Diabetes Volume 69, September 2020 1887

WDFY2 Potentiates Hepatic Sensitivity and Controls Endosomal Localization of the Insulin and IRS1/2

Luyao Zhang,1 Xue Li,1 Nan Zhang,1 Xin Yang,1 Tianyun Hou,1,2 Wan Fu,1 Fengjie Yuan,1 Lina Wang,1 He Wen,2 Yuan Tian,3 Hongquan Zhang,4 Xifeng Lu,5 and Wei-Guo Zhu1,2,3

Diabetes 2020;69:1887–1902 | https://doi.org/10.2337/db19-0699

Endosomes help activate the hepatic insulin-evoked Akt The liver is a crucial insulin tissue target for maintaining signaling pathway, but the underlying regulatory mecha- normal glucose homeostasis: it produces glucose during nisms are unclear. Previous studies have suggested that fasting and stores postprandial glucose (1). In hepatocytes, the endosome-located WD repeat and FYVE insulin binds to the (INSR) to activate its domain–containing 2 (WDFY2) might be involved in meta- intrinsic kinase activity. Insulin receptor sub- bolic disorders, such as diabetes. Here, we generated strates (IRS) are then phosphorylated on tyrosine residues Wdfy2 knockout (KO) mice and assessed the metabolic that serve as anchoring sites for the phosphatidylinositol consequences. These KO mice exhibited systemic insulin 3-kinase (PI3K) p85 regulatory subunit (2). PI3K produces resistance, with increased gluconeogenesis and sup- PIP and subsequent PI34P , which recruit Akt and me- pressed glycogen accumulation in the liver. Mechanisti- 3 2 diate its activation (3,4). cally, we found that the insulin-stimulated activation of Akt2 is the most abundant Akt isoform in insulin- Akt2 and its substrates FoxO1 and GSK-3b is attenuated sensitive tissues (5). Once activated in hepatocytes, in the Wdfy2 KO liver and H2.35 hepatocytes, suggesting that WDFY2 acts as an important regulator of hepatic Akt2 Akt2 functions in several distinct downstream pathways signaling. We further found that WDFY2 interacts with the that modulate metabolism (6). Activated Akt2 phosphor- insulin receptor (INSR) via its WD1–4 domain and localizes ylates FoxO1 to reduce its transcriptional activity on the INSR to endosomes after insulin stimulation. This gluconeogenic enzymes (7) and glycogen synthase kinase- b b process ensures that the downstream insulin receptor 3 (GSK-3 ) to inhibit glycogen synthesis (8). The substrates 1 and 2 (IRS1/2) can be recruited to the endo- endosome has important roles in activating the hepatic somal INSR. IRS1/2-INSR binding promotes IRS1/2 phos- insulin-evoked PI3K-Akt pathway (9). In hepatocytes, phorylation and subsequent activation, initiating downstream INSR binding results in INSR auto- Akt2 signaling in the liver. Interestingly, adeno-associated phosphorylation and internalization to generate intracel- viral WDFY2 delivery ameliorated metabolic defects in db/ lular signaling endosomes; the INSR continues to signal db mice. These findings demonstrate that WDFY2 acti- at endosomal compartments, providing the molecular vates insulin-evoked Akt2 signaling by controlling endo- cue to activate downstream IRS and class I somal localization of the INSR and IRS1/2 in hepatocytes. PI3K (10–12). IRS1 and IRS2 levels increase by 100%– This pathway might constitute a new potential target for 150% in isolated hepatic endosomes following insulin diabetes prevention or treatment. administration. Akt, especially Akt2, is also recruited to

1Key Laboratory of Carcinogenesis and Translational Research, Ministry of Corresponding author: Wei-Guo Zhu, [email protected] fi Education, Beijing Key Laboratory of Protein Posttranslational Modi cations and Received 17 July 2019 and accepted 4 June 2020 Cell Function, Department of Biochemistry and Molecular Biology, School of Basic This article contains supplementary material online at https://doi.org/10.2337/ Medical Sciences, Peking University Health Science Center, Beijing, China figshare.12582722. 2Guangdong Key Laboratory of Genome Instability and Human Disease Prevention, Department of Biochemistry and Molecular Biology, International Cancer Center, © 2020 by the American Diabetes Association. Readers may use this article as Shenzhen University School of Medicine, Shenzhen, China long as the work is properly cited, the use is educational and not for profit, and the 3Shenzhen Bay Laboratory, Shenzhen, China work is not altered. More information is available at https://www.diabetesjournals 4Department of Human Anatomy, Histology, and Embryology, School of Basic .org/content/license. Medical Sciences, Peking University Health Science Center, Beijing, China 5Department of Physiology, Shenzhen University School of Medicine, Shenzhen, China 1888 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020 endosomes, where it exhibits higher specific enzymatic pseudopregnant females to generate chimeric mice; chi- activity than on plasma membranes (13,14). These obser- meric mice were successfully generated from one of the vations support that IRS binding to the INSR in endo- two clones. The chimeras were bred to C57BL/6J mice to somes is essential to initiate the insulin signaling pathway obtain F1 mice carrying the recombined allele containing and regulate insulin-dependent energy homeostasis. The the floxed Neo allele, after which the F1 mice were mated underlying mechanism of endosomal targeting of the INSR with Cre-deleter mice (C57BL/6J background) to remove and IRS1/2, however, is elusive. exon 3 and the Neo cassette. Obtained heterozygous KO Endosomal proteins involved in the insulin signaling mice were interbred to generate heterozygous KO mice, pathway have important roles in mediating cell metabolism which were then interbred to yield homozygous Wdfy2 KO (15–17).FYVEdomain(domainidentified in Fab1p, YOTB, and wild-type (WT) mice (littermate controls). Vac1p, and EEA1)–containing proteins bind with high spec- Murine were confirmed in both males and ificity and avidity to phosphatidylinositol 3-phosphate females at the specified age. Genotyping was performed by (PI3P) (18). WDFY2 is a highly conserved FYVE domain– PCR using genomic DNA isolated from tails using the containing protein: its expression defines a distinct popu- primers detailed in Supplementary Table 2. lation of early endosomes found in close proximity to the Animal Models With Insulin Resistance and Dietary plasma membrane (19,20). The WD repeat protein family Intervention has been implicated in various cellular processes (21). WDFY2 Male db/db mice (3 months old) and lean control mice were itself promotes adipocyte differentiation and insulin-stimulated purchased from the Nanjing Model Animal Research Cen- glucose uptake, which suggests that WDFY2 might orches- ter. Dietary intervention with a high-fat diet (HFD) (60% trate the signaling pathways involved in metabolic disor- calories from fat; Research Diets) or chow diet (11.4% ders (22,23). No studies, however, have focused on WDFY2 calories from fat; Beijing Vital River Laboratory Animal function in the liver. Here, we investigated the role of Technology Co., Ltd) was conducted for 12 weeks from WDFY2 in insulin-evoked glucose metabolism using a sys- 1 month of age in WT C57BL/6 mice. All animal protocols temic Wdfy2 knockout (KO) mouse model. were approved by the Committee for Animal Research of RESEARCH DESIGN AND METHODS Peking University, Beijing, China, and conformed to the Guide for the Care and Use of Laboratory Animals (Na- Antibodies and Reagents tional Institutes of Health [NIH] publication no. 86-23, An anti-WDFY2 rabbit polyclonal antiserum was raised – revised 1985). All mice were maintained in a temperature- against a conserved 17 amino acid peptide at the COOH controlled barrier facility with a 12-h light/dark cycle and terminus of murine and human WDFY2. The serum was were given free access to food and water in the Center for recovered after boosting six times, and the antibody was Experimental Animals at Peking University. affinity purified (Medical & Biological Laboratories Co. Ltd., Japan). For commercial antibodies, see Supplementary Table Animal Experiments 1. Palmitic, stearic, oleic and linoleic acids, D-glucose, insulin, Male mice were used in this study. Glucose tolerance and puromycin were purchased from Sigma-Aldrich. tests (GTT), insulin tolerance tests (ITT), and pyruvate tolerance tests (PTT) were performed as previously de- Wdfy2 Generation of KO Mice and Genotyping Strategy scribed (16). The serum concentrations of metabolic fl Wdfy2 A targeting vector was designed to ank exon 3 with factors were measured with an Ultrasensitive Mouse loxP sites. The fragment included a sequence upstream of Insulin ELISA kit (Mercodia), a triglyceride assay kit –fl exon 3 (502 base pairs [bp]), the exon 3 anked sequence (FUJIFILM Wako Pure Chemical Corporation), and a cho- (875 bp), and a sequence downstream of exon 3 (497 bp) of lesterol assay kit (FUJIFILM Wako Pure Chemical Cor- Wdfy2 genomic DNA; it was ligated into the pNLF vector poration). Hepatic glycogen was measured with a Liver/ (Beijing Biocytogen Co., LTD, China), which has two loxP Muscle Glycogen Assay Kit (Nanjing Jiancheng Bioengi- fl sites and an FRT- anked neomycin (Neo) resistance cas- neering Institute). sette. The targeting vector covered 5.2 kb sequence up- stream of Wdfy2 exon 3 and 7.4 kb sequence downstream Histological Analysis of exon 3 and included a diphtheria toxin A negative Liver tissue was isolated from euthanized mice, fixed in 4% selection marker; it was subcloned from a BAC clone paraformaldehyde overnight, embedded in paraffin, and cut (RP23-178J15; Invitrogen) from the C57BL/6J mouse into sections of 5-mm thickness. The sections were analyzed genomic BAC library. After linearization, the targeting by hematoxylin-eosin or periodic acid Schiff (PAS) staining. vector was electroporated into C57BL/6J embryonic stem (ES) cells (Beijing Biocytogen Co., LTD). G418-re- Plasmid Construction sistant embryonic stem clones were screened for homol- The GFP-INSR (mouse) (MG51062-ACG) plasmid was ogous recombination by PCR, and targeted clones were purchased from Sino Biological. The -WDFY2 plasmid confirmed by Southern blotting with the indicated probes was provided by Dr. Karin Moelling (Institute of (Beijing Biocytogen Co., LTD). Two positive clones were Medical Virology, University of Zurich, Zurich, Switzerland). injected into BALB/c blastocysts and implanted into INSR (human) cDNA was amplified and cloned into a diabetes.diabetesjournals.org Zhang and Associates 1889 p3xFLAG-CMV-10 vector (Addgene). Full-length WDFY2 and were prepared using an anti-Flag M2 affinity gel (Bimake), fragments (WD1–4domain,1–195 aa; WD5–7 domain, 196– following the manufacturer’s instructions. The Flag 400 aa) were cloned into pGEX-6p1 or pET28a vectors peptide (0.2 mg/mL) (Bimake) was applied to the column (Addgene). The INSR cytoplasmic domains (962–1,382 to elute the Flag protein complex. Fractions of the bead aa) were cloned into the pGEX-6p1 vector. Retroviruses volume were resolved by SDS-PAGE, and the gels were containing PITA-Flag-WDFY2 or a WD5–7fragment stained with Coomassie brilliant blue stain (Solarbio); the were produced using the 293T packaging cell line (ATCC). bands were excised for liquid chromatography–tandem mass spectrometry sequencing. Cell Culture and DNA Transfection HepG2 (human hepatic cancer cells), H2.35 (murine hep- RNA Extraction and Real-time PCR atocytes), and 293T (human renal cells) cell lines were Total RNA was isolated using TRIzol reagent (APPLYGEN). m purchased from ATCC and cultured according to ATCC cDNA was synthesized from 2 g RNA using a Quantscript guidelines. All cells were maintained in a 37°C incubator RT kit (TOYOBO). Relative expression was deter- mined by real-time PCR (25) using the primers detailed in with a humidified, 5% (v/v) CO2 atmosphere. HepG2 and 293T cells were transfected with Lipofectamine 2000 accord- Supplementary Table 3. ing to the manufacturer’s instructions. H2.35 cells were Immunofluorescence transduced by a retrovirus system (22). Immunofluorescence was performed as previously described Generation of Wdfy2 KO Cell Lines (19). CRISPR/Cas9-based gene-editing Wdfy2 KO H2.35 cells – Adeno-Associated Virus Construction for WDFY2 were generated via the Lipofectamine 2000 mediated Overexpression transfection of single guide RNA (sgRNA) constructs in cDNAs encoding full-length human WDFY2 or the WD5– an SpCas9-2A-Puro vector (Addgene) (24). The following 7 fragment were inserted into a pHBAAV2/9-TBG-GFP Wdfy2 sgRNAs were designed using online software (http:// vector and then subcloned in Escherichia coli.Forpack- 9 crispr.mit.edu): sequence 1, 5 -GGGTGTCATCAGCGTCTCGG- aging of the adeno-associated virus (AAV), the AAV 9 9 9 3 ;sequence2,5-ATCCTGCTGCAGCGGGTCGA-3 . vectors were transfected into AAV-293 cells using Lip- ofectamine 2000. After propagation, the recombinant Protein Extraction AAVs were purified on a column (Biomiga) and the titer Murine tissues were homogenized in radioimmunopreci- was determined by end point assay (Hanbio Biotechnol- pitation assay lysis buffer (50 mmol/L Tris-HCl, pH 7.4; ogy Co., LTD). 150 mmol/L NaCl; 1% NP-40; and 0.1% SDS) supple- mented with a 1% protease inhibitor cocktail (Roche) Statistical Analyses and 1% phosphatase inhibitors (APPLYGEN). Whole cell The data represent the means 6 SD of at least three extracts were isolated as previously described (25). independent experiments. Statistical significance was cal- culated by two-tailed unpaired Student t test for two Subcellular Fractionation groups or one or two-way ANOVA followed by Tukey Plasma membrane and endosomal fractions were isolated post hoc test for more than two groups. P , 0.05 was from cell lysates by discontinuous sucrose-gradient ultra- considered statistically significant. centrifugation (26). Data and Resource Availability PI3K Assay The data sets generated and/or analyzed during the cur- Extracted proteins were immunoprecipitated with the rent study are available from the corresponding author indicated antibodies as previously described (16). PI3K upon reasonable request. activity was determined using a PI3K ELISA kit (Echelon ’ Biosciences) according to the manufacturer s instructions. RESULTS

Western Blotting and Immunoprecipitation Hepatic WDFY2 Expression Is Decreased by Saturated Fatty Acids and High Glucose Levels Western blotting and immunoprecipitation were per- To investigate the role of WDFY2 in metabolism, we first formed as previously described (25). detected WDFY2 expression levels in insulin target tissues. Protein Purification and In Vitro Binding Assay WDFY2 was highly expressed in the liver and adipose Protein purification and in vitro binding assay were per- tissues but expressed at lower levels in skeletal muscles A B formed as previously described (25). (Fig. 1 and ). We thus aimed to determine whether physiological metabolites influence WDFY2 expression in Coomassie Brilliant Blue Staining and Mass the liver and visceral white adipose tissue (Vis. WAT). We Spectrometry fed 1-month-old C57BL/6 mice an HFD or a normal diet HepG2 cells were transfected with Flag-WDFY2 or an (chow) for 12 weeks and then detected WDFY2 protein and empty vector for 48 h. Anti-Flag immune affinity columns mRNA levels in the liver and Vis. WAT. WDFY2 expression 1890 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020

Figure 1—Hepatic WDFY2 expression is decreased by saturated fatty acids and high glucose levels. Western blot (A) and real-time PCR (B) analysis of WDFY2 expression in peripheral insulin target tissues. The data are normalized to b-actin. BAT, brown adipose tissue; SM, skeletal muscle; Sub. WAT, subcutaneous white adipose tissue. (n 5 3.) Western blots (C) and quantified WDFY2 expression levels (D) in livers isolated from mouse models of insulin resistance and metabolic disorders. The comparisons are made with respective age-matched and sex- matched controls (n 5 6 for all groups). E: Real-time PCR analysis of Wdfy2 mRNA levels in mouse models of metabolic disorders. The data are normalized to b-actin (n 5 6 for all groups). Western blots (F) and quantified WDFY2 expression levels (G) in H2.35 hepatocytes treated with 100 mmol/L palmitic, stearic, oleic, or linoleic acids for 24 h, respectively (n 5 3). H: Real-time PCR analysis of Wdfy2 mRNA levels in H2.35 hepatocytes treated with free fatty acids. The data are normalized to b-actin (n 5 3). Western blots (I) and quantified WDFY2 expression levels (J) in H2.35 hepatocytes treated with different concentrations of glucose for 24 h (n 5 3). K: Real-time PCR analysis of Wdfy2 mRNA levels in H2.35 hepatocytes treated with increasing concentrations of glucose. The data are normalized to b-actin (n 5 3). The data represent the means 6 SD. P values were calculated by Student t test (C–E) and one-way ANOVA with Tukey post hoc test (F–H)(*P , 0.05, **P , 0.01, ***P , 0.001 vs. the respective control). N.S., not significant. diabetes.diabetesjournals.org Zhang and Associates 1891

Figure 2—Loss of Wdfy2 leads to insulin resistance and glucose intolerance in mice. GTT were performed in 3-month-old (A) and 8-month- old (B) WT and Wdfy2 KO mice at the indicated time points (left). The area under the curve (AUC) for GTT is shown (right) (n 5 5–6 mice for each genotype). ITT were performed in 3-month-old (C) and 8-month-old (D) WT and Wdfy2 KO mice at the indicated time points (left). The area under the curve for ITT is shown (right) (n 5 5 mice for each genotype). E: Serum insulin levels of 3-month-old and 8-month-old WT and Wdfy2 KO mice were measured after 6 h of fasting (n 5 5–10 mice for each genotype). Serum triglycerides (F) or cholesterol levels (G) in 3-month-old and 8-month-old WT and Wdfy2 KO mice maintained under random fed conditions (n 5 5 mice for each genotype). The data represent the means 6 SD. P values were calculated by Student t test (*P , 0.05, **P , 0.01, ***P , 0.001 vs. the respective control). N.S., not significant.

was significantly decreased in the livers of mice with HFD- WDFY2 expression levels in the liver and Vis. WAT are induced obesity (Fig. 1C–E, left lanes). WDFY2 was also decreased in metabolic disorder mice, but the level of this reduced in the Vis. WAT of mice with HFD-induced obesity decrease is most pronounced in the liver. but to a lesser extent than observed in the liver (Supple- As the liver is critical for regulating the impact of insulin mentary Fig. 1A–C, left lanes). The same pattern of on metabolic homeostasis, we focused our subsequent changes was detected in the livers and Vis. WAT of studies on the liver. Mice with HFD-induced obesity and 3-month-old diabetic (db/db) mice (Fig. 1C–E and Supple- db/db mice exhibited elevated levels of serum metabolic mentary Fig. 1A–C, right lanes). These results indicate that factors, including glucose and nonesterified fatty acids 1892 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020

Figure 3—WDFY2 inhibits gluconeogenesis but potentiates glycogen synthesis. A: PTT were performed in 3-month-old WT and Wdfy2 KO mice at the indicated time points (left). The area under the curve (AUC) for the PTT is shown (right) (n 5 5 mice for each genotype). B: Real-time PCR analysis of G6pc and Pck1 mRNA levels in WT and Wdfy2 KO livers isolated from mice fasted overnight (fasted) or fasted and then refed for 4 h (refed). The data are normalized to b-actin (n 5 6 mice in each group). C: Representative histological liver sections from 3-month-old WT and Wdfy2 KO mice stained with PAS, evidencing carbohydrate deposits in hepatocytes. Scale bars, 50 mm(n 5 8 mice for each genotype). D: Hepatic glycogen content, determined by anthrone reaction in livers obtained from 3-month-old mice in fasted and refed conditions (n 5 8 mice in each group). The data represent the means 6 SD. P values were calculated by Student t test (A) and two-way ANOVA with Tukey post hoc test (B and D)(*P , 0.05, **P , 0.01, ***P , 0.001 vs. the respective control).

(27). We queried whether these metabolites affected mice (Supplementary Fig. 2A and B). Crossing WDFY2 het- WDFY2 expression in the H2.35 hepatocyte cell line. erozygous mice produced litters with the expected Mendelian We thus treated H2.35 cells with exogenous palmitic, ratios and normal body size. Furthermore, Wdfy2 KO mice stearic, oleic, and linoleic acids and then detected WDFY2 were viable and fertile, with no differences in body weight protein levels. Interestingly, only the saturated palmitic and (Supplementary Fig. 2C). GTT, however, showed impaired stearic fatty acids reduced WDFY2 expression levels com- glucose reduction in Wdfy2 KO compared with WT mice pared with untreated cells (Fig. 1F–H). We then exposed (Fig. 2A and B). Consistently, ITT showed significantly reduced H2.35 cells to different glucose concentrations and found insulin sensitivity in Wdfy2 KO than in WT mice (Fig. 2C and that WDFY2 expression increased in response to low glucose D). Moreover, Wdfy2 KO mice had significantly higher serum but decreased in response to high glucose (Fig. 1I–K). These insulin levels than WT mice under fasting conditions (Fig. 2E). observations demonstrate that WDFY2 is enriched in the Thesechangesweredetectableinmiceaged3and8months liver and its expression is downregulated by high levels of old, indicating that WDFY2 has a continuous influence on glucose or saturated fatty acids. WDFY2 might, therefore, be glucose metabolism (Fig. 2A–E). These results demonstrate involved in the pathogenesis of metabolic diseases, such as that loss of Wdfy2 at the whole body level leads to impaired type 2 diabetes. insulin sensitivity and glucose intolerance. Next, we performed a biochemical analysis of lipid Loss of Wdfy2 Leads to Insulin Resistance and Glucose metabolism. We detected no changes in serum triglyceride Intolerance in Mice but a mild increase in serum cholesterol levels in 3-month- To determine whether WDFY2 is involved in the pathogenesis old Wdfy2 KO mice (Fig. 2F and G). However, we detected of metabolic diseases, we generated systemic WDFY2-deficient significantly increased serum triglyceride and cholesterol diabetes.diabetesjournals.org Zhang and Associates 1893

Figure 4—WDFY2 specifically activates insulin-evoked endosomal Akt2 signaling. A and B: WT and Wdfy2 KO 3-month-old mice were fasted overnight and then injected intraperitoneally with either insulin (0.75 units/kg body wt) or saline for 15 min. Representative Western blots (A) and the associated quantifications (B) show phosphorylated (p-)Akt, FoxO1, and GSK-3b levels in liver lysates (n 5 10 mice in each group). C and D: WT and Wdfy2 KO H2.35 hepatocytes were serum starved overnight and then treated with 10 nmol/L insulin for 15 min. Representative Western blots (C) and the associated quantifications (D) show phosphorylated Akt, FoxO1, and GSK-3b levels in hepatocyte lysates (n 5 5). E and F: WT and Wdfy2 KO 3-month-old mice were fasted overnight and then injected intraperitoneally with either insulin (0.75 units/kg body wt) or saline for 15 min. Representative Western blots (E) and the associated quantifications (F) show phosphorylated Akt1 and Akt2 levels in the liver lysates (n 5 8 mice in each group). G and H: WT and Wdfy2 KO H2.35 hepatocytes were serum starved overnight and then treated with 10 nmol/L insulin for 15 min. Representative Western blots (G) and the associated quantifications (H) show phosphorylated Akt1 and Akt2 levels in hepatocyte lysates (n 5 5). I–L: Analysis of total and phosphorylated Akt1 and Akt2 levels in the endosomes (ENs) and plasma 1894 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020 levels in 8-month-old Wdfy2 KO mice (Fig. 2F and G). activation of Akt and its substrates FoxO1 and GSK-3b in WDFY2 thus has a slight impact on lipid metabolism in murine livers and hepatocytes. young mice that becomes more prominent with age. WDFY2 Selectively Regulates Akt2 but Not Akt1 WDFY2 Inhibits Gluconeogenesis but Potentiates Signaling in Endosomes Glycogen Synthesis in Liver We next queried whether other functions under Akt We next aimed to determine liver metabolic function in control, such as growth and proliferation, are affected Wdfy2 Wdfy2 KO mice. The liver contributes to postprandial by loss. We assessed the activity of TSC2 and — circulating glucose by suppressing hepatic gluconeogenesis PRAS40 two downstream Akt effectors that control pro- — and increasing the rate of glycogen synthesis (28). Results tein synthesis and are involved in cell growth (29) and from a PTT in 3-month-old Wdfy2 KO mice showed a sig- found that phosphorylation of these proteins was Wdfy2 nificant increase in hepatic glucose output compared with unaffected by loss. These data suggest that cell – fl Wdfy2 WT mice (Fig. 3A). Consistently, loss of Wdfy2 markedly growth related substrates are not in uenced by C D attenuated the suppressive effects of insulin on two gluco- (Supplementary Fig. 4 and ). neogenic : glucose 6-phosphatase (G6pc) and phos- Both Akt1 and Akt2 are expressed in the liver, and Akt2 ; phoenolpyruvate carboxykinase (Pck1)(Fig.3B). We also accounts for 85% of total liver Akt (30). Akt2 is the ’ observed reduced positivity to PAS staining in 3-month-old predominant Akt isoform to mediate insulin s control of Akt22/2 Wdfy2 KO liver sections (Fig. 3C), indicative of decreased glucose metabolism. mice exhibit a strong met- – glycogen deposits compared with WT mice. The results of an abolic with type 2 diabetes like symptoms that Akt12/2 anthrone reaction confirmed these findings (Fig. 3D). are not seen in mice (31,32). We thus examined fi Hepatic glucose metabolism disorders lead to lipid the effects of WDFY2 loss on isoform-speci c Akt phos- Wdfy2 Wdfy2 heterotopic deposition with age (27). Consistently, we phorylation in KO mice liver and KO H2.35 observed that 8-month-old Wdfy2 KO mouse livers were hepatocytes. Only Akt2 phosphorylation levels were sig- fi Wdfy2 heavier than those of WT mice of the same age (Supple- ni cantly suppressed in the insulin-stimulated KO mentary Fig. 3A), and hematoxylin-eosin staining showed liver and H2.35 hepatocytes compared with WT models E–H mild but obvious hepatic steatosis (Supplementary Fig. (Fig. 4 ). Of note, insulin-stimulated Akt2 phosphory- 3B). Accordingly, we found increased triglyceride and lation was more dramatically reduced than total Akt Wdfy2 cholesterol content in the livers of Wdfy2 KO mice com- phosphorylation in KO models. Total Akt1 and pared with WT mice (Supplementary Fig. 3C). These data Akt2 protein and mRNA levels remained unchanged (Sup- G H show that WDFY2 helps regulate hepatic glucose metab- plementary Fig. 4 and ), and Akt2 expression was Wdfy2 I olism by decreasing hepatic glucose production and in- unaffected in KO WAT (Supplementary Fig. 4 J creasing hepatic glycogen accumulation. and ). Because WDFY2 mainly localizes to the early endosome WDFY2 Activates Insulin-Evoked Akt Signaling in the membrane by binding PI3P via its FYVE domain (18), we Murine Liver and in H2.35 Hepatocytes investigated the effects of WDFY2 on Akt subcellular Given the important role of the PI3K-Akt axis in the insulin distribution in WT and Wdfy2 KO H2.35 cells. Compared signaling pathway (3), we hypothesized that WDFY2 might with Akt2 phosphorylation in Wdfy2 KO H2.35 cells, Akt2 be involved in activating this axis in hepatocytes. We first in the endosomal fraction of WT cells was markedly studied Akt phosphorylation in response to insulin in liver phosphorylated at Ser473 in response to insulin (Fig. 4I extracts from Wdfy2 KO and WT mice. Loss of WDFY2 and J). However, both WT and KO cells showed a similar attenuated insulin-stimulated Akt phosphorylation at level of elevated Akt2 phosphorylation in the plasma Ser473 and Thr308 and reduced FoxO1 phosphorylation membrane in response to insulin treatment (Fig. 4I and at Ser253 and GSK-3b phosphorylation at Ser9 compared K). Akt1 was exclusively phosphorylated in the plasma with WT controls; total Akt, FoxO1, and GSK-3b expression membrane after insulin stimulation,andAkt1phosphorylation remained unchanged (Fig. 4A and B). We observed the same was unaffected by loss of Wdfy2 (Fig. 4I and L). WDFY2 thus patterns in phosphorylation in response to feeding (Sup- selectively regulates Akt2 signaling in endosomes. plementary Fig. 4A and B). We then generated Wdfy2 KO H2.35 hepatocytes and again detected inhibited kinase WDFY2 Affects Insulin-Evoked Signaling Events activation compared with WT cells (Fig. 4C and D), whereas Upstream of Akt Erk1/2 phosphorylation was unaffected (Supplementary Fig. WDFY2 colocalizes and interacts with Akt in 3T3-L1 4C and D). WDFY2 thus potentiates the insulin-evoked adipocytes (23), but whether this interaction exists in

membrane (PM) from WT and Wdfy2 KO H2.35 hepatocyte lysates. Representative Western blots (I) and the associated quantifications (J–L) show phosphorylated Akt1 and Akt2 levels in the endosomes and plasma membrane fraction (n 5 6). The data represent the means 6 SD. P values were calculated by two-way ANOVA with Tukey post hoc test (**P , 0.01, ***P , 0.001 vs. the respective control). N.S., not significant. diabetes.diabetesjournals.org Zhang and Associates 1895

Figure 5—WDFY2 interacts with the INSR via the WD1–4 fragment. A: WT and Wdfy2 KO H2.35 hepatocytes were serum starved overnight and treated with 10 nmol/L insulin for 10 min. PI3K activity associated with IRS1 or IRS2 is expressed as the fold change relative to the baseline levels in the WT group (n 5 4). B and C: WT and Wdfy2 KO H2.35 hepatocytes were serum starved overnight and treated with 10 nmol/L insulin for 5 min. Representative Western blots (B) and the associated quantifications (C) show the insulin-induced tyrosine phosphorylation levels of the INSR, IRS1, and IRS2 in hepatocyte lysates (n 5 3). D and E: HepG2 cells were transiently transfected with Myc- WDFY2 and Flag-INSR or empty vectors. The interaction between exogenous WDFY2 and INSR was analyzed by immunoprecipitation (IP) assay. F: The interaction between endogenous WDFY2 and the INSR in liver and hepatocytes was analyzed by IP assay. G and H: HepG2 cells were transiently transfected with Myc-WDFY2 and GFP-INSR. Colocalization of WDFY2 (red) with the INSR (green) appeared as yellow fluorescence in the merged images (G) and was quantified using Manders’ colocalization coefficient (H). n 5 3 with 50 cells per condition. Scale bars, 20 mm. I(a): Schematic of full-length and truncated WDFY2 (FYVE: domain identified in Fab1p, YOTB, Vac1p, and EEA1). WD, 1896 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020 hepatocytes is unknown. After transfecting H2.35 cells domain, and the deletion of single amino acids, larger frag- with Myc-tagged WDFY2, we found no detectable associ- ments, or individual blades elicits no marked reduction in ation between Myc-WDFY2 and Akt1 or Akt2 in hepato- its binding capability with proteins (34,35). We therefore cytes (Supplementary Fig. 4K). WDFY2 might, therefore, generated truncation mutants embracing larger portions indirectly regulate Akt phosphorylation in hepatocytes. of WDFY2 [Fig. 5I(a)]andfoundthattheINSRinteracted Insulin induces Akt activation by stimulating IRS1 and with the C-terminal deletion mutant GST-WD1–4but IRS2 tyrosine phosphorylation, which in turn triggers PI3K not with the N-terminal deletion mutant GST-WD5–7 activation (33). We investigated whether WDFY2 regulates (Fig. 5J). these upstream signaling events in hepatocytes. We found The INSR is a transmembrane protein, and the cyto- that the insulin-induced elevation of PI3K activity was plasmic domain of its b subunit can interact with several markedly reduced in Wdfy2 KO H2.35 cells compared intracellular proteins (36). We next generated a construct with WT cells (Fig. 5A). Although WDFY2 protein deficiency consisting of the INSR cytoplasmic domain (INSR CD), impaired IRS1 and IRS2 tyrosine phosphorylation in re- which included part of the juxtamembrane segment, the sponse to insulin, it had no effect on the insulin-stimulated entire tyrosine kinase domain, and the C-terminal tail [Fig. tyrosine phosphorylation of INSR and total INSR, IRS1, and 5I(b)]. WDFY2 interacted with the INSR CD peptide (Fig. IRS2 protein expression in hepatocytes (Fig. 5B and C). 5K). Binding assays with purified proteins (Supplementary Decreased tyrosine phosphorylation of IRS1/2 was also Fig. 5J) also showed that only full-length WDFY2 could observed in Wdfy2 KO mice liver tissue (Supplementary interact with the endogenous INSR (Supplementary Fig. Fig. 5A and B). We also investigated the effects of WDFY2 5K). These data support an intracellular association be- on subcellular IRS1/2 phosphorylation in WT and Wdfy2 KO tweenWDFY2andtheINSRandsuggestthatWDFY2 H2.35 cells. We found that insulin-stimulated IRS1/2 tyro- can directly interact with the INSR via the WD1–4 sine phosphorylation was decreased in the endosomal frac- fragment. tion, but not in the plasma membrane, of Wdfy2 KO cells compared with WT cells (Supplementary Fig. 5C–G). These WDFY2 Interacts With the INSR and Localizes It in Early results suggest that WDFY2 promotes insulin signaling by Endosomes to Recruit and Activate IRS1/2 acting downstream of the INSR along the PI3K-Akt signal- We next performed coimmunoprecipitation experiments ing pathway. to explore whether WDFY2 interacts with direct INSR substrates, IRS1/2. Although IRS1/IRS2 and WDFY2 did WDFY2 Interacts With the INSR via a WD1–4 Fragment not interact in H2.35 cells under basal conditions, they did We next examined whether WDFY2 interacts with insulin coimmunoprecipitate after insulin administration (Supple- signaling molecules. By affinity purification and mass mentary Fig. 6A). GST pull-down assay, however, showed spectrometry, we found that the INSR was a major that the association between WDFY2 and nonphosphory- WDFY2-associated protein (Supplementary Fig. 5H). Ex- lated IRS1/2 was indirect (Supplementary Fig. 6B). These ogenous and endogenous coimmunoprecipitation assays data suggest that WDFY2 regulates IRS1/2 activation via indicated that the INSR could interact with WDFY2 in the a mechanism other than directly binding both the sub- liver and in hepatocytes (Fig. 5D–F). We also investigated strate and the kinase. whether WDFY2 and the INSR colocalized under basal and After insulin binding, the insulin receptor kinase (IRK) insulin-stimulation conditions. Under serum-starved con- b-subunit is phosphorylated, resulting in IRK activation ditions, we observed that the INSR was mainly located on and rapid internalization into endosomes (13). We thus the plasma membrane and showed only a weak level of examined the localization of the INSR upon insulin stim- colocalization with WDFY2. After insulin stimulation, the ulation. We extracted endosome fractions from WT and INSR internalized and distinctly colocalized with WDFY2 Wdfy2 KO mice livers with or without insulin stimulation (Fig. 5G and H). and then detected the INSR levels. In the WT liver, the To determine whether the interaction between WDFY2 endosomal INSR was significantly increased after insulin and INSR is direct, we performed a glutathione S-transferase stimulation; this increase was suppressed in Wdfy2 KO (GST) pull-down assay. His-tagged INSR interacted with GST- mice (Fig. 6A and B). We also found that the interaction tagged WDFY2 but not with GST alone (Supplementary Fig. between WDFY2 and the INSR occurred in endosomes and 5I). WDFY2 contains seven WD40 repeats and an FYVE significantly increased after insulin stimulation (Fig. 6C

tryptophan-aspartic acid dipeptide. I(b): Schematic of the INSR b subunit. The CD contains the juxtamembrane (JM), tyrosine kinase (TK), and CT (C-terminal) domains. FnIII, fibronectin domains. J: GST, GST-WDFY2, GST-WD1–4, or GST-WD5–7 was incubated with whole-cell lysates of H2.35 cells. All the samples were resolved by SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining or immunoblotting (IB) with the indicated antibodies (*specific protein bands). K: GST or GST–INSR CD was incubated with H2.35 whole-cell lysates. All samples were resolved by SDS-PAGE, followed by Coomassie brilliant blue staining or Western blotting with the indicated antibodies (*specific protein bands). The data represent the means 6 SD. P values were calculated by two-way ANOVA with Tukey post hoc test (A–C) or Student t test (H) (*P , 0.05, **P , 0.01, ***P , 0.001 vs. the respective control). N.S., not significant. diabetes.diabetesjournals.org Zhang and Associates 1897

Figure 6—WDFY2 interacts with the INSR and localizes it in early endosomes (ENs) to recruit and activate IRS1/2. A–D: The endosomes fraction obtained from WT and Wdfy2 KO 3-month-old mice that were fasted overnight and injected intraperitoneally with either insulin (0.75 units/kg body wt) or saline for 15 min. Representative Western blots (A) and the associated quantifications (B) show the INSR levels in the liver endosomes fraction (n 5 4 mice in each group). C: The stimulation-dependent interaction between WDFY2 and the INSR was analyzed by immunoprecipitation (IP) with the indicated antibodies. D: Quantification of the relative levels of the interaction between WDFY2 and the INSR (n 5 3 mice in each group). E–I: The plasma membrane (PM) and endosomes fraction obtained from WT and Wdfy2 KO 3-month-old mice that were fasted overnight and injected intraperitoneally with either insulin (0.75 units/kg body wt) or saline for 15 min. E: Subcellular fractions enriched in endosomes and the plasma membrane were analyzed by Western blotting with the indicated antibodies. F–I: Quantification of the relative levels of IRS1 and IRS2 in the endosomes and plasma membrane fractions (n 5 3 mice in each group). J and K: Wdfy2 KO H2.35 hepatocytes were transduced with an empty vector, Flag-WDFY2, or Flag-WD5–7 by retrovirus system for 48 h. WT and Wdfy2 KO H2.35 1898 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020 and D). Because IRS binding with the INSR and the hyperglycemia, hyperinsulinemia, and hyperlipidemia, while phosphorylation of IRS occurs both on the plasma mem- overexpression of the WDFY2 truncation mutant had no obvi- brane and in endosomes upon insulin stimulation (13), we ous effect (Fig. 7E–H).ThesedataindicatethatAAV-mediated explored whether the IRS1/2 recruitment to endosome WDFY2 overexpression can alleviate insulin resistance was affected by WDFY2 depletion. We found that IRS1/2 and glucose intolerance in mice with metabolic disorders. recruitment to endosomes following insulin treatment was Finally, we infused WT mice with WDFY2 AAV and markedly suppressed in the Wdfy2 KO liver, yet IRS1/2 monitored WDFY2 expression relative to WT WDFY2 mice localization on the plasma membrane was unaffected (Fig. after 21 days (Supplementary Fig. 7D). The GTT and ITT 6E–I). These data indicate that WDFY2 interacts with the showed that AAV-mediated WDFY2 delivery enhanced INSR and localizes it to endosomes after insulin stimula- both insulin resistance and glucose intolerance in WT tion; then, INSR recruits IRS1/2 to endosomes. mice (Supplementary Fig. 7E and F). Furthermore, the We further confirmed the role of the WDFY2-INSR fasting insulin levels of WT mice overexpressing hepatic interaction in evoking downstream IRS1/2 and Akt acti- WDFY2 were significantly decreased (Supplementary Fig. vation by performing a rescue test. Here, overexpressing 7G). These results demonstrate that increased WDFY2 full-length WDFY2 fully rescued the impaired interaction expression in the liver ameliorates glucose utilization in between INSR and IRS in Wdfy2 KO H2.35 hepatocytes, healthy animals. whereas overexpressing the WD5–7 mutant plasmid had no effect on the interaction (Supplementary Fig. 6C and DISCUSSION D). Inhibition of IRS1/2 and Akt phosphorylation as a re- WDFY2 interacts with the INSR via its WD1–4 domain and sult of Wdfy2 KO was also alleviated upon the overexpres- localizes it in early endosomes to recruit downstream sion of full- length, but not WD5–7 mutant, WDFY2 (Fig. IRS1/2 following insulin stimulation. In this way, 6J and K). These results indicate that after insulin stim- WDFY2 mediates normal insulin signaling transduction ulation, the INSR is unable to localize to the endosome and in the liver and compromising the INSR-WDFY2 interac- phosphorylate IRS1/2 without associating with WDFY2. tion ultimately impairs downstream PI3K-Akt signaling. Hepatic WDFY2 Overexpression Attenuates Subsequently, phosphorylation of Akt2 and its direct Hyperglycemia and Insulin Resistance in Wdfy2 KO substrates is decreased, hepatic glucose production is in- Mice and db/db Mice appropriately promoted, and glycogen accumulation is We finally evaluated whether we could rescue the meta- suppressed. This novel mechanism explains how endo- bolic changes observed in our Wdfy2 KO mice with adeno- somal WDFY2 activates the hepatic insulin-evoked Akt2 associated viral WDFY2 or WD5–7 mutant. Ectopic signaling pathway (Fig. 8). full-length and mutant WDFY2 expression was detectable WDFY2 protein is highly expressed in the liver (37), but in the liver tissue of Wdfy2 KO mice at day 21 (Fig. 7A) but prior studies had not analyzed WDFY2 function in the liver not in the skeletal muscle and adipose tissue (Supplemen- in depth. We found that hepatic WDFY2 is dynamically tary Fig. 7A). Interestingly, GTT and ITT showed that AAV- suppressed in the liver of dietary and genetic mouse mediated WDF2 delivery alleviated both insulin resistance models of diabetes. Hepatic WDFY2 levels decreased and glucose intolerance in Wdfy2 KO mice compared with upon treatment with saturated but not unsaturated fatty Wdfy2 KO mice infused with a control AAV. WD5–7mutant acids, providing rationale for a link between WDFY2 and expression, however, could not rescue the impaired glucose metabolic diseases. Using our systemic Wdfy2 KO mouse and insulin tolerance (Fig. 7B and C). Similarly, in Wdfy2 KO model, we found that mice lacking WDFY2 exhibited in- mice with viral WDFY2 overexpression, the circulating in- sulin resistance, glucose intolerance, and impaired hepatic sulin levels at baseline were much lower than those in the glucose regulatory function when fed a normal diet. control Wdfy2 KO and Wdfy2 KO mice with viral WD5–7 Whether these described metabolic changes occur in fe- overexpression (Fig. 7D). We also found that the expression male mice now needs further study. of gluconeogenic genes (G6pc and Pck1)decreasedandthe Insulin resistance in Wdfy2 KO male mice is mainly due accumulation of liver glycogen increased after refeeding only to WDFY2 loss in the liver. Restoring WDFY2 liver but in the Wdfy2 KO mice with viral WDFY2 overexpression not adipose expression improved glucose homeostasis (Supplementary Fig. 7B and C). and insulin sensitivity. Interestingly, WDFY2 loss in adi- We also infused db/db mice (with frank diabetes) with pose tissue can influence glucose metabolism, as WDFY2 the full-length or mutant WDFY2 AAV. Here, full-length knockdown in 3T3-L1 adipocytes impairs glucose uptake WDFY2 overexpression in the liver alleviated fasting and adipogenesis (22,23). Adipocyte dysfunction causes

hepatocytes were stimulated with 10 mmol/L insulin for 5 min or unstimulated before lysis. Representative Western blots (J) and the associated quantifications (K) show the tyrosine phosphorylation levels of IRS1 and IRS2 and phosphorylation level of Akt in hepatocyte lysates (n 5 3). The data represent the means 6 SD. P values were calculated by Student t test (C and D), one-way ANOVA with Tukey post hoc test (J and K), or two-way ANOVA with Tukey post hoc test (A, B, and F–I) (**P , 0.01, ***P , 0.001 vs. the respective control). IB, immunoblotting; N.S., not significant; T-Akt, total Akt. diabetes.diabetesjournals.org Zhang and Associates 1899

Figure 7—Hepatic WDFY2 overexpression attenuates hyperglycemia and insulin resistance in Wdfy2 KO mice and db/db mice. A: Immunoblotting was performed to detect the protein levels in the liver tissue of 2-month-old Wdfy2 KO mice at day 21 after infection with an AAV encoding WDFY2 and WD5–7 mutant (n 5 5 mice for each group). B and C: GTT (B) and ITT (C) were performed in 3-month-old Wdfy2 KO mice, Wdfy2 KO mice with WDFY2 overexpressed, and Wdfy2 KO mice with WD5–7 overexpressed at the indicated time points (left). The area under the curve (AUC) for the GTT is shown (right) (n 5 5 mice for each group). D: Serum insulin levels in 3-month-old Wdfy2 KO mice, Wdfy2 KO mice with WDFY2 overexpressed, and Wdfy2 KO mice with WD5–7 overexpressed under fasting conditions (n 5 5 mice for each group). E–H: Two-month-old db/db mice were infected with AAV encoding WDFY2 or WD5–7 for 42 days. The fasting glucose (E), fasting serum insulin (F), fasting serum triglyceride (G), and fasting serum cholesterol (H) levels were determined (n 5 4 mice for each group). The data represent the means 6 SD. P values were calculated by one-way ANOVA with Tukey post hoc test (*P , 0.05, **P , 0.01, ***P , 0.001 vs. the respective control). CTR, empty vector control; N.S., not significant. 1900 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020

Figure 8—A model for the role of WDFY2 in insulin-evoked Akt2 signaling. After insulin stimulation, the INSR internalizes into endosomes in hepatocytes. Here, WDFY2 interacts with the INSR to localize it to early endosomes so that downstream IRS1/2 can be recruited to the endosomal INSR. If WDFY2 is absent, the INRS loses its localization in the early endosomes to interact and phosphorylate its substrates and mediate downstream metabolism-associated Akt2 activation in endosomes. P, phosphorylation.

decreased adipokine production, which can potentiate in- nuclear exclusion and reduced transcriptional activity (40). sulin sensitivity (38,39). GSK-3b phosphorylation relieves the inhibitory effect on Mechanistically, insulin stimulation activates Akt- downstream glycogen synthase activity (41). This path- mediated FoxO1 phosphorylation, resulting in FoxO1 way explains the increased levels of gluconeogenesis and diabetes.diabetesjournals.org Zhang and Associates 1901 decreased glycogen synthesis seen in the Wdfy2 KO mouse INSR upon insulin administration. We found that the liver. Akt2 has a role in glucose homeostasis (32,42), INSR cytoplasmic domain bound to the WDFY2 WD1–4 – preferentially associating with PI34P2 at the plasma mem- domain; a WD5 7 truncation mutant could localize on the brane and in endosomes, while Akt1 and Akt3 associate endosome membranes but could not interact with the with PI345P3 exclusively at the plasma membrane (43,44). endosomal INSR. Overexpressing this mutant in Wdfy2 PI34P2, which is abundant in early endocytic membranes, KO H2.35 hepatocytes and murine liver could not rescue promotes Akt2 activation in endosomes (45). WDFY2 has impaired insulin signaling in cells or insulin resistance in a distinct and crucial role in endosomal Akt2 signaling mice. Given the function of WDFY2 in endocytosis, activity and specifically regulates metabolism-related sub- WDFY2 might also have a broader role in internalization, strates, such as FoxO1 and GSK-3b (43,46). As a result of such as the internalization of plasma membrane–derived WDFY2 endosomal localization, Akt2 signaling in endo- PIP3 or PI34P2. A defect in the internalization of plasma somes is preferentially impaired such that the associated membrane–derived PIP3 or PI34P2 to endosomes also metabolic processes are mainly influenced in Wdfy2 KO selectively impacts the endosomal pool of phosphorylated H2.35 cells. Interestingly, Wdfy2 KO has no effect on the PI34P2 and thus Akt2 (19,43). phosphorylation of Akt substrates, which are mainly in- It is worth noticing that our study only demonstrated the volved in cell growth and proliferation. This finding is in function of WDFY2 in the liver; thus, insulin-stimulated line with previous studies showing that their phosphory- Akt2 activation may not require WDFY2 in skeletal muscle. lation is not regulated by endosomal Akt2 (47,48). Insulin- In addition, the mechanism of WDFY2 transcriptional mediated phosphorylation of Akt1 might be slightly regulation by metabolites, and its regulation in other affected by WDFY2 deficiency; further studies are required organs, now remains to be studied. Because WDFY2 over- to clarify this point. A previous study showed that Akt2 expression in the liver can reverse hyperglycemia, hyper- could bind to WDFY2 and was protected from degradation insulinemia, insulin resistance, and glucose intolerance in in adipocytes (23); our results, however, indicated that db/db mice, our data could have translational relevance. total Akt protein levels were unaltered in the Wdfy2 KO Indeed, increasing WDFY2 liver expression might consti- mouse liver and Wdfy2 KO H2.35 hepatocytes. We did not tute a new therapeutic concept for metabolic disorders. detect an association between WDFY2 and Akt2 in hep- Future work will now assess the role of WDFY2 in human atocytes. This discrepancy could be due to differences in patient samples. the models used, tissues investigated, and/or approaches used to detect the interaction. Most crucially, changes in WDFY2 protein expression might be tissue specific. Acknowledgments. The authors thank Dr. Ying Zhao (Peking University) Although we did not detect an association between and Dr. George Liu (Peking University) for critical comments on the manuscript. WDFY2 and Akt by coimmunoprecipitation in H2.35 cells, The authors also appreciate Dr. Karin Moelling (University of Zurich, Switzerland) it is still possible that WDFY2 regulates hepatic insulin- and Silvia Corvera (University of Massachusetts Medical School) for sharing the evoked Akt signaling. Insulin induces Akt activation by WDFY2 plasmids. Finally, the authors thank Dr. Jessica Tamanini (ETediting, stimulating INSR and IRS tyrosine phosphorylation, which Shenzhen, and Shenzhen University, China) for proofreading the manuscript. in turn triggers PI3K activation (49). We found that INSR Funding. This work was supported by grants from National Key R&D Program of China (2017YFA0503900), National Natural Science Foundation of China activation was not affected by Wdfy2 KO in H2.35 hep- fi (81720108027, 81530074), Science and Technology Program of Guangdong atocytes but IRS1/2 or PI3K activation was signi cantly Province in China (2017B030301016), Shenzhen Municipal Commission of impaired. In particular, insulin-stimulated IRS1/2 phos- Science and Technology Innovation (JCYJ20170818092450901), Discipline Con- phorylation was decreased in the endosomes, but not struction Funding of Shenzhen [(2016)1452], and Shenzhen Bay Laboratory plasma membrane, in Wdfy2 KO cells. WDFY2 might (SZBL2019062801011). thus help regulate IRS1/2 activation by the INSR. The Duality of Interest. No potential conflicts of interest relevant to this article WDFY2 WD repeats preferentially bind the INSR in re- were reported. sponse to insulin stimulation. After insulin stimulation, Author Contributions. L.Z. and W.-G.Z. conceived the project and the INSR on the plasma membrane is phosphorylated and designed most of the experiments. L.Z. performed most of the experimental internalized to endosomes. The INSR continues to signal at work. X. Li and N.Z. contributed to protein purification. X.Y., T.H., and L.W. endosomal compartments, providing the molecular cue to contributed to animal study. W.F. and F.Y. performed construction of plasmids. H.Z. and X. Lu helped with the design of some experiments. L.Z. and W.-G.Z. wrote activate IRS1/2 and class I PI3K to generate PIP at 3 the manuscript. W.-G.Z. supervised the project. L.Z., H.W., Y.T., X. Lu, and W.-G.Z. endosomes (11,12). PIP3 is a short-lived lipid messenger critically read the manuscript. W.-G.Z. and L.Z. are the guarantors of this work and, that generates PI34P2 in endosomes dephosphorylated by as such, had full access to all the data in the study and take responsibility for the inositol phosphatases (43). Endosomes are the major site integrity of the data and the accuracy of the data analysis. of IRS1/2 activation by the INSR (50). As an INSR-binding partner, WDFY2 retains the INSR within the early endo- References some so that downstream IRS1/2 can be recruited to the 1. Accili D. Lilly lecture 2003: the struggle for mastery in insulin action: from endosomal INSR and activated. Under basal conditions, triumvirate to republic. Diabetes 2004;53:1633–1642 nonphosphorylated IRS1/2 is distributed in the cytoplasm 2. Shaw LM. The insulin receptor substrate (IRS) proteins: at the intersection of but is rapidly recruited to endosomes to interact with the metabolism and cancer. Cell Cycle 2011;10:1750–1756 1902 WDFY2 Activates Insulin-Evoked Akt2 Signaling Diabetes Volume 69, September 2020

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