br Acknowledgments br Introduction Within the classical secr

Acknowledgments
Introduction Within the classical secretory pathway, transmembrane and soluble cargos travel via the endoplasmic reticulum (ER) and Golgi apparatus en route to their final destinations [1]. However, at a post-Golgi level, trafficking routes can diverge. In this context, the trans-Golgi network (TGN) is conventionally regarded as the main cargo sorting station; together with a broad range of accessory proteins, the TGN regulates accurate packaging of cargo into the right transport vesicle and delivery along correct trafficking routes. The assembly of cargo-laden vesicles at the TGN is initiated by the ARF family of small GTPases consisting of ARF, SAR (secretion-associated and Ras-related), and ARL (ARF-like) proteins [2]. Mammalian 4-P-PDOT sale possess 6 ARF proteins and more than 20 ARF-like proteins, whose intracellular roles are poorly understood [3]. Among them, ARFRP1 (ADP-ribosylation factor-related protein 1), which localizes in the activated GTP-bound form to the TGN, has been functionally implicated in vesicle trafficking of VSVG (vesicular stomatitis virus G) [4,5], E-cadherin [6], Vangl2 (vang-like 2) [7], and the glucose transporters GLUT4 [8] and GLUT2 [9]. Furthermore, conditional adipocyte-specific deletion of Arfrp1 resulted in a severe lipodystrophic phenotype of newborn mice underlining the pivotal role of ARFRP1 for the development of functional adipose tissue depots [10]. To explore the impact of ARFRP1 as part of the vesicle trafficking machinery on the secretory capacity of mature adipocytes, we generated a mouse model with an inducible fat-specific disruption of the Arfrp1 gene (Arfrp1). Here, we demonstrate that the loss of Arfrp1 from differentiated adipocytes diminishes adiponectin secretion and plasma membrane localization of the insulin receptor associated with detrimental effects on adipocyte metabolism and glucose homeostasis. These findings were attributed to defective endosomal-mediated exocytosis that was monitored in vitro following Arfrp1 suppression.
Material and methods
Results
Discussion In the current study, we have identified adiponectin secretion and insulin receptor surface targeting to largely rely on the action of functional ARFRP1 at the trans-Golgi in mature adipocytes. Both cargo molecules utilize trans-endosomal routes for cell surface delivery resulting in either secretion (adiponectin) or plasma membrane exposure (insulin receptor). Mice with an inducible deletion of Arfrp1 in adipocytes reveal significantly less adiponectin release and insulin receptor surface localization causing hepatic insulin resistance and impaired glucose homeostasis as well as reduced adipose insulin signaling promoting lipolytic activity and inhibiting adipose tissue expansion. Adiponectin, a classically secreted adipokine depending on proper ER/Golgi function, has been reported to exit the cell via endosomal-mediated secretion pathways [26]. Initially, folding and assembly of adiponectin into higher-order complexes (trimer, hexamer, high-molecular weight adiponectin) involves a complex set of regulatory steps in the ER and Golgi apparatus [17,27–29]. Once adiponectin reaches the TGN, a pool of adiponectin molecules is packaged into GGA1 (Golgi-localized, gamma adaptin ear containing, ARF-binding protein 1)-coated vesicles for further delivery to endosomes [30]. GGA1 is a monomeric coat adapter known to be activated at the trans-Golgi network where it mediates the sorting of selected cargo-containing transport vesicles from the TGN to endosomes [31]. In agreement with that, cellular adiponectin has been shown to partially co-localize with Rab11, a marker of recycling endosomes as well as with Rab5 and EEA1 (early endosome antigen 1) representing early endosomes [18,32]. Based on assays employing the transferrin receptor (TfR) and its ligand transferrin (Tf), which have been widely used to trace endocytic and recycling processes, we demonstrated in the current study ARFRP1 to be critically involved in the process of endosomal-mediated exocytosis (Figure 3C,D). Consistently, adipocyte-specific ablation of Arfrp1 in mice (Figure 2A,B) or 3T3-L1 cells (Figure 2C,D) promoted an endosomal enrichment of adiponectin, showing increased co-localization with Rab11-positive recycling endosomes when Arfrp1 was depleted in 3T3-L1 adipocytes (Figure 2E,F). In accordance with accumulating adiponectin in fat cells, the endosomal transit of the Tf–TfR complex was impaired in Arfrp1-deficient HeLa cells as indicated by accumulation of Tf–TfR at the cell periphery in these cells (Figure 3A,B). From these data, we conclude that the loss of Arfrp1 in adipocytes selectively impairs secretion of adiponectin via trans-endosomal routes. Further supportive of this assumption is the finding that a fraction of intracellular adiponectin overlaps with TfR-positive membranes in 3T3-L1 adipocytes, implying that adiponectin release occurs via the TfR-positive endosomal compartment [26]. Beside this, adiponectin was also described to exit the cell constitutively [18]. However, this route of secretion is principally intact in Arfrp1-depleted cells (Figure S5) and might explain the residual adiponectin that could be detected in plasma and fat explant supernatant of Arfrp1 mice.