The Adaptor Protein APPL2 Controls Glucose-Stimulated Insulin Secretion Via F-Actin Remodeling in Pancreatic Β-Cells

The adaptor protein APPL2 controls glucose-stimulated insulin secretion via F-actin remodeling in pancreatic β-cells

Baile Wanga,b,1, Huige Linc,1, Xiaomu Lid, Wenqi Luc, Jae Bum Kime, Aimin Xua,b,f,2, and Kenneth K. Y. Chengc,2

aState Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, Hong Kong, China; bDepartment of Medicine, The University of Hong Kong, Hong Kong, China; cDepartment of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong, China; dDepartment of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai 200032, China; eDepartment of Biological Sciences, Institute of Molecular Biology and Genetics, Center for Adipose Tissue Remodeling, Seoul National University, Seoul 08826, South Korea; and fDepartment of Pharmacology & Pharmacy, The University of Hong Kong, Hong Kong, China

Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved September 28, 2020 (received for review August 19, 2020) Filamentous actin (F-actin) cytoskeletal remodeling is critical for have been observed in islet β-cells of diabetic mice and patients glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells, with type 2 diabetes, respectively (3, 4). Early studies indicated and its dysregulation causes type 2 diabetes. The adaptor protein that cortical F-actin acts as a barrier to prevent fusion of the APPL1 promotes first-phase GSIS by up-regulating soluble N-ethyl- insulin granule with the plasma membrane under basal glucose maleimide-sensitive factor attachment protein receptor (SNARE) condition (<5 mM) (5). In response to high glucose stimulation, protein expression. However, whether APPL2 (a close homology F-actin is depolymerized to allow movement and fusion of in- of APPL1 with the same domain organization) plays a role in β-cell sulin granules for exocytosis (6). The guanosine triphosphatases functions is unknown. Here, we show that APPL2 enhances GSIS (GTPase) of the Rho family, including cell division cycle 42 by promoting F-actin remodeling via the small GTPase Rac1 in pan- (Cdc42) and Rac1, are key players to control insulin granule β β creatic -cells. -cell specific abrogation of APPL2 impaired GSIS, trafficking via F-actin remodeling and required for GSIS (7, 8). leading to glucose intolerance in mice. APPL2 deficiency largely Adaptor proteins containing the NH -terminal BAR domain, a MEDICAL SCIENCES abolished glucose-induced first- and second-phase insulin secre- 2 central PH domain, and a COOH-terminal phosphotyrosine- tion in pancreatic islets. Real-time live-cell imaging and phalloidin bindingdomain(PTB)1and2(APPL1andAPPL2),apairof staining revealed that APPL2 deficiency abolished glucose-induced F-actin depolymerization in pancreatic islets. Likewise, knockdown endosomal and signaling molecules with the same domain orga- of APPL2 expression impaired glucose-stimulated F-actin depoly- nization and high protein sequence identity, are originally identi- merization and subsequent insulin secretion in INS-1E cells, which fied as the interacting partners of the small GTPase Rab5 (9). were attributable to the impairment of Ras-related C3 botulinum Subsequent studies showed that APPL1 and APPL2 positively and toxin substrate 1 (Rac1) activation. Treatment with the F-actin de- negatively, respectively, control glucose homeostasis via adipo- polymerization chemical compounds or overexpression of gelsolin nectin and insulin signaling (10–12). In pancreatic β-cells, APPL1 (a F-actin remodeling protein) rescued APPL2 deficiency-induced augments first-phase GSIS by up-regulating SNARE protein ex- defective GSIS. In addition, APPL2 interacted with Rac GTPase ac- pression through the insulin signaling cascade (13). APPL1 also tivating protein 1 (RacGAP1) in a glucose-dependent manner via enhances the potentiating effect of adiponectin on GSIS (14). The the bin/amphiphysin/rvs-pleckstrin homology (BAR-PH) domain of APPL2 in INS-1E cells and HEK293 cells. Concomitant knockdown of Significance RacGAP1 expression reverted APPL2 deficiency-induced defective GSIS, F-actin remodeling, and Rac1 activation in INS-1E cells. Our Dysregulation of cytoskeletal remodeling could result in de- data indicate that APPL2 interacts with RacGAP1 and suppresses fective GSIS and cause type 2 diabetes. Previous studies have its negative action on Rac1 activity and F-actin depolymerization reported the role of small GTPases including Rac1 and Cdc42 in β thereby enhancing GSIS in pancreatic -cells. the regulation of F-actin remodeling, whereas the upstream regulatory pathway remains poorly understood. Here, we glucose-stimulated insulin secretion | type 2 diabetes | Rac1 | F-actin identify the adaptor protein APPL2 as an upstream regulator of depolymerization | APPL2 Rac1 activation. APPL2 promotes F-actin remodeling by antag- onizing the inhibitory effect of RacGAP1 on Rac1 activation, SIS is a highly regulated and dynamic process in pancreatic which eventually enhances GSIS. Our findings fill the overall Gβ-cells. Glucose enters the β-cell and is, subsequently, me- puzzle of F-actin remodeling with a crucial piece and provide tabolized in mitochondria to produce ATP. The increased ATP/ insights into type 2 diabetes with disrupted actin dynamics. ADP ratio leads to the closure of ATP-sensitive potassium (KATP) channels, resulting in membrane depolarization, calcium influx, Author contributions: A.X. and K.K.Y.C. designed research; B.W., H.L., X.L., and W.L. performed research; J.B.K. contributed new reagents/analytic tools; B.W., H.L., and X.L. and ultimate insulin secretion (also known as first-phase GSIS) (1). analyzed data; and B.W., H.L., A.X., and K.K.Y.C. wrote the paper. This rapid insulin secretion is followed by a gradual and prolonged The authors declare no competing interest. second-phase GSIS which requires multiple coupling factors and This article is a PNAS Direct Submission. translocation of insulin granules from intracellular storage pools to Published under the PNAS license. the plasma membrane for exocytosis (2). Both first- and second- 1B.W. and H.L. contributed equally to this work. phase GSIS are diminished in type 2 diabetes (1), yet the under- 2To whom correspondence may be addressed. Email: [email protected] or kenneth.ky. lying pathogenic pathways remain poorly understood. [email protected]. Secretory vesicle trafficking and cytoskeleton reorganization This article contains supporting information online at https://www.pnas.org/lookup/suppl/ are essential for GSIS in pancreatic β-cells (2). Higher micro- doi:10.1073/pnas.2016997117/-/DCSupplemental. tubule density and compromised cellular cytoskeletal structure

www.pnas.org/cgi/doi/10.1073/pnas.2016997117 PNAS Latest Articles | 1of9 Downloaded by guest on September 30, 2021 loss-of-function APPL1 mutants are identified in the family with a Furthermore, an earlier and severe glucose intolerance was ob- high prevalence of diabetes, and APPL1 expression in the human served in RIP-APPL2 KO mice when they were fed with HFD pancreatic islet is positively correlated with GSIS (15). In addition, (SI Appendix, Fig. S1 A and B). Similar to the observation in the APPL1 protects pancreatic β-cells from apoptosis and inflamma- STC-fed group, HFD-fed RIP-APPL2 KO mice also displayed tion in the type 1 diabetic mouse model by inhibiting nuclear defective GSIS but similar insulin sensitivity when compared to factor NF-κB activation (16). their WT littermates and RIP-Cre controls (SI Appendix, Fig. Although the protective effects of APPL1 on β-cells are well S1 C and D). These data suggest that APPL2, like APPL1, is established, the role of its close homolog APPL2 on β-cell function essential for GSIS in pancreatic β-cells. Since WT littermates has never been explored. By using the β-cell specific APPL2 and RIP-Cre controls displayed similar glucose tolerance and knockout (KO) mouse model and the insulinoma cell line, we, insulin secretory ability, we only included WT littermates as here, show that APPL2 is essential for both first- and second- controls for all of the subsequent analyses. phase GSIS in pancreatic β-cells. Mechanistically, APPL2 regulates F-actin remodeling and Rac1 activity by interacting with Both First- and Second-Phase Glucose-Stimulated Insulin Secretions RacGAP1 (also known as MgcRacGAP, CYK-4, or Rac- Are Abolished in APPL2 Deficient Islets. We next assessed the effect GAP50C), a GTPase-activating protein (GAP) that inactivates of APPL2 deficiency on insulin secretion using ex vivo ap- Rac1 during cytokinesis (17, 18). proaches. Insulin secretion in response to low concentration of glucose stimulation (2.8 mM, basal) did not differ between the Results islets isolated from RIP-APPL2 KO mice and WT controls fed β-Cell Specific Deletion of APPL2 Results in Glucose Intolerance and with STC or HFD (Fig. 2 A and B and SI Appendix, Fig. S1E). In Impaired GSIS. Our recent study demonstrated that APPL2 is contrast, under high glucose condition (16.7 mM), the islets from expressed in both pancreatic islets and exocrine cells (19), but RIP-APPL2 KO mice fed with STC or HFD exhibited a dramatic whether this adaptor protein plays a role in β-cell function, in reduction of insulin secretion when compared to those isolated particular, in the regulation of GSIS, is unknown. To address this from WT littermates (Fig. 2 A and B and SI Appendix, Fig. S1E). question, we generated β-cell specific APPL2 KO mice (so-called On the other hand, potassium chloride ([KCl], which directly floxed/floxed RIP-APPL2 KO mice) by crossing APPL2 mice with the induces membrane depolarization and subsequent insulin se- transgenic mice expressing Cre recombinase under the control of cretion)-induced insulin secretion was similar between the two rat insulin promoter (19). Six-wk-old RIP-APPL2 KO mice, their genotypes (Fig. 2A and SI Appendix, Fig. S1E), indicating that wild-type (WT) littermates, and RIP-Cre controls were fed with the defect is specific to glucose stimulation and/or at the a standard chow (STC) or a high fat diet (HFD) for 10 wk. Al- downstream of calcium influx. Next, we determined whether though STC-fed RIP-APPL2 KO mice displayed normal glucose APPL2 deficiency affects first- and/or second-phase GSIS in tolerance at the age of 12 wk, they showed delayed glucose ex- isolated islets using a perfusion system as we previously de- cursion and diminished serum level of insulin during a glucose scribed (13, 20). Similar to the observation in the static insulin tolerance test (GTT) at age 16 wk when compared to WT lit- secretion assay, dynamic insulin secretion in response to low termates and RIP-Cre controls (Fig. 1 A–C). Insulin sensitivity glucose stimulation (2.8 mM) was comparable between islets did not differ among the three groups of mice at age 14 wk from RIP-APPL2 KO mice and their WT controls (Fig. 2C). (Fig. 1D). The above data indicate that glucose intolerance in Under high glucose condition (16.7 mM), insulin secretion from RIP-APPL2 KO mice was primarily due to defective GSIS. APPL2 deficient islets during first- (first 9 min of high glucose

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Fig. 1. β-cell specific deletion of APPL2 impairs glucose-stimulated insulin secretion and induces glucose intolerance. Male RIP-APPL2 KO mice, their WT littermates, and RIP-Cre controls fed with STC were used. (A) GTT, 2g/kg in 6 h fasted 16-wk-old mice. (B) The area under the curve (AUC) of GTT performed at different ages. (C) Insulin secretion during GTT in A.(D) Insulin tolerance test in the 14-wk-old mice. n = 5 for each group. All data are presented as the mean ± SD. Significance was determined using one-way ANOVA with Bonferroni correction. *P < 0.05, **P < 0.01 (KO vs. WT), #P < 0.05, and ##P < 0.01 (KO vs. RIP-Cre).

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.2016997117 Wang et al. Downloaded by guest on September 30, 2021 A B C 4.5 12 WT * WT * WT KO 6 KO * KO * 3.0 8 * 4 * * Glucose

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Fig. 2. The defective glucose-stimulated insulin secretion in APPL2 deficient islets is attributed to impaired glucose-induced F-actin remodeling. Pancreatic islets isolated from 16-wk-old male RIP-APPL2 KO mice and their WT littermates under a STC diet were used. (A) Static insulin secretion under basal (2.8 mM), glucose (16.7 mM), and KCl (50 mM) treatment conditions. n = 5–9. (B) The statics GSIS was repeated as A, and insulin secretion normalized with total intracellular insulin content. n = 7–9. (C) Dynamic insulin secretion in response to glucose stimulation (16.7 mM). Note that the islets were maintained in the conditioned medium with 2.8-mM glucose as the basal level. n = 4–5. (D) AUC for the first phase (0–9 min) and second phase (10–42 min) of insulin secretion in C. n = 4–5. (E) The islets were stimulated with glucose (16.7 mM) for 10 min, followed by staining with Alexa Fluor 488 phalloidin (green). The Right is a quantification of fluorescence intensity of the phalloidin signal relative to WT basal. n = 8–12. (Scale bar: 50 μM.) (F–H) The islets were incubated in Krebs buffer containing 2.8-mM glucose (basal) and SPY555-actin probe for 90 min, followed by 16.7-mM glucose (glucose) stimulation for 10 min. (F) Repre- sentative images of F-actin visualized by SPY555-actin probe (red) in the islets at the basal level and after high glucose stimulation for 10 min. Yellow rectangles indicate the regions with most obvious difference, while white rectangles indicate the regions without obvious difference before and after glucose stimulation. (Scale bar: 25 μM.) (G) Quantification of the dynamic change in the SPY555-actin signal intensity in the islets upon glucose stimulation. The control means the WT islets without high glucose stimulation. (H) The islets were pretreated with latrunculin A or vehicle for 1 h, followed by stimulation with 16.7-mM glucose for 30 min. Insulin secretion in the conditioned medium was measured. n = 5. All data are presented as the mean ± SD. Significance was determined using Student’s t test or one-way ANOVA with Bonferroni correction. *P < 0.05 and **P < 0.01. Representative images were shown.

stimulation) and second-phase (from 10 to 42 min of high glu- the plasma membrane (labeled by the Cellmask Green Plasma cose stimulation) was significantly diminished (Fig. 2 C and D). Membrane Stain) (SI Appendix, Fig. S4 A–C). Glucose stimulation had no obvious effect on the subcellular localization of APPL2 Regulates GSIS by Modulating F-Actin Remodeling. Dimin- APPL2 in MIN6 cells (SI Appendix, Fig. S4 A–C). ished GSIS could be due to reduced β-cell mass and insulin Although APPL1 deficiency reduces SNARE protein expres- content, changes in β- and α-cell areas, defective glucose me- sion and the number of insulin granules docking to the plasma tabolism, and/or its downstream signal transduction in β-cells. membrane (13), the islets from RIP-APPL2 KO mice displayed First, we examined islet morphology and mass as well as β- and similar expressions of both APPL1 and SNARE proteins in- α-cell areas by hematoxylin and eosin staining and immunoflu- cluding synaptosomal-associated protein 25, vesicle-associated orescence staining of insulin and glucagon, respectively. There membrane protein 2 , and syntaxin-1 when compared to those was no difference in islet morphology and mass as well as β- and isolated from WT controls (SI Appendix, Fig. S3 C and D). α-cell proportion between RIP-APPL2 KO mice and WT con- Electron microscope analysis revealed that the islets from RIP- trols (SI Appendix, Fig. S2 A–E). In addition, APPL2 deficiency APPL2 KO mice had more insulin granules with smaller diam- did not affect glucose-stimulated ATP production and calcium eters, and the proportion of immature insulin granules was influx in the isolated islets (SI Appendix, Fig. S3 A and B). As slightly increased (SI Appendix, Fig. S5 A–E). The total number APPL2 transduces signals via the early endosome and the cell of docked insulin granules in APPL2 deficient islets was com- membrane, we examined the effect of glucose stimulation on parable to their WT controls. Further analysis showed that there APPL2 subcellular localization in MIN6 β-cells. Our confocal was a significant increase in immature docked insulin granules imaging results showed that APPL2 distributed in the cytosol and and a trend of decrease in mature docked insulin granules in nucleus of MIN6 cells (SI Appendix, Fig. S4 A–C). A small APPL2 deficient islets compared to those in WT controls (SI portion of APPL2 was found to be localized in the early endo- Appendix, Fig. S5 F and G). In addition, the volume density of somes (labeled by the Rab5 antibody), insulin granules (labeled mature and immature docked insulin granules was significantly by the insulin antibody), and not much APPL2 could be found at reduced and increased, respectively, in APPL2 deficiency islets

Wang et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 30, 2021 (SI Appendix,Fig.S5F and H).ThesedataindicatethatAPPL2reg- Cdc42 in INS-1E cells with down-regulation of APPL2 using pull- ulates GSIS via a mechanism differing from that controlled by APPL1. down activation assays as recently described (28). Consistent with We and others previously demonstrated that APPL2 regulates previous studies (7, 29), glucose significantly increased both Rac1 small GTPase activity through direct interaction with small and Cdc42 activities in INS-1E cells transfected with the scrambled GTPase, such as Rab5 and Rab31 or the GAP protein TBC1 control (Fig. 3 G and H). However, knockdown of APPL2 ex- domain family member 1 (TBC1D1) (21, 22). On the other hand, pression resulted in an impairment of Rac1 activation but had no the BAR domain containing proteins are known to regulate actin effect on Cdc42 activity upon glucose stimulation (Fig. 3 G and H). remodeling (23). Thus, we hypothesized that APPL2 regulates To confirm the defective GSIS and F-actin remodeling are due to insulin secretion via GTPase activity and F-actin remodeling. To defective Rac1 activation, we transfected APPL2-knockdowned test this, we examined the distribution of F-actin in islets from INS-1E cells with a vector expressing a constitutively active Rac1 RIP-APPL2 KO mice by phalloidin staining and live-cell imag- mutant (Q61L) or an empty vector (as a control) (Fig. 4A). Rac1- ing. Consistent with previous studies (6, 24), phalloidin staining Q61L is not responsible for its upstream GAP and hence exhibits a revealed that intensity of F-actin was dramatically reduced in constitutively GTP-bound form (30). Consistently, knockdown of islets isolated from WT mice in response to high glucose stim- APPL2 expression diminished glucose-stimulated F-actin depoly- ulation (Fig. 2E), whereas genetic deletion of APPL2 abolished merization and insulin secretion, but such unresponsiveness to this glucose-induced effect (Fig. 2E). To further confirm this glucose was largely reversed by ectopic expression of the constitu- finding, we monitored F-actin remodeling in islets using a real- tively active Q61L mutant (Fig. 4 B and C and SI Appendix,Fig. time live-cell imaging approach. The islets from RIP-APPL2-KO S8B). These data suggest that APPL2 appears to control GSIS via mice and WT controls were incubated with a SPY555-actin Rac1-mediated F-actin remodeling. probe, a membrane permeable fluorescent probe that specifi- cally binds to endogenous F-actin filaments. We first validated The Interaction between APPL2 and RacGAP1 Is Crucial for GSIS and this method by observing the change in F-actin in WT islets F-Actin Remodeling. In our previous study, we have employed co- treated with latrunculin A, a F-actin depolymerization com- immunoprecipitation (IP) and mass spectrometry analysis to pound. Consistent with previous studies (25, 26), islets isolated identify the interacting partners of APPL2 in the HEK293 cells from WT controls exhibited obvious morphological changes and (21). Interestingly, we found that APPL2 was coimmunopreci- a small cluster of F-actin signal upon stimulation with latrunculin pitated (co-IP) with Rac GTPase activating protein 1 (Rac- A for an hour (SI Appendix, Fig. S6A). In line with our phalloidin GAP1), a GAP known to inactivate Rac1 during cytokinesis (17, staining data, the intensity of the F-actin signal was decreased in 18). In addition, a recent proteomics analysis indicates that the WT islets upon glucose stimulation in a time-dependent manner, interaction between Rac1 and RacGAP1 is observed in INS-1E but this glucose action was less pronounced in APPL2 deficiency cells, and RacGAP1 is expressed in human islets (31). Therefore, islets (Fig. 2 F and G). Notably, the F-actin signal did not ob- we hypothesized that APPL2 regulates Rac1 activity by antago- viously change in islets maintained at a low glucose condition for nizing GAP activity of RacGAP1 via the protein–protein inter- 10 min (Fig. 2G and SI Appendix, Fig. S6B), indicating that the action. We first validated and characterized the APPL2- reduction of the F-actin signal in WT and APPL2 deficiency is- RacGAP1 interaction by co-IP and immunoblotting analyses in lets was not due to photobleaching. These findings suggest that cells cotransfected with FLAG-tagged APPL2 and HA-tagged the defective GSIS observed in APPL2 deficient islets may be RacGAP1. IP of FLAG-tagged APPL2 protein resulted in co- due to aberrant F-actin depolymerization. To confirm this, we IP of HA-tagged RacGAP1 protein in HEK293 cells and vice treated the islets from RIP-APPL2 KO mice and WT controls versa (Fig. 5 A and B). In addition, the APPL2-RacGAP1 in- with the F-actin depolymerizing compounds latrunculin A or teraction also occurred in INS-1E cells and was enhanced by cytochalasin B. In line with the above data, defective GSIS was glucose stimulation in a time-dependent manner (Fig. 5 C and observed in APPL2 deficient islets, but this defect could be D). Further analysis indicated that the BAR-PH domain of partially rescued by treatment with latrunculin A or cytochalasin APPL2 was the functional unit for the RacGAP1 interaction, B (Fig. 2H and SI Appendix, Fig. S7A). while the BAR, the PH, or the PTB domain alone only displayed We also ascertained whether the inhibitory effects of APPL2 a very weak or even no interaction with the RacGAP1 protein silencing on the β-cell function also occurs in INS-1E pancreatic (Fig. 5 E and F). Apart from the interaction with RacGAP1, our insulinoma cells. We found that small interfering RNA (siRNA)- co-IP assay revealed that APPL2 also interacted with Rac1 (SI mediated knockdown of APPL2 expression abolished the effect Appendix, Fig. S9 A and B). of glucose on insulin secretion (Fig. 3 A and B), accompanied by To delineate whether APPL2 regulates GSIS and Rac1 defective F-actin depolymerization as determined by live-cell activation via RacGAP1, we employed siRNAs for concomi- imaging using a SPY555-actin probe and phalloidin staining tant knockdown of APPL2 and RacGAP1 in INS-1E cells. (Fig. 3 C–E and SI Appendix, Fig. S8A). Previous studies showed Protein expression of APPL2 and RacGAP1 were markedly that the F-actin remodeling protein gelsolin promotes GSIS by decreasedinINS-1EcellstransfectedwithsiRNAagainst inducing F-actin depolymerization in β-cells (27). We tested APPL2 and/or RacGAP1 compared with the cells transfected whether induction of F-actin depolymerization by overexpression with the scrambled control (Fig. 6A). As expected, knock- of gelsolin is able to rescue the defective GSIS in INS-1E cells down of APPL2 expression led to impairment of GSIS, Rac1 with APPL2 silencing. To this end, we cotransfected the plasmid activation, and F-actin depolymerization, whereas these im- encoding green fluorescent protein (GFP)-tagged gelsolin or a pairments were largely reversed when RacGAP1 expression GFP empty vector together with siRNA against APPL2 or was concomitantly down-regulated (Fig. 6 B–D and SI Ap- scrambled control into INS-1E cells for 48 h, followed by static pendix,Fig.S8C). These data suggest that APPL2 regulates GSIS assay. This analysis showed that overexpression of gelsolin GSIS and Rac1-mediated F-actin remodeling by suppression was able to revert APPL2 deficiency-induced defective GSIS (SI of RacGAP1. Appendix, Fig. S7 B and C). Taken together, the findings from islets and INS-1E cells suggest that APPL2 deficiency impairs Discussion GSIS by abrogating F-actin remodeling. Dysregulation of cytoskeletal remodeling causes impairment of fusion and trafficking of insulin granules to the plasma mem- Activation of Rac1 Rescues the Defective GSIS and F-Actin Remodeling in brane, leading to defective GSIS in type 2 diabetes. The small APPL2-Deficient β-Cells. Since F-actin remodeling is tightly regulated GTPase Rac1 positively controls GSIS via F-actin cytoskeletal by Rac1 and Cdc42 (6), we measured the activity of Rac1 and remodeling, but its upstream regulatory mechanism remains

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.2016997117 Wang et al. Downloaded by guest on September 30, 2021 Basal Glucose A Scramble siAPPL2 B C ** 800 APPL2 Scramble siAPPL2 600

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Fig. 3. Knockdown of APPL2 attenuates glucose-stimulated Rac1 activation and subsequent F-actin remodeling in INS-1E cells. (A–F) INS-1E cells were transfected with siRNA against scramble or APPL2 for 48 h. (A) Immunoblotting of APPL2 in the transfected cells. The Lower is the densitometric analysis for the relative abundance of APPL2 normalized with GAPDH. n = 5. (B) Static GSIS in the transfected cells. n = 7–8. (C) Representative images from live-cell imaging in INS-1E cells labeled with a SPY555-actin probe (red) under a basal condition (2.8 mM glucose) and upon glucose (16.7 mM) stimulation for 10 min were shown. (D) Representative images of the photobleaching experiment in INS-1E cells under low glucose (2.8 mM) condition for 10 min. (C, D) Yellow rectangles indicate the regions with the most obvious difference while white rectangles indicate the regions without obvious difference before and after glucose stimulation. (Scale bar: 10 μm.) (E) Quantification of the dynamic change in SPY555 signal intensity in an individual cell upon glucose stimulation and in the photobleaching control group in C and D.(F) Quantification of the relative fluorescence signal intensity in an individual cell before and after glucose stimulation for 10 min. (E, F) n = 8 for the control group and n = 14–25 for the other groups. (G) Rac1 activity in the transfected cells under the basal condition or after high glucose (16.7 mM) stimulation for 3 or 10 min. n = 4–6. (H) Cdc42 activity in the transfected cells under the basal level or after high glucose stimulation for 3 min. n = 10. All data are presented as the mean ± SD. Student’s t test or one-way ANOVA with Bonferroni correction for multiple com- parisons was used. *P < 0.05 and **P < 0.01. Representative images were shown.

poorly characterized. In this study, we show that the BAR-PH promoting effect on insulin secretion. Our previous study showed domain containing protein APPL2 regulates Rac1-mediated that APPL1 deficiency impairs first- but not second-phase GSIS F-actin depolymerization and insulin secretion by interacting by downregulating SNARE proteins via an Akt-dependent with RacGAP1 in pancreatic β-cells (SI Appendix, Fig. S10). pathway in pancreatic β-cells, whereas the current study indi- APPL2 deficiency in pancreatic β-cells leads to a dramatic re- cates that APPL2 deficiency abolishes both first- and second- duction of GSIS and glucose intolerance in mice, and such im- phase GSIS, at least, in part, via modulating the RacGAP1- pairment is more pronounced in the dietary-induced obese Rac1 pathway. APPL1 has also been shown to increase the mouse model. promoting effect of adiponectin on GSIS (14). In the current APPL1 and APPL2 are a pair of Yin and Yang proteins that study, we show that APPL2 deficiency has no obvious effect on positively and negatively regulate insulin and adiponectin actions SNARE protein expression in pancreatic islets. Therefore, the in multiple cell types, respectively (10, 32, 33). APPL1 and regulatory pathways of APPL2 and APPL1 on GSIS appear to be APPL2 form a heterodimer and/or homodimer via their BAR- distinct and independent of each other, although the effect of PH domains and have distinct structures and electrostatic sur- APPL2 deficiency on insulin and adiponectin signaling in pan- faces (22, 34). Both APPL1 and APPL2 can bind to the adipo- creatic β-cells remains to be further determined. nectin receptors but exert opposite effects on adiponectin- Rac1 is a master regulator of cytoskeletal remodeling and is induced AMPK activation and subsequent glucose uptake and important for insulin granule fusion and trafficking for subsequent nitric oxide production in myotubes and endothelial cells, re- secretion in pancreatic β-cells (2). Inactivation of Rac1 by genetic spectively (11, 32, 33). As an insulin sensitizer, APPL1 enhances ablation, the pharmacological inhibitor, or siRNA abolishes GSIS in Akt activation by blocking the interaction between Akt and its insulinoma cell lines or islets (8, 29, 36). The activity of Rac1 is endogenous inhibitor Tribble 3 (13, 35). On the other hand, regulated by its upstream regulators including guanine exchange overexpression of APPL2 suppresses insulin-stimulated glucose factors ([GEFs], positive regulator), Rho GDP-dissociation inhibi- uptake in myotubes at a step downstream of Akt (21). On the tors ([Rho-GDIs], negative regulator) and GAPs (negative regula- contrary, in pancreatic β-cells, both APPL1 and APPL2 exert a tor), respectively. Two GEFs including T-lymphoma invasion and

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1.1 ## ** siAPPL2 + Empty siAPPL2 + Q61L 1.0 Basal Glucose Basal Glucose 0.9 F-actin signal F-actin

Relative intensity of intensity Relative 0.8

0.7 Basal Glucose

Photobleaching control Time: 0 min 10 min

Scramble + Empty

Fig. 4. APPL2 regulates glucose-stimulated insulin secretion and F-actin remodeling via Rac1 activation. (A–C) INS-1E cells were cotransfected with siRNA against the scrambled control or APPL2 and an empty vector or plasmid encoding Rac1-Q61L mutant (active form of Rac1) for 48 h. The transfected cells were subjected to immunoblotting analysis (A), static GSIS (B), and (C) live-cell imaging of F-actin remodeling using a SPY555-actin probe (red) as described in Fig. 3. The chart on the Right is the quantification of the relative fluorescence signal intensity in an individual cell before (basal) and after glucose stimulation (16.7 mM) for 10 min. (B) n = 6. (C) n = 10 for the photobleaching control group, and n = 12–14 for the other groups. (Scale bar: 10 μm.) All data are presented as the mean ± SD. Significance was determined using one-way ANOVA with Bonferroni correction. **P < 0.01 (Scramble + Empty vs. siAPPL2 + Empty) and ##P < 0.01 (siAPPL2 + Empty vs. siAPPL2 + Q61L). Representative images were shown.

metastasis-inducing protein 1 (Tiam1), and guanine nucleotide ex- islets. Further investigation on whether activation of Rac1 or inac- change factor (Vav2) have been shown to promote GSIS via Rac1 tivation of RacGAP1 is able to rescue the defective first- and activation in pancreatic β-cells (37–39). Rho-GDIα and Rho-GDI-β second-phase GSIS in APPL2 deficient islets is required. Although are expressed in INS-1E cells (40) and exhibit distinct effects on concomitant knockdown of RacGAP1 almost completely rescues GSIS, although both of them inhibit Rac1 and Cdc42 activation the defective Rac1 activation and F-actin depolymerization in (40–42). To date, no GAP has been identified to inactivate Rac1 APPL2 down-regulated INS-1E cells (Fig. 6), its rescue effect on and control GSIS in β-cells. Although a recent proteomics study insulin secretion is partial. These data indicate that an additional identified RacGAP1 as a potential interacting partner of Rac1 in pathway, such as insulin and adiponectin signaling, might mediate INS-1E 832/13 cells (31), its role in β-cell function remains un- the APPL2 actions in pancreatic β-cells. Taken in conjunction, our known. RacGAP1 has been shown to be abundantly expressed in data suggest that APPL2 orchestrates GSIS, at least, in part, via the human and rodent islets and is known to inactivate Rac1 by pro- RacGAP1-Rac1 signaling axis-mediated F-actin remodeling. moting hydrolysis of GTP bound to GDP during cytokinesis (17, 18, The BAR domain proteins control multiple biological pathways 31, 43). Similar to the pancreatic β-cells lack of Rac1 (8), APPL2 including F-actin remodeling, activity of GTPases, and vesicle deficiency only affects glucose- but not KCl-induced insulin secre- trafficking and fusion (44, 45). We and others have demonstrated tion, and such a defect can be reversed by treatment with latrun- that the BAR domain proteins affect the activity of GAP via culin A, cytochalasin B, or overexpression of gelsolin. Knockdown protein–protein interaction (21, 46). Deletion of BAR domain in of APPL2 expression impairs Rac1 activation and F-actin remod- Arf-GAP with the SH3 domain, ANK repeat, and PH domain- eling induced by glucose. The defects in APPL2 knockdown cells containing protein 1 (ASAP1) increases its GAP activity (46). We are largely reversed by simultaneous down-regulation of RacGAP1 previously reported that APPL2 prevents insulin-elicited phos- or ectopic expression of the constitutively active Rac1 mutant. phorylation of the GAP domain of TBC1D1 at threonine 596 via Upon glucose stimulation, the interaction between APPL2 and protein–protein interaction, which, in turn, inhibits glucose uptake RacGAP1 is enhanced in INS-1E cells. In addition, it is worth in skeletal muscles (21). Our current study indicates that the BAR- noting that Rac1 deficient islets display impairment of second- but PH domain but not the BAR, the PH, or the PTB domain alone not first-phase GSIS (8). On the contrary, genetic deletion of mediates the interaction of APPL2 with RacGAP1. We speculated APPL2 abolishes both first- and second-phase GSIS in pancreatic that the BAR-PH domain of APPL2 is able to antagonize the

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.2016997117 Wang et al. Downloaded by guest on September 30, 2021 A HA-RacGAP1 - + + B FLAG-APPL2 - + + FLAG-APPL2 + + - HA-RacGAP1 + + -

FLAG HA IP: IP: HA FLAG HA FLAG

FLAG HA Total Total lysate lysate HA FLAG

C D IP: IgG RacGAP1 FLAG-APPL2 - + + + + + Glucose 0 0 2 5 10 30 min HA-RacGAP1 + + + + + + APPL2 Glucose 0 0 2 5 10 30 min IP HA RacGAP1 IP: FLAG FLAG APPL2

HA Total Total RacGAP1 lysate lysate FLAG βactin

EFHA-RacGAP1

FLAG- WT BAR PH PTB APPL2: BAR-PH BAR PH HA BAR BAR BAR PTB MEDICAL SCIENCES PH PH IP: FLAG PTB PTB FLAG

Total lysate HA

Fig. 5. APPL2 interacts with RacGAP1 in a glucose-dependent manner in INS-1E cells. HEK293 cells (A, B) or INS-1E cells (C) were cotransfected with plasmids encoding FLAG-tagged APPL2 and HA-tagged RacGAP1 for 48 h. INS-1E cells were stimulated with glucose (16.7 mM) for the indicated time points. The transfected cells were subjected to IP using an antibody against HA tag (A) or FLAG tag (B and C). (D) INS-1E cells stimulated with glucose (16.7 mM) for the indicated time points were subjected to IP using an anti-RacGAP1 antibody or nonspecific IgG as a negative control, followed by immunoblotting analysis as indicated. (E) Schematic of FLAG-tagged full-length (FL) APPL2 and its truncated mutants. (F) HEK293 cells were transfected with indicated APPL2 plasmids and HA-RacGAP1, followed by IP and immunoblotting analysis. Representative images were shown from three independent experiments.

inhibitory effect of RacGAP1 on Rac1 activation via protein– insulin granules to the plasma membrane for exocytosis. There- protein interaction. Apart from APPL1 and APPL2, several other fore, the APPL2-RacGAP-Rac1 signaling axis is essential for BAR domain proteins including protein interacting with C-kinase tight regulation of GSIS and subsequent glucose homeostasis. 1, islet cell autoantigen 69 kDa, and arfaptin-1 have been impli- cated in insulin secretion by controlling biogenesis, maturation, and trafficking of insulin granules (47–49). Deletion of these BAR Materials and Methods domain containing proteins leads to generation of small non- Animal Studies. All animals were sex and age matched, and littermates were functional and immature insulin granules in pancreatic β-cells, used, as indicated in the figure legends. Animals were allocated to their which in turn causes glucose intolerance in mouse models (19, experimental groups according to their genotypes. Therefore, no randomi- 47–49). Of note, APPL2, Arfaptin-1, ICA69, and PICK1 belong to zation was used unless otherwise noted. The investigators were not blinded floxed/floxed the classical crescent-shape BAR domain subfamily. Indeed, we to the experimental groups. Generation of APPL2 mice, RIP-APPL2 also found that APPL2 somehow affects the docking of mature KO mice, and RIP-Cre control mice have been described in our previous insulin granules to the plasma membrane. This change might ex- publications (19, 21). All mice were kept in cages in a 12h/12h light/dark cycle plain the defective first-phase GSIS in the APPL2 deficiency islet, and had free access to water and either STC (Purina) or 45% HFD (Cat. no. D12451, Research Diets). GTT and GSIS were performed in 6-h-fasted mice yet further experiments are required to delineate the underlying (fed with STC) and overnight-fasted mice (fed with HFD) after intraperito- cause. Collectively, these findings indicate that the BAR domain neal (i.p.) injection of D-glucose (2 g/kg) as previously described (13, 20). For protein family plays an essential role in the regulation of insulin the insulin tolerance test, mice were i.p. injected with human recombinant secretion via multiple pathways and levels. insulin (Actrapid HM Novo Nordisk) after 6-h fasting. Blood samples were To summarize, our current study highlights the importance of taken from the tail vein for the measurement of glucose and insulin levels β APPL2 in the regulation of GSIS in pancreatic -cells. In re- using a glucose meter and an insulin enzyme-linked immunosorbent assay sponse to glucose, APPL2 interacts with RacGAP1, which in (ELISA) kit (Cat. no. 32380, Antibody and Immunoassay Services, The Uni- turn inhibits the conversion of active GTP-bound Rac1 to inac- versity of Hong Kong [HKU]), respectively. All animal experimental protocols tive GDP-bound Rac1 (SI Appendix, Fig. S10). Activation of were approved by the animal ethics committee of HKU and The Hong Kong Rac1 depolymerizes F-actin, allowing fusion and trafficking of Polytechnic University (PolyU).

Wang et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 30, 2021 Scramble Scamble A B siAPPL2 C siAPPL2 siAPPL2+siRacGAP1 siAPPL2+siRacGAP1 siAPPL2+ 800 ** ## 1.75 Scramble siAPPL2 siRacGAP1 ** 600 1.50 APPL2 # 1.25 1.00 RacGAP1 400 0.75 GAPDH 200 0.50 Insulin Secretion Insulin

(ng/mg protein/20 min) protein/20 (ng/mg 0.25

0 change) activity (fold Rac1 0.00 Basal Glucose Basal Glucose

D Basal Glucose Photobleaching control Time: 0 min 10 min

Scramble Scramble

1.2 siAPPL2 ## 1.0 **

0.8 Control siAPPL2 + Scramble siRacGAP1 siAPPL2 siAPPL2+siRacGAP1

Relative intensity of F-actin signal F-actin of intensity Relative 0.6 Basal Glucose

Fig. 6. APPL2 regulates Rac1 activity and F-actin remodeling via RacGAP1. (A–D) INS-1E cells were cotransfected with siRNA against APPL2, RacGAP1, and/or the scrambled control for 48 h, followed by immunoblotting analysis of APPL2, RacGAP1, and GAPDH (A), static GSIS (B), measurement of Rac1 activity at the basal level or after glucose stimulation for 3 min (C), and live-cell staining of F-actin (D) as described in Fig. 3. Images taken from INS-1E cells transfected with the scrambled control kept in a Krebs buffer with 2.8-mM glucose for 10 min were used as the photobleaching control. The chart on the Right is the quantification of the relative fluorescence signal intensity in an individual cell before (basal) and after glucose stimulation (16.7 mM) for 10 min.(B) n = 11–16. (C) n = 6–8. (D) n = 8 for the photobleaching control group and n = 14–25 for the other groups. (Scale bar: 10 μm.) All data are presented as mean ± SD. Significance was determined using one-way ANOVA with Bonferroni correction. **P < 0.01 (Scramble vs. siAPPL2), #P < 0.05, and ##P < 0.01 (siAPPL2 vs. siAPPL2 + siRacGAP1). Representative images were shown.

Islet Isolation and Insulin Secretion Assay. Mice were fasted for 4 h and killed content and insulin secreted in the conditional medium were measured by cervical dislocation. The pancreas was perfused with collagenase P (1.4 mg/mL, using the insulin ELISA kit. Cat. no. 11213865001, Roche) via the bile common duct and subsequently digested at 37 °C for 20 min. The digested pancreas was then filtered Real-Time Live-Cell Imaging. F-actin fluorescent probe SPY555-actin (Cat. no. through 500- and 70-μm cell strainers. The captured islets at 70-μmcell CY-SC202, Cytoskeleton) was used to stain and visualize F-actin in islets and strainers were washed with solution G (Hanks’ balanced salt solution [Cat. INS-1E cells (50). Islets isolated from RIP-APPL2 KO mice and their WT con- no. 14065056, ThermoFisher Scientific] with 0.1% bovine serum albumin trols or INS-1E cells were seeded into an 8-well Nunc Lab-Tek II chambered [BSA]) and cultured in Roswell Park Memorial Institute (RPMI) 1640 with coverglass with a no. 1.5 borosilicate glass bottom (ThermoFisher Scientific) 10% fetal bovine serum (FBS) overnight. The isolated islets with similar size and cultured for overnight. Islets or INS-1E cells transfected with siRNA were manually picked under a microscope and then washed twice with against APPL2 (siAPPL2), RacGAP1 (siRacGAP1), scrambled control, or plas- Krebs buffer containing 0.1% fatty acid-free BSA and 2.8-mM glucose for 1 mid vectors encoding the Rac1-Q61L mutant or empty vector for 48 h were h, followed by stimulation with different stimulants for various time periods incubated in Krebs buffer containing 2.8-mM glucose and SPY555-actin as specified in each figure legend. In Fig. 2H, the islets were pretreated with probe (1,000× dilution according to manufacturer instruction) at 37 °C for 0.5-μM latrunculin A (Cat. no. 76343–93-6, Cayman Chemical) or dimethyl 90 min. After that, a dynamic change in F-actin signal was recorded at the sulfoxide (DMSO) (as a vehicle control) for 1 h. In SI Appendix, Fig. S7A,the basal level and then switched to glucose (16.7 mM) stimulation for 10 min islets were pretreated with 5-μM cytochalasin B (Cat. no. C6762, Sigma- using the live-cell confocal imaging systems with temperature, oxygen, and Aldrich) or DMSO for 1.5 h. For the determination of dynamic insulin se- carbon dioxide control (University Life Science, PolyU, or Centre for Imaging cretion, the isolated islets were incubated with Krebs buffer for 30 min and and Flow Cytometry Core, HKU). Another batch of cells or islets with SPY555- perfused with Krebs buffer containing 2.8-mM glucose for 6 min, and the actin labeling were subjected to recording of the F-actin signal for 10 min in perfusate was, then, switched to Krebs buffer containing 16.7-mM glucose. the Krebs buffer containing 2.8-mM glucose, which was served as the pho- Eluted fractions were collected at 3-min intervals for 42 min. The first- and tobleaching control. The F-actin images in INS-1E cells and islets were cap- second-phase insulin secretions were defined as 0–9 and 10–42 min, re- tured and recorded every 20 s or every 70 s using a ZEISS LSM 900 confocal spectively. Insulin secreted in each fraction was measured using the insulin microscope or a Leica TCS SP8 MP multiphoton/confocal microscope, re- ELISA kit and normalized with total number of islets or intracellular insulin spectively. For the analysis in islets, data were averaged from, at least, 11 content as indicated in the figure legend. To extract insulin from pancreatic islets from three animals per each genotype. F-actin fluorescence intensity in islets, the isolated islets were incubated with acid ethanol (1.5% hydrogen an individual islet was quantified by ImageJ and normalized with islet size. chloride in 70% ethanol) at −20 °C for overnight and then sonicated. The For the analysis in INS-1E cells, data were averaged from, at least, 12 cells per lysate was incubated overnight at −20 °C and centrifuged at 10,000 g at 4 °C. experiment group and, at least, eight cells from the photobleaching group The pancreatic extract was neutralized with equal volume of 1-M from three independent experiments. Fluorescence signal intensity of F-actin 2-amino-2-hydroxymethyl-1,3-propanediol buffer (pH 7.5). Extracted insulin in an individual cell was quantified using ZEN software. Relative intensity of

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.2016997117 Wang et al. Downloaded by guest on September 30, 2021 the F-actin signal before and after glucose stimulation for 10 min or a dynamic (HKU) for their assistance on live-cell imaging experiments. This research was change in F-actin signal was shown and specified in each figure legend. funded by Hong Kong Research Grant Council General Research Grant (17101815), National Natural Science Foundation of China (NSFC) (Grants Data Availability. All study data are included in the article and supporting 81471015 and 91857119), National Key Research and Development Program information. of China (Grant 2016YFC1305003), and Hong Kong Research Grant Council Area of Excellence (AoE/M-707/18). JBK was supported by the National ACKNOWLEDGMENTS. We thank Dr. Michael Yuen from University Life Research Foundation of 868 Korea (NRF) grant funded by the Korea Science (PolyU) and Dr. Miao Chen from Imaging and Flow Cytometry Core government (MSIT) (NRF-2020R1A3B2078617).

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