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

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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 regu- lates 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.
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