp1 adÀ/À adipocytes [9]. SNARE proteins (VAMP2, syntaxin-4, and SNAP23) have been implicated in the insulin-induced translocation of vesicles contain- ing the GLUT4 glucose transporter to the plasma membrane of adi- pocytes [11–16] indicating that GLUT4 translocation follows typical membrane fusion rules. Because SNAP23 distribution was altered in adipocytes of Arfrp1 adÀ/À mice [9] we reasoned that GLUT4 targeting might be modified in adipocytes lacking Arfrp1, thereby allowing further dissection of the different vesicular GLUT4 compartments. Since Arfrp1 adÀ/À mice die early due to the lack of white adipose tissue [9] we studied GLUT4 localization in adipocytes from 18.5 days old Arfrp1 adÀ/À embryos which did not show impaired growth or survival. In addition, we used 3T3-L1 adi- pocytes in which expression of Arfrp1 was depleted by siRNA. 2. Materials and methods 2.1. Arfrp1 adÀ/À mice Generation of Arfrp1 flox/flox mice and the fat cell-specific deletion of Arfrp1 was described previously [8,9]. Animals were housed in a con- trolled environment (20 ± 2 °C, 12 h/12 h light/dark cycle) and had free access to water and standard chow diet. All animal experiments were approved by the ethics committee of the Ministry of Agriculture, Nutrition and Forestry (State of Brandenburg, Germany). 2.2. Antibodies We used the polyclonal antiserum against recombinant GST- ARFRP1 [1,4]. Antiserum against GLUT4 [9] was used for immuno- 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.059 * Corresponding author. Fax: +49 33200 88334. E-mail address: schuermann@dife.de (A. Schürmann). 1 These authors contributed equally to this paper. Biochemical and Biophysical Research Communications 394 (2010) 896–903 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc