Diabetes Volume 68, February 2019 377 WASH Regulates Glucose Homeostasis by Facilitating Glut2 Receptor Recycling in Pancreatic b-Cells Li Ding, Lingling Han, John Dube, and Daniel D. Billadeau Diabetes 2019;68:377–386 | https://doi.org/10.2337/db18-0189 WASH is an endosomal protein belonging to the Wiskott- family 2 (SLC2A) genes (8). Glut2 is well established as the Aldrich syndrome protein superfamily that participates in principal membrane Glut with low affinity in rodent endosomal receptor trafficking by facilitating tubule fis- pancreatic b-cells (9,10), and previous studies using sion via activation of the ubiquitously expressed Arp2/3 a transgenic mouse model showed that Glut2-null mice complex. While several studies have begun to elucidate generated by homologous recombination provoked severe an understanding of the functions of WASH in cells lines, glycosuria and died at around the weaning period with the in vivo function of WASH has not been fully eluci- a diabetic phenotype (11). Importantly, pancreatic-specific dated, since total body deletion in mice leads to early expression of Glut2 in Glut2-null mice restored normal embryonic lethality. To circumvent this problem, we have glucose-stimulated insulin secretion (GSIS) and glucose- PATHOPHYSIOLOGY used a WASH conditional knockout mouse model to stimulated insulin biosynthesis (12). In addition, Glut2 investigate the role of WASH in the pancreas. We find protein levels in pancreatic islets are strongly reduced that pancreas-specific deletion of WASH leads to im- with loss of GSIS in numerous animal models of diabe- paired blood glucose clearance and reduced insulin re- tes (13–17). Although the mechanism for Glut2 protein lease upon glucose stimulation. Furthermore, WASH fi depletion results in impaired trafficking of Glut2 in pan- expression (18), posttranscriptional modi cation (19), fi creatic b-cells as a consequence of an intracellular in vitro traf cking (20), and in vivo subcellular trans- fi accumulation of Glut2 and overall decreased levels of location (21) has been identi ed, the in vivo regulation Glut2 protein. Taken together, these results indicate that of Glut2 in pancreatic islets is still unclear. WASH participates in pancreatic b-cell glucose sensing WASH (Wiskott-Aldrich syndrome protein and SCAR and whole-body glucose homeostasis. Thus, patients homolog) (22) is a member of the Wiskott-Aldrich syn- harboring mutations in components of the WASH com- drome protein (WASP) family that promotes branched plex could be at risk for developing type 2 diabetes. F-actin generation through activation of the Arp2/3 com- plex (23). WASH forms a multiprotein complex with FAM21, SWIP, strumpellin, and CCDC53 that is targeted Diabetes is a term used to describe a metabolic disorder of to endosomes through an interaction of FAM21 with multiple etiology characterized by high blood glucose levels VPS35, a component of the endosomal coat complex resulting from insulin secretion defects (type 1 diabetes), known as the retromer (24–26). Several in vitro studies insulin action failure (type 2 diabetes), or both (1–5). have demonstrated an important role for WASH in the Insulin release involves a sequence of well-controlled recycling of plasma membrane receptors through the events in b-cells that start from environmental stimula- endosomal system in a manner dependent on the gener- tions (sensing) and end with releasing of secretory gran- ation of branched F-actin by the Arp2/3 complex. These ules containing insulin (action). Glucose is known to be include, for example, receptors such as integrins, growth the strongest stimulator for insulin release in pancre- factor receptors, lipid transporters, and solute carriers atic b-cells (6,7). There are 14 facilitative diffusion glu- (27–31). The mechanism by which this diverse cadre of cose transporters (Glut) encoded by the solute carrier receptors is trafficked into WASH-dependent sorting Division of Oncology Research and Schulze Center for Novel Therapeutics, Mayo L.D. and L.H. contributed equally to this work. Clinic, Rochester, MN © 2018 by the American Diabetes Association. Readers may use this article as Corresponding author: Daniel D. Billadeau, [email protected] long as the work is properly cited, the use is educational and not for profit, and the Received 12 February 2018 and accepted 31 October 2018 work is not altered. More information is available at http://www.diabetesjournals .org/content/license. This article contains Supplementary Data online at http://diabetes .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0189/-/DC1. 378 The Role of WASH in Glucose Homeostasis Diabetes Volume 68, February 2019 domains depends on their interaction with sorting nexin ImmunoResearch Laboratories) were used as secondary anti- 27 (SNX27), which binds to the cytoplasmic tails of bodies at a 1:300 dilution. receptors via its postsynaptic density 95/discs large/zonus occludens-1 (PDZ) domain and directly couples to the Animals and Animal Care WASHflox/flox Pdx1 retromer subunit VPS26 and the WASH complex member and -Cre mice have previously been fi FAM21 (31) or via an interaction of SNX17-receptor described (28,33). Pancreas-speci cWASHcKOmice WASHflox/flox complexes with the retriever (32). were generated by crossing mice with Pdx1 Pdx1 WASHflox/flox We recently showed that patients with mutations -Cre mice to produce -Cre; animals. WASHflox/flox in CCDC22 fail to appropriately trafficLDLRand These animals were crossed with mice. Unless WASHflox/flox fi ATP7A resulting in substantially elevated levels of serum otherwise indicated, mice are classi ed as WT Pdx1 WASHflox/flox fi cholesterol/LDL and copper, respectively. Significantly, mice and -Cre; mice are classi ed as cKO. patients with mutations in strumpellin were also found Control experiments were performed using littermate WASHflox/flox – to have high levels of circulating cholesterol and LDL (29). WT animals. Mice were housed in a 12 h Thus, defective trafficking of receptors through WASH 12 h light-dark cycle barrier facility. All procedures were endosomal sorting domains can have a physiological im- approved by the Mayo Clinic Institutional Animal Care and pact beyond the intellectual disability associated with Use Committee. mutations in CCDC22 and strumpellin. Using our pre- Islet Isolation viously described WASH conditional knockout (cKO) mice Islet isolation was performed following an established (30), we asked whether WASH might be involved in pan- protocol (34). Briefly, islets were isolated by intraductal fi creas development or function using a pancreas-speci c collagenase (Sigma-Aldrich) perfusion and digestion. Islets Cre mouse model. Interestingly, WASH deletion did not were handpicked using dithizone (Sigma-Aldrich) detec- affect body weight, fasting blood glucose, or pancreas tion of zinc granules. After isolation, islets were placed in tissue development compared with wild-type (WT) ani- RPMI plus 10% FBS and cultured at 37°C and 5% CO2 for mals. However, WASH cKO mice showed decreased insulin future experiments. release and delayed glucose clearance. Significantly, total and plasma membrane Glut2 levels were significantly re- Glucose Tolerance, Insulin Sensitivity Tests, Plasma duced in cKO compared with WT mice leading to dimin- Insulin Level, and Pancreatic Insulin Content ished glucose uptake. Taken together, these results Measurement identify that WASH plays an important and unique phys- Oral and intraperitoneal glucose tolerance tests (OGTTs iological role in pancreatic b-cell glucose sensing and in- and ipGTTs) were performed on mice, which had fasted for sulin secretion through trafficking of Glut2. 12 h (8:00 P.M. to 8:00 A.M.). Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after oral or RESEARCH DESIGN AND METHODS intraperitoneal administration of glucose (2 g/kg body wt). Antibodies Blood samples from the tail vein were collected simulta- m Antibodies to human WASH, mouse WASH, and FAM21 neously in the presence of aprotinin (2 g/mL) and EDTA 2 have previously been described (24–26). Antibody to insulin (1 mg/mL). Serum was harvested and stored at 70°C. For was obtained from Cell Signaling Technologies (Beverly, the insulin tolerance test, mice were fasted for 4 h (8:00 A.M. MA); antibody to b-actin and GFP were from Sigma-Aldrich to 12:00 A.M.) and injected with 1 IU/kg body wt human (St. Louis, MO); antibody to Glut2 was from Proteintech crystalline insulin (Eli Lilly, Indianapolis, IN). Blood glucose Group (Rosemont, IL) and Abcam (Cambridge, MA); anti- levels were determined by use of a Glucometer (Bayer Con- body to glucagon-like peptide 1 receptor (GLP-1R) was from tour) with blood collected from the tail vein. Levels of plasma insulin were measured using an ELISA (cat. no. EZRMI-13K Developmental Studies Hybridoma Bank (University of for insulin; Millipore). For measurement of pancreatic insulin Iowa, Iowa), Proteintech Group, and Santa Cruz Biotechnol- content, the pancreas tail was isolated, homogenized in acid ogy (Dallas, TX); antibody to Glut1 was from Abcam and Cell alcohol, and extracted overnight at 220°C. The solution was Signaling Technologies; and antibody to Lamp1 (CD107a) centrifuged to remove debris and neutralized and insulin was from BD Pharmingen (San Jose, CA). For immunohis- content was determined by ELISA. tochemical and immunofluorescence staining, the following primary antibodies were used: rabbit anti-human WASH Reagents, Cell Culture, Transfection, and 2-NBDG (1:500), rabbit anti-mouse WASH (1:500), mouse anti-insulin