For control experiments, cells were electroporated with 20 nmol scrambled siRNA (5-GACUGUACGUGAUAGAUUGUU-3). in brown adipose tissue fromArfrp1ad/mice. Accordingly, basal lipolysis was increased after knockdown ofArfrp1in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of Ginsenoside Rb2 lipolysis. The GTPase ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) (34) is a member of the family of ARFs that operate as GTP-dependent molecular switches in the regulation of intracellular protein traffic and in Golgi function (18,19). ARFRP1 in 3T3-L1 Ginsenoside Rb2 adipocytes was identified in a PCR-based screen of differentially expressed transcripts. In addition, it is expressed in brown adipose tissue, liver, kidney, intestine, and lung (34). GTP-bound ARFRP1 specifically binds the guanine nucleotide exchange factor of ARF1, mSec7-1/cytohesin, and inhibits ARF-controlled pathways (35). ARFRP1 is associated withtrans-Golgi membranes (47) and CASP8 controls the recruitment of proteins such as ARL1 (ARF-like 1) and its effector golgin-245 totrans-Golgi membranes (31,39,47). Due to defective E-cadherin targeting and adhesion defects, conventionalArfrp1/embryos die during early gastrulation (27,48). Adipose tissue plays an important role for energy homeostasis. Adipocytes of brown adipose tissue (BAT) generate heat via mitochondrial uncoupling of lipid oxidation (37). White adipose tissue (WAT) is the major organ for regulated storage of triglycerides, which are used as metabolic energy to be mobilized during fasting and extended exercise (8,41). Triglycerides are stored in lipid droplets (LDs) formed at membranes of the endoplasmic reticulum (ER), and their assembly depends on the rate of triglyceride synthesis (24). In addition, droplets increase in size by fusion which is catalyzed by the SNAREs, SNAP23, syntaxin-5, and VAMP4 (6,21,29). LDs are surrounded by a phospholipid monolayer which binds a number of proteins, particularly the PAT family proteinsperilipin,ADRP (adipocyte differentiation-related protein [adipophilin]), andTIP47 (tail-interacting protein of 47 kDa) and are recognized as dynamic organelles (3,7,9,29,32). In addition, proteins involved in sorting and trafficking events in the cell and interacting with other organelles also associate with LDs (22,29,30). The high expression of ARFRP1 Ginsenoside Rb2 in adipose tissue suggested that the GTPase plays an important role in the regulation of adipocyte-specific processes. Thus, in order to define this role, we generated and characterized mice with a fat cell-specific deletion ofArfrp1. We found that triglyceride storage was almost abolished, LDs were smaller, and lipolysis was enhanced. Furthermore, ultrastructural analysis indicated that ARFRP1 controls the interaction of small lipid-loaded particles with storage LDs. == MATERIALS AND METHODS == == Generation ofArfrp1ad/mice. == For tissue-specific disruption ofArfrp1(Arfrp1ad/mice),Arfrp1flox/floxmice (48) were intercrossed with transgenic mice expressing the Cre recombinase under the control of theFabp4/aP2promoter/enhancer (aP2-Cre) (17). PCR-based genotyping ofArfrp1ad/mice was performed with the following primers:aP2-Cre, 5-TCTCACGTACTGACGGTGG-3 and 5-ACCAGCTTGCATGATCTCC-3; for the upstream 5loxPsite, 5-CAGGGTCAGGGATTTTAACAG-3; and for the downstream 3loxPsite, 5-GAAAGCAACTTGGGAACCTG-3. Deletion of exons 2 to 4 ofArfrp1was verified as described previously (48). Ginsenoside Rb2 The animals were housed in a controlled environment (20 2C, 12 h/12 h of a 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). == Antibodies. == We used the polyclonal antiserum against recombinant GST-ARFRP1 as described previously (32,44). For Western blot analysis of adiponectin, we used polyclonal antiadiponectin antibody (ab3455; Abcam) in a dilution of 1 1:500. Polyclonal antiserum against GLUT4 was described previously (32) and used for immunohistochemistry in a dilution of 1 1:1,000. Polyclonal antiserum against FATP1 was described previously (43) and used in a dilution of 1 1:800. Anti-UCP1 antiserum (Abcam, Cambridge, United Kingdom) was used in a dilution of 1 1:1,000. Anti-SNAP23 was purchased from Abcam and used in a dilution of 1 Ginsenoside Rb2 1:500 for immunohistochemistry and for Western blotting in a dilution of 1 1:1,000. Antisera against perilipin, ADRP (Progen Biotechnik GmbH, Wieblingen, Germany), and TIP47 (AnaSpec, San Jose, CA) were used for immunohistochemistry in a dilution of 1 1:5,000, 1:250, and 1:100, respectively, and for Western blotting in a dilution of 1 1:2,000 with perilipin. The anti-HSL and anti-pHSL antibodies were purchased from Cell Signaling (Boston, MA) and used in a dilution of 1 1:1,000 for Western blotting and of 1 1:800 for cytochemistry. Anti-Cav1 antiserum (Serotec, Oxford, United Kingdom).