Diabetes Volume 68, February 2019 377

WASH Regulates 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 belonging to the Wiskott- family 2 (SLC2A) (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 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 . 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 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 . 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 Uptake (1:300), mouse anti–chromogranin A (1:200), rabbit anti- All the chemicals were obtained from Sigma-Aldrich un- Glut2 (1:100), rat anti-mouse Lamp1 (1:100), rabbit anti– less otherwise specified. INS-1 cells were a gift from GLP-1R (1:50), and rabbit anti-Glut1 (1:100). For immuno- Dr. Weizhen Zhang (University of Michigan, Ann Arbor, fluorescence staining, Alexa Fluor 488 donkey anti-mouse MI) and cultured in RPMI-1640 containing 11.1 mmol/L IgG, Alexa Fluor 568 donkey anti-rabbit IgG (Life Technol- glucose and supplemented with 10% FBS, 1 mmol/L py- ogies), and Alexa Fluor 633 donkey anti-rat IgG (Jackson ruvate, 10 mmol/L HEPES, 50 mmol/L 2-mercaptoethanol, diabetes.diabetesjournals.org Ding and Associates 379

100 units penicillin/mL, and 100 g streptomycin/mL. b-cell mass was assessed by immunohistochemistry staining 2 2 WASH / mouse embryonic fibroblasts (WASHout of insulin. Area of insulin-positive cells were measured and MEFs) have previously been described (28). Stealth siRNAs normalized to total pancreatic area using ImageJ (National (sequences provided in Supplementary Table 1) were pur- Institutes of Health, Bethesda, MD). b-Cellmassisexpressed chased from Thermo Fisher Scientific and transfected with in grams after normalization to total pancreas mass. Lipofectamine RNAiMAX reagent (Thermo Fisher Scien- tific) according to the manufacturer’s protocol. A set of two Western Blot Analysis and Quantitative RT-PCR siRNAs was used per target. Glut2-GFP plasmid was a gift Snap frozen pancreata or isolated islets from mice of from Dr. Jeffrey E. Pessin (Albert Einstein College of desired genotypes were homogenized or lysed in radio- Medicine, Bronx, New York) and transfected using Lipofact- immunoprecipitation assay buffer (Abcam) or islet lysis amine 2000 according to the manufacturer’s instructions buffer (36). Protein extracts were prepared, separated by fl (Invitrogen, Carlsbad, CA). For 2-(N-(7-nitrobenz-2-oxa- SDS-PAGE, transferred to polyvinylidene uoride mem- 1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) uptake, brane, and immunoblotted as previously described (35). fi the INS-1 cells transfected with control siRNA (siControl) Protein bands of interest were quanti ed by calculating or target siRNAs were resuspended in glucose-free KRBH an integrated density value for each band using buffer for 30 min and then supplemented with 30 mmol/L ImageJ. Pancreatic total RNA was isolated using Trizol fi concentration of the glucose analog 2-NBDG (Thermo and further puri ed with an RNeasy Mini Kit (Qiagen, Fisher Scientific) for 1, 2.5, and 5 min and washed and Valencia, CA). Reverse transcription was performed with run on a FACSCanto II flow cytometer (BD Bioscience), and the Superscript III RT-PCR Kit (Invitrogen). Quantitative the data were analyzed using FlowJo (TreeStar). The mean PCR was performed with the SYBR Green PCR Master Mix fluorescence intensity was definedasthegeometricmeanof the given fluorescent probe.

GSIS INS-1 cells or isolated islets were preincubated in KRBH buffer: 136 mmol/L NaCl, 4.7 mmol/L potassium chloride (KCl), 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 5 mmol/L NaHCO3, 1 mmol/L CaCl2, 10 mmol/L HEPES, and 0.5% BSA, pH 7.4) containing 3 mmol/L glucose at 37°C for 1 h. After aspiration of the buffer, INS-1 cells or isolated islets were incubated in fresh KRBH buffer supplemented with 3 mmol/L glucose, 25 mmol/L glucose, 20 mmol/L KCL, or 10 nmol/L glucagon-like peptide 1 (GLP-1) (Sigma-Aldrich), at 37°C for 30 min. The supernatant was collected and centrifuged for later use. The unreleased insulin within the INS-1 cells or isolated islets was extracted by the acid alcohol method. The insulin collected from the supernatant medium and remaining islets were first diluted and then measured by ELISA for calculating the percentage of insulin secretion.

Morphometry, Immunohistochemistry, and Immunofluorescence Mice were anesthetized using isoflurane (Piramal Critical Care, Bethlehem, PA), followed by cervical dislocation. The whole pancreas was quickly removed and fixed overnight in 4% PFA with gentle shaking, embedded in paraffin, and cut into 5-mm-thick sections. Human and mouse pancreas sections were subjected to hematoxylin-eosin staining, im- munohistochemistry, or immunofluorescence staining as previously described (35). This reference encompasses the Figure 1—WASH expression is highly concentrated in pancreatic islets in both humans and mice. A: Shown are the representative Hoechst staining as well. Confocal images were collected immunohistochemistry (IHC) staining of WASH in pancreas tissue with an LSM-710 laser scanning confocal microscope with from humans (upper panel) and mice (lower panel) under 310 (bars, a633 water Plan-Apochromat objective lens using ZEN 200 mm) or 340 (bars, 50 mm) magnification lens. B: Representative fl 2009 software (Carl Zeiss, Oberkochen, Germany). Islet cell immuno uorescence staining of WASH (red) and CgA (green) in pan- creatic tissue from humans and mice was examined. Nuclei were size and number were assessed by hematoxylin-eosin stain- counterstained with Hoechst 33342 fluorescent stain (blue). Shown ing of pancreas tissue section at 100-mmintervals,and are representative results from six individual islets. Bars, 20 mm. 380 The Role of WASH in Glucose Homeostasis Diabetes Volume 68, February 2019 using the ABI StepOnePlus Sequence Detection System a substantial reduction of WASH protein, as well as the (Applied Biosystems, Carlsbad, CA). Gapdh and Rplp0 were WASH complex member FAM21 (Fig. 2A and B). Consis- used as housekeeping genes for normalization of tent with the immunoblotting results, WASH staining was expression. The double D Ct method was used to analyze greatly reduced in CgA-positive endocrine cells in cKO mice gene expression. Experiments were performed in triplicate compared with WT mice (Fig. 2C). Thus, we have generated using three independent cDNAs. Primer sequences are pancreas-specific WASH knockout mice, allowing in vivo provided in Supplementary Table 1. study of the physiological role of WASH in the pancreas.

Statistical Analysis WASH cKO Mice Display a Normal Pancreatic Data are expressed as mean 6 SEM and analyzed by Development repeated-measures ANOVA and unpaired Student t test Given the essential role of pancreas in body weight and using GraphPad Prism software (GraphPad Software, La glucose homeostasis regulation, we first examined the Jolla, CA). A value of P ,0.05 denotes statistical significance. body weight and fasting blood glucose of 8- and 16-week-old WT and cKO mice. As shown in Fig. 3A RESULTS and B, there was no difference in total body weight or WASH Expression Is Highly Concentrated in Pancreatic fasting glucose between WT and cKO mice. Since there was Islets in Both Humans and Mice no alteration of pancreas weight in cKO mice (data not Pancreatic tissue sections from humans or mice were shown), we next evaluated the expression of several stained for WASH protein expression. WASH expression mRNAs that are enriched in the pancreas including is weakly cytoplasmic in the human and mouse exocrine a-amylase, insulin, glucagon (Gcg), somatostatin, and pancreas but is highly concentrated in the islets (Fig. 1A). cytokeratin-19 (CK19). As shown in Fig. 3C, deletion of For further confirmation of this expression pattern of WASH did not affect the expression level or ratio of the WASH, double immunofluorescent staining was per- tested genes. WASH deletion also had no effect on islet size formed to colocalize the signal of WASH with the endo- (Fig. 3D and E), b-cell mass, average islet size or islet crine cell marker chromogranin A (CgA) in the pancreas of number (Supplementary Fig. 1). Finally, we extracted humans and mice. As shown in Fig. 1B, WASH showed pancreatic insulin and observed no significant alteration strong immunoreactivity in both human and mouse islets of insulin content between WT and cKO animals (Fig. 3F). that also stained positive for CgA. These data indicate that Taken together, these data suggest that deletion of WASH WASH is expressed in the human and mouse pancreas but during pancreas development does not overtly affect pan- significantly accumulated in islets of Langerhans. creas development or impact body weight.

Generation of Pdx1-cre;WASHflox/flox Compound Impaired Glucose Clearance and Insulin Release Mouse Strain in cKO Mice To delineate the physiological role of WASH in the pan- As there is no apparent developmental deficiency in islets creas, we generated pancreas-specific WASH cKO mice by from cKO mice, we next tested pancreas function by fl fl crossing Pdx1-cre mice with WASH ox/ ox mice. Analysis of performing an OGTT, ipGTT, and insulin tolerance test. lysates from the pancreas of WT and cKO animals showed Interestingly, we found glucose was cleared more slowly in

Figure 2—Generation of Pdx1-cre;WASHflox/flox compound mouse strain. A: Pdx1-cre;WASHflox/flox mice were used to generate transgenic mice as indicated in RESEARCH DESIGN AND METHODS. WASH and FAM21 in pancreatic tissue from WT and cKO mice were examined by Western blotting using specific antibodies. b-Actin was used as a loading control. Shown are representative results from six experiments. B: Average signal intensities of WASH and FAM21 were analyzed and expressed as mean 6 SEM. n =6.*P , 0.05 cKO vs. WT mice C: Immunofluorescence staining of WT and cKO mice was analyzed by specific antibody of WASH (red) and CgA (green). Shown are representative results from six individual islets. Bars, 20 mm. diabetes.diabetesjournals.org Ding and Associates 381

showed that T cells lacking WASH had reduced Glut1 endosome-to-membrane recycling (30). We therefore in- vestigated whether the impaired OGTT, ipGTT, and insulin responses to glucose are due to an alteration of Glut2 expression in cKO mice. Interestingly, total Glut2 protein levels were lower in islets obtained from cKO mice com- pared with WT mice (Fig. 5A and B). In addition, we found that the mRNA expression of Glut2 was also substantially reduced in cKO mice relative to WT mice (Fig. 5C). We further found, using immunohistochemistry and immu- nofluorescence staining, that a large proportion of Glut2 is expressed on the surface of insulin-positive b-cells within WT islets, whereas Glut2 levels were lower in cKO mice and less Glut2 was detected on the surface of the insulin- positive b-cells (Fig. 5D and E). Moreover, consistent with serum insulin level changes during OGTT in vivo, we observed a significant reduction of GSIS in islets of cKO mice relative to WT mice ex vivo (Fig. 5F). Finally, as insulin is also secreted in response to different secreta- gogues such as KCl and hormonal factors (e.g., glucagon, GLP-1) and the magnitude of this response is modulated by a variety of receptors and transporters (6), we next examined the contribution of WASH loss in pancreas to the mRNA, protein expression level, or membrane local- ization of several Gluts and GLP-1R. To this end, we found that only Glut2 mRNA was significantly decreased (Sup- plementary Fig. 2A). Additionally, only Glut2 protein levels were down in the WASH knockout islets, whereas Glut1 — Figure 3 WASH cKO mice display a normal pancreatic develop- and GLP-1R were unaffected (Supplementary Fig. 2B–E). ment. A and B: Shown were the body weight and fasting blood glucose level in 8- and and 16-week-old WT and cKO mice. C: Real- Furthermore, islets from WT and cKO mice secreted time PCR quantification of mice pancreatic cytokeratin 19 (CK19), similar levels of insulin upon GLP-1 stimulation in the amylase, insulin, glucagon (Gcg), and somatostatin (SS) from WT and presence of either KCl or 3 mmol/L glucose (Supplemen- cKO mice was examined. D: Freshly isolated primary mouse islets F from WT or cKO mice were cultured in RPMI-1640 medium. Photos tary Fig. 2 ). Taken together, these data indicate that showed dithizone (diphenylthiocarbazone, DTZ)-stained normal WASH deletion decreases overall Glut2 protein levels islets architecture highlighted with red dot line. Bars, 200 mm. and appears to alter GSIS in islets as a result of reduced E: Size of isolated islets was quantified by ImageJ. F: Pancreatic plasma membrane localization of Glut2, whereas other insulin protein was extracted from pancreas tissue extracts, mea- sured by ELISA, and normalized with total protein level. Results were Gluts and GLP-1R were unaffected in either expression or expressed as mean 6 SEM. n = 6. wks, weeks. function.

WASH Is Necessary for GSIS in INS-1 Cells Through cKO mice relative to WT mice in both OGTT and ipGTT Trafficking of Glut2 (Fig. 4A–D). This observation indicates a significant im- To further examine the role of WASH in Glut2 trafficking pairment in glucose tolerance in WASH cKO mice. On the and its physiological function as part of glucose sensing other hand, there was virtually no difference in insulin in vitro, we used RNA interference–mediated suppression sensitivity in peripheral tissues between WT and cKO of WASH and Glut2 in INS-1 cells. Consistent with our mice (Fig. 4E and F). In addition, serum insulin release in vivo cKO data, knockdown of WASH in INS-1 cells during the glucose load remained significantly lower in reduced Glut2 protein levels similar to those observed in cKOmicethaninWTmice(Fig.4G–J). Thus, defective Glut2 siRNA (siGlut2)-treated cells (Fig. 6A and B), and IF insulin release in response to glucose loading in cKO mice showed that WASH was dramatically reduced in insulin- may explain, at least in part, glucose intolerance in this positive INS-1 cells (Fig. 6C). Consistent with the notion model. that decreased Glut2 total or plasma levels in WASH cKO mice might be the reason for de- WASH Contributes to Glut2 Expression in b-Cells creased glucose sensitivity and insulin release in vivo, The widely expressed Glut isoform Glut2 is thought to siWASH-treated INS-1 cells had a significant decrease in have highest expression among all other Gluts in rodent glucose uptake and subsequent GSIS comparable with pancreatic islet b-cells and is required for GSIS and glucose siGlut2-treated cells (Fig. 6D and E). Moreover, transfec- homeostasis (10,11,37,38). Previous studies in T cells tion of a Glut2-GFP plasmid into WASH-depleted cells 382 The Role of WASH in Glucose Homeostasis Diabetes Volume 68, February 2019

Figure 4—Impaired glucose clearance and insulin release in cKO mice. Blood glucose levels (A) and area under the curve (AUC) (B)of 8-week-old WT and cKO mice fed normal chow diet were measured at indicated time points after oral administration of glucose (2 g/kg body wt). Blood glucose levels (C) and AUC (D) of 8-week-old WT and cKO mice fed normal chow diet were measured at indicated time points after intraperitoneal administration of glucose (2 g/kg body wt). Blood glucose levels (E) and AUC (F) of WT and cKO mice fed normal chow diet were measured at indicated time points after intraperitoneal injection of insulin (1 IU/kg body wet). Serum levels of insulin (G and I) and AUC (H and J) of WT and cKO mice fed normal chow diet were measured at indicated time points after oral or intraperitoneal administration of glucose (2 g/kg body wt). Results were expressed as mean 6 SEM. *P , 0.05 cKO vs. WT mice. n = 6. ITT, insulin tolerance test. showed a substantial loss of Glut2-GFP in the plasma WASHout MEFs (Fig. 7C). Significantly, while Glut2-GFP membrane (Fig. 6F and G). These findings further support accumulated in the plasma membrane of WT MEFs, we the idea that WASH plays an important role in glucose found an increased incidence of Glut2-GFP colocalization homeostasis through endosome-to-membrane trafficking with LAMP1 in WASH knockout MEFs (Fig. 7D and E). of Glut2. Taken together, these data indicate that WASH is critical for the appropriate trafficking of Glut2 and that in the Glut2 Is Degraded in Lysosomes in the Absence of absence of WASH, Glut2 is aberrantly trafficked to the WASH lysosome and degraded. We have previously shown that T cells derived from WASH knockout mice accumulated Glut1 in lysosomes due to defective endosome–to–plasma membrane recycling DISCUSSION (30). Since our results above showed that depletion of Prior cell line studies have linked WASH to the recycling of WASH resulted in reduced cell surface and total levels several cell surface receptors through its interaction with of Glut2 (Fig. 6A and F), we next investigated whether the CCC retriever and retromer complexes, which direct Glut2 is degraded and accumulates in lysosomes in the the trafficking of SNX17- and SNX27-dependent cargo, absence of WASH. To test this, we used INS-1 cells treated respectively (23,39). Herein we show that WASH is with siControl or siWASH and transfected them with enriched in the islets of Langerhans where it plays an a Glut2-GFP expression vector. The cells were subse- important role in pancreatic b-cell regulation of Glut2 quently treated with cycloheximide for 2 or 4 h to block trafficking and whole-body glucose homeostasis. Mecha- new protein synthesis, and Glut2 levels were measured by nistically we found that WASH spares Glut2 from lyso- immunoblotting for GFP. Although Glut2-GFP levels somal degradation and promotes its endosome–to–plasma started lower in the siWASH-treated cells, Glut2-GFP membrane recycling. In the absence of WASH, Glut2 exhibited faster degradation kinetics than control cells becomes associated with the lysosome and is degraded (Fig. 7A and B). To determine whether Glut2 accumulated following internalization leading to decreased GSIS and in lysosomes, we transfected Glut2-GFP into WT and ultimately defective glucose homeostasis. diabetes.diabetesjournals.org Ding and Associates 383

Figure 5—WASH contributes to Glut2 expression in b-cell of islets. A: Glut2 and WASH in pancreatic islets lysate from WT and cKO mice were examined by Western blotting using specific antibodies. b-Actin was used as a loading control. Shown are representative results from six experiments. B: Average signal intensity of Glut2 was analyzed and expressed as mean 6 SEM. n =6.C: Real-time PCR quantification of pancreatic Glut2 from WT and cKO mice was examined. D: Immunohistochemistry (IHC) staining of Glut2 (left) and immunofluorescence staining of insulin (green) and Glut2 (red) (right) from WT and cKO mice were analyzed. Shown are representative results from six independent animals. Bars, 50 mm in left panels and 20 mm in right panels. E: Immunohistochemistry staining of Glut2 was analyzed by the Color Deconvolution plugin in ImageJ software, and integrated intensity was expressed as mean 6 SEM. n =5.B, C, and E:*P , 0.05 cKO vs. WT mice. F: Supernatant and residual insulin content from WT or cKO mice islets treated with 3 mmol/L or 25 mmol/L glucose was measured by ELISA, calculated, and expressed as mean 6 SEM. n =7.*P , 0.05, WT mouse islets treated with 25 vs. 3 mmol/L glucose; #P , 0.05, cKO vs. WT mouse islets treated with 25 mmol/L glucose.

Obesity and type 2 diabetes have reached epidemic WASH, Glut2 protein levels are reduced and that an proportions in Western societies (40). The identification exogenously expressed Glut2-GFP construct is more and characterization of the genes involved in type 2 di- quickly degraded following the inhibition of protein syn- abetes have added to our understanding of the pathways thesis. Moreover, the Glut2-GFP construct becomes local- regulating b-cell function. Recently, genome-wide associ- ized with Lamp1+ vesicles within the cytoplasm of MEFs ation studies and biochemical studies have identified lacking WASH suggesting that the increased degradation is a member of the vacuolar protein sorting-10 (VPS10) a result of defective endosome–to–plasma membrane re- family of receptors, SORCS1, and the retromer component trieval Glut2. It remains to be determined whether Glut2 VPS26 as risk factors for type 2 diabetes (41–43). contains a COOH-terminal PDZ binding motif like Glut1, Interestingly, both SNX27 and retromer were found to but our data would be consistent with a similar mechanism maintain the total and cell surface levels of Glut1 in HeLa of endosomal retrieval of Glut2 that involves WASH, cells through the interaction of the SNX27 PDZ binding retromer, and sorting nexin 27. domain with VPS26 and the Glut1 PDZ binding motif While Glut2 is no doubt the most abundant Glut in found at its COOH terminus. Significantly, loss of either rodent islets (10–12), its role in human b-cell development SNX27 or the retromer subunit VPS35 results in Glut1 and function seems more complicated (10,44). In mice endosomal trapping and lysosomal degradation (31). In lacking Glut2, there are major defects in GSIS and mouse addition, using WASH knockout T cells we previously pups die within 2–3 weeks owing to severe diabetes. In demonstrated that WASH regulates Glut1 protein levels, human beings, some mutations in SLC2A2 encoding Glut2 glucose uptake, and T-cell proliferation (30). We now are responsible for the Fanconi-Bickel syndrome, an au- provide evidence that Glut2 preferentially accumulates tosomal recessive disorder associated with defective car- intracellularly in the absence of WASH in several model bohydrate metabolism (45). In contrast to mice, patients systems. In addition, we show that in the absence of with this rare disorder are not reported to require insulin 384 The Role of WASH in Glucose Homeostasis Diabetes Volume 68, February 2019

Figure 6—WASH is necessary for GSIS in INS-1 cells through trafficking of Glut2. A: Glut2 and WASH in INS-1 cells of siControl (siCtrl) and target siRNAs were examined by Western blotting using specific antibodies. b-Actin was used as a loading control. Shown are representative results from six experiments. B: Average signal intensity of WASH and Glut2 was analyzed and expressed as mean 6 SEM. n =6.*P , 0.05 siWASH vs. siControl; #P , 0.05 siGlut2 vs. siControl. C: Immunofluorescence staining of insulin (green) and WASH (red) from siControl and siWASH was analyzed. Bars, 20 mm. D: siControl- and target siRNA–transfected INS-1 cells were incubated with 30 mmol/L fluorescent glucose analog 2-NBDG, and uptake after indicated time points was assayed by flow cytometry. The mean fluorescence intensity from each group was expressed as mean 6 SEM. n =6.*P , 0.05 siWASH vs. siControl; #P , 0.05 siGlut2 vs. siControl. E: Insulin secretion from siControl- or target siRNA–transfected INS-1 cells treated with 3 mmol/L glucose (LG) or 25 mmol/L glucose (HG) was measured by ELISA, calculated, and expressed as mean 6 SEM. n =6.*P , 0.05 siWASH vs. siControl; #P , 0.05 siGlut2 vs. siControl. F: siControl- or siWASH- transfected INS-1 cells were transfected with Glut2-GFP plasmid, and GFP signal from different groups was examined by fluorescence microscope. Bars, 20 mm. G: Membrane and total cell fluorescent density of GFP in siControl- or siWASH-transfected INS-1 cells were quantified by ImageJ and expressed as mean 6 SEM. n = 50. *P , 0.05 siWASH vs. siControl. treatment but, rather, display severe glycosuria due to using WASH RNA interference (48). Although the WASH deficient glucose reabsorption by the kidney (46). This knockout islets do not lose their insulin secretion response discrepancy between humans and rodents is related to to KCl, the contribution of WASH to the trafficking of Glut2 tissue expression. Glut2 is the predominant Glut in KATP channel Kir6.2 needs further investigation. One rodent islets, while Glut1 and Glut3 are more prevalent in possible explanation for the reduction of Glut2 mRNA is human b-cells (10,47). Surprisingly, we note that only the feedback response of disrupted GSIS in WASH cKO Glut2 mRNA and protein expression was decreased in mice, since it is known that impaired GSIS is associated islets from WASH cKO mice, while other Gluts and with markedly reduced expression of Glut2 (14,15). Lastly, GLP-1R remained unchanged. As a result, we detected Glut1 and Glut3 mRNA expression in the mouse pancreas impaired insulin secretion upon high glucose stimulation is barely detectable compared with Glut2 (10); thus, it is but not GLP-1, which is consistent with a previous study not surprising that Glut1 and Glut3 mRNA as well as Glut1 diabetes.diabetesjournals.org Ding and Associates 385

Figure 7—Glut2 is degraded in lysosomes in the absence of WASH. A: siControl (siCtrl)-transfected or siWASH-transfected INS-1 cells were first transfected with Glut2-GFP plasmid and then treated with 20 mg/mL cycloheximide (CHX) for 2 and 4 h, and lysates were immunoblotted with GFP and WASH. Shown are representative results from three independent experiments. B: Glut2-GFP degradation was quantified from immunoblots via densitometry from three independent experiments and expressed as mean 6 SEM. *P , 0.05 siWASH vs. siControl. C:WASHinWASHflox/flox and WASHout MEF cells was examined by immunoblotting, and b-actin was used as a loading control. Shown are representative results from three experiments. D:WASHflox/flox and WASHout MEF cells were transfected with Glut2-GFP plasmid (green) and stained with LAMP1 (red). E: Pearson correlation coefficients of GFP with LAMP1 from WASHflox/flox and WASHout MEF cells transfected with Glut2-GFP plasmid were analyzed by Coloc2 in ImageJ. *P , 0.05 WASHout vs. WASHflox/flox MEF cells. n = 15. hrs, hours. protein expression levels are not affected in islets from Duality of Interest. No potential conflicts of interest relevant to this article WASH deletion mice. Whether Glut1 and Glut3 expres- were reported. sion, localization, or function is similarly affected in Author Contributions. L.D., L.H., and J.D. contributed to the study patients carrying mutations that impact WASH activity concept and design; data acquisition, analysis, and interpretation; drafting the manuscript; and critical revision of the manuscript for important intellectual is of interest, as they might have defective GSIS and be content. D.D.B. contributed to the study concept and design; data acquisition, more prone to develop glucose intolerance. analysis, and interpretation; drafting the manuscript; critical revision of the In summary, our study provides evidence that WASH is manuscript for important intellectual content; technical support; and study expressed in human and mouse pancreas and highly supervision. D.D.B. is the guarantor of this work and, as such, had full access concentrated in pancreatic endocrine islets. In addition, to all the data in the study and takes responsibility for the integrity of the data and WASH contributes to glucose uptake and insulin release the accuracy of the data analysis. through its critical role in the trafficking of the membrane References Glut Glut2 both in vivo and in vitro. Interesting, although 1. Alberti KG, Zimmet PZ. Definition, diagnosis and classification of diabetes mutations in the genes encoding the CCC complex member mellitus and its complications. Part 1: diagnosis and classification of diabetes CCDC22 and strumpellin are associated with intellectual mellitus provisional report of a WHO consultation. Diabet Med 1998;15:539–553 disability syndrome, we have previously demonstrated that 2. Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease these patients also have undiagnosed pathologies including pathogenesis and treatment. Lancet 2001;358:221–229 elevated levels of circulating cholesterol and LDL, which could 3. Cavaghan MK, Ehrmann DA, Polonsky KS. Interactions between insulin make them prone to heart disease. Thus, patients carrying resistance and insulin secretion in the development of glucose intolerance. J Clin mutations in the WASH complex components strumpellin or Invest 2000;106:329–333 SWIP, or the CCC complex member CCDC22, might be at 4. Kahn SE. Clinical review 135: the importance of beta-cell failure in the a higher risk for developing diabetes. development and progression of type 2 diabetes. J Clin Endocrinol Metab 2001;86: 4047–4058 5. Notkins AL. Immunologic and genetic factors in type 1 diabetes. J Biol Chem Funding. This work was supported by the Mayo Foundation for Medical 2002;277:43545–43548 Education and Research. D.D.B. was supported by National Institutes of Health 6. Bell GI, Polonsky KS. Diabetes mellitus and genetically programmed defects grant R01DK107733. in beta-cell function. Nature 2001;414:788–791 386 The Role of WASH in Glucose Homeostasis Diabetes Volume 68, February 2019

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