3748 Diabetes Volume 63, November 2014

Kenneth K.Y. Cheng,1,2 Weidong Zhu,2 Bin Chen,2 Yu Wang,1,3 Donghai Wu,4 Gary Sweeney,5 Baile Wang,1,2 Karen S.L. Lam,1,2 and Aimin Xu1,2,3

The Adaptor APPL2 Inhibits Insulin-Stimulated Glucose Uptake by Interacting With TBC1D1 in Skeletal Muscle

Diabetes 2014;63:3748–3758 | DOI: 10.2337/db14-0337

Insulin stimulates glucose uptake by promoting the Insulin maintains glucose homeostasis by facilitating the trafficking of GLUT4 to the plasma membrane in muscle uptake of postprandial glucose into adipose tissues and cells, and impairment of this insulin action contributes to skeletal muscle, the latter of which accounts for ;75% of hyperglycemia in type 2 diabetes. The adaptor protein total glucose disposal (1). The binding of insulin to its recep- APPL1 potentiates insulin-stimulated Akt activation and tors elicits tyrosine phosphorylation of insulin receptor sub- downstream actions. However, the physiological func- strates, which in turn activates phosphatidylinositol 3-kinase tions of APPL2, a close homolog of APPL1, in regulating (PI3K), leading to the membrane recruitment and activation glucose metabolism remain elusive. We show that of Akt. Activated Akt subsequently induces the translocation insulin-evoked plasma membrane recruitment of GLUT4 of GLUT4 from intracellular vesicular compartments to the and glucose uptake are impaired by APPL2 overexpres- plasma membrane for glucose uptake. In type 2 diabetic sion but enhanced by APPL2 knockdown. Likewise, patients, the ability of insulin to stimulate glucose uptake is conditional deletion of APPL2 in skeletal muscles significantly impaired, owing in part to the defective insulin- enhances insulin sensitivity, leading to an improvement SIGNAL TRANSDUCTION dependent recruitment of GLUT4 to the plasma membrane in glucose tolerance. We identified the Rab-GTPase– (2,3). In rodents, genetic ablation of GLUT4 in skeletal muscle activating protein TBC1D1 as an interacting partner of APPL2. Insulin stimulates TBC1D1 phosphorylation on causes insulin resistance and diabetes (4), whereas overex- serine 235, leading to enhanced interaction with the pression of GLUT4 alleviates hyperglycemia and insulin re- db/db BAR domain of APPL2, which in turn suppresses insulin- sistance in diabetic mice (5). GLUT4 translocation is evoked TBC1D1 phosphorylation on threonine 596 in tightly controlled by insulin signaling cascades and a se- cultured myotubes and skeletal muscle. Substitution of ries of small GTPase (6). Studies have demon- – serine 235 with alanine diminishes APPL2-mediated in- strated that the Rab-GTPase activating proteins (GAPs) hibition on insulin-dependent TBC1D1 phosphorylation Tre-2/Bub2/Cdc16 domain family, member 1 (TBC1D1) on threonine 596 and the suppressive effects of TBC1D1 andmember4(TBC1D4[alsoknownasAS160])may on insulin-induced glucose uptake and GLUT4 transloca- integrate insulin signaling and Rab-GTPase activity, tion to the plasma membrane in cultured myotubes. There- thereby regulating GLUT4 trafficking and glucose up- fore, the APPL2–TBC1D1 interaction is a key step to fine take (6). However, the detailed intracellular signaling tune insulin-stimulated glucose uptake by regulating the events that confer insulin-elicited glucose uptake are membrane recruitment of GLUT4 in skeletal muscle. still not fully characterized.

1State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Received 25 February 2014 and accepted 23 May 2014. Kong, Hong Kong This article contains Supplementary Data online at http://diabetes 2 Department of Medicine, The University of Hong Kong, Hong Kong .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0337/-/DC1. 3Department of Pharmacology & Pharmacy, The University of Hong Kong, Hong K.K.Y.C. and W.Z. contributed equally to this study. Kong 4The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine © 2014 by the American Diabetes Association. Readers may use this article as and Health, Chinese Academy of Sciences, Guangzhou, China long as the work is properly cited, the use is educational and not for profit, and 5Department of Biology, York University, Toronto, Ontario, Canada the work is not altered. Corresponding author: Aimin Xu, [email protected], or Kenneth K.Y. Cheng, [email protected]. diabetes.diabetesjournals.org Cheng and Associates 3749

Mounting evidence suggests that the adaptor protein antibody against TBC1D1 (#5929), and mouse monoclo- APPL1, which comprises an NH2-terminal Bin/amphiphysin/ nal antibodies against phosphotyrosine and GLUT4 were Rvs (BAR) domain, a central pleckstrin (PH) from Cell Signaling Technology. Rabbit anti-HA polyclonal domain, and a COOH-terminal phosphotyrosine-binding and mouse monoclonal antibodies against FLAG and (PTB) domain, is an insulin-sensitizing molecule in multiple c-Myc were from Sigma, and a rabbit polyclonal antibody insulin-responsive tissues (7). Genetic disruption of APPL1 against TBC1D1 (ab56191) was from Abcam. Rabbit causes insulin resistance and defective glucose-stimulated polyclonal antibodies against APPL1, APPL2, phospho- insulin secretion, leading to glucose intolerance in mice TBC1D1 (serine [Ser] 235 and threonine [Thr] 596), (8). In contrast, transgenic overexpression of APPL1 pre- phospho-Akt (Ser-473), and recombinant proteins of full- vents obesity-induced deleterious effects on glucose homeo- length and truncated APPL1 and APPL2 were obtained stasis and endothelial and cardiac functions (8–10). Hepatic from Antibody and Immunoassay Services, The University overexpression of APPL1 improves hyperglycemia, glucose of Hong Kong (HKU). Human recombinant insulin was intolerance, and insulin sensitivity in db/db diabetic mice, from Novo Nordisk. The PI3K inhibitor LY294002 and whereas hepatic silencing of APPL1 causes insulin resistance cytochalasin B were from Sigma, and 2-deoxy-[3H]-glucose and hyperglycemia in lean mice (11). In pancreatic b-cells, and [14C]-mannitol were from PerkinElmer. APPL1 promotes glucose-stimulated insulin secretion by N Animal Studies upregulating the expression of soluble -ethylmaleimide- To generate APPL2 transgenic (APPL2 Tg) mice, human sensitive factor attachment protein receptor proteins APPL2 cDNA was cloned into a transgenic vector under in an Akt-dependent manner (8). At the molecular level, the control of cytomegalovirus immediate early b-actin the interaction between APPL1 and Akt prevents Akt promoter (9). The transgenic mice were generated as from binding to the pseudokinase tribble 3 (TRB3), described previously (9) and were screened by PCR anal- thereby promoting Akt to the plasma membrane for fur- ysis with genotyping primers as listed in Supplementary ther activation in hepatocytes, endothelial cells, and pan- Table 1. creatic b-cells (8,9,11,12). Furthermore, APPL1 potentiates APPL2 knockout (KO) mice were generated by Shang- the insulin-sensitizing effects of adiponectin on promotion hai Nanfang Center for Model Organisms. The targeting of glucose uptake by direct interaction with the two adipo- construct containing loxP sites flanking exon 5 of the nectin receptors (13). APPL2 and the FRT-flanked selection cassette up- APPL2 is a close homolog of APPL1 abundantly expressed stream of the loxP sites was electroporated into embryonic in skeletal muscle. These two adaptor proteins share 52% stem cells, followed by selection of positive embryonic stem identity and 72% similarity in amino acid sequence and the clones, microinjection, and chimera identification as de- same domain organization (7). APPL1 and APPL2 appear to scribed previously (9). To generate muscle-specificAPPL2 play a similar role in mediating growth factor–induced cell floxed fi fi KO mice and their wild-type (WT) littermates, APPL2 proliferation in broblasts and apoptosis in zebra sh (14,15). mice were crossed with transgenic mice expressing Cre Ontheotherhand,aninvitrostudydemonstratedthatthese recombinase under the control of muscle creatine kinase two proteins possess distinct or even opposite functions in promoter (The Jackson Laboratory), and their genotypes regulating glucose and lipid metabolism (16). Structural anal- were identified by PCR analysis using the primers listed ysis revealed that APPL2 incorporates two homodimers, in Supplementary Table 1. whereas APPL1 incorporates only one homodimer in the Both APPL2 Tg and APPL2 KO mice were backcrossed asymmetric unit (17–19). Although APPL1 binds to the onto a C57BL/6 genetic background for at least seven adiponectin receptors and increases adiponectin-induced generations and housed in a room with temperature glucose uptake and fatty acid oxidation, APPL2 inhibits (23 6 1°C) and light (12-h light-dark cycle) control. adiponectin actions in muscle cells (13,16). Four-week-old male APPL2 Tg mice, muscle-specific Although the insulin-sensitizing effects of APPL1 are APPL2KO,andtheirWTlittermateswerefedwitha well characterized, little is known about the physiological standard chow (Purina Mills) comprising 20% kcal from role of APPL2 in insulin signaling and glucose metabolism. protein, 10% kcal from fat, and 70% kcal from combined In this study, we provide both in vitro and in vivo evidence simple carbohydrates. Glucose tolerance test (GTT) and that APPL2 is a negative regulator of insulin-stimulated insulin tolerance test (ITT) were performed in 16-h– and glucose transport in skeletal muscle. Furthermore, we 6-h–fasted animals as previously described (8). All animal identify TBC1D1 as an interacting partner and a down- experimental protocols were approved by the Animal stream effector that mediates the suppressive effect of Ethics Committee of HKU. APPL2 on insulin-elicited plasma membrane translocation of GLUT4. RNA Interference Preparation and Transfection The sequences of RNA interference (RNAi) duplex oligos RESEARCH DESIGN AND METHODS against APPL1, APPL2, and scrambled control (Invitrogen) Materials are listed in Supplementary Table 1. These oligos were Rabbit monoclonal antibodies against total Akt, GAPDH, transfected into C2C12 or L6 cells by electroporation b-actin and insulin receptor-b (IRb), rabbit polyclonal according to the manufacturer’s protocol (Bio-Rad). 3750 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

Mutagenesis, Generation, and Purification of GLUT4 translocation to the plasma membrane compared Adenoviruses with cells with ectopic expression of luciferase controls. The adenoviruses encoding APPL1 and luciferase were gen- On the contrary, overexpression of APPL2 inhibited such erated in our previous study (11). To construct adenoviral insulin actions (Fig. 1B and C). A similar result was also vectors for overexpression of APPL2 or TBC1D1, cDNA found in C2C12 myotubes (data not shown). encoding human APPL2 or human TBC1D1 were cloned To verify these in vitro findings in cultured cells, we into pAdeasy-1 adenoviral backbone vector (Stratagene) as evaluated the impact of APPL1 and APPL2 overexpression described previously (11). PCR-based site-directed muta- on insulin-stimulated glucose uptake in skeletal muscle in genesis was performed to introduce S235A, S237A, and mice. Transgenic mice with overexpression of FLAG- T596D mutations in human TBC1D1 by using the muta- tagged human APPL1 driven by cytomegalovirus-b-actin genic primers as previously described (12). C2C12 and L6 promoter were generated in our previous study (9,10). We myotubes were infected with various adenoviruses at used a similar strategy to generate transgenic mice with a multiplicity of infection of 50. overexpression of human APPL2 (Supplementary Fig. 1A), fi B Analysis of Glucose Uptake and GLUT4 Translocation which was con rmed by PCR (Supplementary Fig. 1 ) and in Muscle Cells and Isolated Skeletal Muscles immunoblotting (Supplementary Fig. 1C). The expression Glucose uptake assays were performed in C2C12 and L6 levels of APPL2 in EDL and soleus muscles of the trans- myotubes using 2-deoxy-[3H]-glucose as tracer as de- genic mice were elevated approximately four- to sixfold scribed in our previous study (20). For ex vivo glucose compared with WT controls (Supplementary Fig. 1C). uptake assay, mice fasted for 4 h were anesthetized. Iso- Consistently, ex vivo studies in isolated muscles showed lated extensor digitorum longus (EDL) or soleus muscles that transgenic expression of APPL2 suppressed, whereas were stimulated without or with insulin 60 mU/mL fol- APPL1 enhanced, insulin-stimulated glucose uptake in lowed by measurement of 2-deoxy-[3H]-glucose uptake (21). EDL muscle compared with WT controls (Fig. 1D). A sim- For determination of GLUT4 translocation, an antibody- ilar result was also observed in soleus muscle (data not coupled densitometric assay was used to measure the shown). content of surface Myc-GLUT4 in L6 myotubes stably APPL2 Tg mice exhibited a trend of increased fasting expressing Myc-tagged GLUT4 as described in previous glucose and insulin levels (Supplementary Fig. 1D and E) studies (20,22). and displayed a modest, but significant impairment in both glucose tolerance and insulin sensitivity compared Immunoprecipitation and Mass Spectrometry with WT littermates (Supplementary Fig. 1F and G). Of C2C12 myotubes or human embryonic kidney (HEK) 293 note, overexpression of APPL2 had no effect on protein cells were subjected to immunoprecipitation as described abundance of GLUT4 in EDL muscles (Supplementary Fig. in our previous study (12). The immunocomplexes were 2). eluted and subjected to immunoblotting analysis with Transfection of L6 myotubes with the duplex RNAi various antibodies as specified in each figure legend or fi against rat APPL1 and APPL2 led to a reduction of APPL1 by mass spectrometry analysis for identi cation of inter- and APPL2 expression by 76% and 81%, respectively, acting partners of APPL2 as previously described (23). compared with scrambled controls (Fig. 1E). Of note, Statistical Analysis knockdown of APPL2 expression potentiated, whereas sup- All experiments were performed routinely, with four to pression of APPL1 expression inhibited, insulin-stimulated six repeats in each group. Data are presented as mean 6 glucose uptake and plasma membrane recruitment of SE. Statistical significance was determined by Student GLUT4 (Fig. 1F and G). Likewise, the potentiating effects t test or two-way ANOVA (for the experiments that in- of APPL1 and the inhibitory effects of APPL2 on insulin- volved two factors) followed by Bonferroni post hoc tests. evoked glucose uptake were observed in C2C12 myotubes In all statistical comparisons, P , 0.05 indicated a signif- (data not shown). icant difference. APPL2 Is a Key Regulator of Glucose Homeostasis in RESULTS Mice APPL1 and APPL2 Exert Opposite Effects on Insulin- To test whether deletion of APPL2 in skeletal muscles Stimulated Glucose Uptake in Muscle Cells protects mice from glucose intolerance, we generated fl To compare the effects of APPL1 and APPL2 on glucose muscle-specific APPL2 KO mice by crossing APPL2 oxed uptake and metabolism, we used the adenoviral gene mice with transgenic mice expressing Cre recombinase delivery system for overexpression of these two adaptor under the control of muscle creatine kinase promoter, proteins in L6 myotubes. The APPL1 and APPL2 protein which resulted in the disruption of the APPL2 gene at levels in cells with ectopic expression of both APPL1 and exon 5 (Fig. 2A). Immunoblotting analysis confirmed APPL2 were increased by approximately threefold relative the dramatic reduction of APPL2 protein in EDL and to their endogenous levels in L6 myotubes (Fig. 1A). Con- soleus muscles but not in the brain of APPL2 KO mice sistent with a previous study (24), ectopic overexpression (Fig. 2B). The residual expression of APPL2 in EDL and of APPL1 enhanced insulin-stimulated glucose uptake and soleus muscles in APPL2 KO mice is perhaps due to its diabetes.diabetesjournals.org Cheng and Associates 3751

Figure 1—Opposite actions of APPL1 and APPL2 on insulin-stimulated glucose uptake and GLUT4 translocation in myotubes and skeletal muscles. L6 myotubes were infected with adenovirus-encoding luciferase (Luci), APPL1, or APPL2 for 24 h followed by serum starvation for 6h.A: The starved cells were subjected to immunoblotting using a rabbit polyclonal antibody against APPL1 or APPL2 or a rabbit monoclonal against b-actin. B: Insulin-stimulated glucose uptake was measured in the infected cells using 2-deoxy-[3H]-glucose. C: Insulin-stimulated GLUT4 translocation was measured by colorimetric assay in L6 myotubes stably expressing Myc-tagged GLUT4 infected with the indicated adenoviruses. The reference values for 2-deoxy-[3H]-glucose uptake and membrane-bound Myc-GLUT4 are 4.82 6 0.41 pmol/min/mg protein and 4.12 6 0.35 fmol/mg protein in noninsulin-treated cells expressing luciferase control, respectively. Data are fold changes relative to basal levels in luciferase-expressing cells. D: Insulin-stimulated glucose uptake was measured in EDL muscles isolated from 20-week-old APPL1 Tg and APPL2 Tg mice and WT controls using 2-deoxy-[3H]-glucose and normalized with [14C]- mannitol as described in RESEARCH DESIGN AND METHODS. E and F: L6 myotubes were transfected with RNAi against APPL1, APPL2, or scrambled control for 24 h followed by serum starvation for 6 h. The starved cells were subjected to immunoblotting (E) or measurement of insulin-stimulated glucose uptake (F). G: Insulin-stimulated GLUT4 plasma membrane recruitment in the L6 myotube stable cell line expressing Myc-tagged GLUT4 transfected with RNAi as indicated. The reference values for glucose uptake and membrane-bound Myc- GLUT4 are 4.53 6 0.31 pmol/min/mg protein and 3.8 6 0.42 fmol/mg protein in noninsulin-treated cells transfected with RNAi against scrambled control, respectively. All experiments were repeated at least three times, and representative images are shown. *P < 0.05 (n =6) by Student t test.

ubiquitous expression in other nonmyocyte cells (16). Ge- mass spectrometry identified several putative interacting netic ablation of APPL2 in muscles had no obvious effects partners of APPL2, including heat shock protein (HSP) on food intake, body weight, and fasting glucose and in- 70, HSP90, APPL1, centaurin delta 1, TBC1D1, and son of sulin levels (Supplementary Table 2). GTT revealed that sevenless homolog 1. Among these APPL2-interacting APPL2 KO mice exhibited a significant improvement of proteins, TBC1D1, a member of the TBC1 Rab-GTPase glucose tolerance in response to glucose challenge com- family of proteins abundantly expressed in skeletal pared with WT littermates (Fig. 2C and D). Serum insulin muscles, is an important regulator of insulin signaling levels during the GTT were similar between the two groups and glucose metabolism (25,26). Of note, our coimmuno- of mice (Fig. 2E). Insulin sensitivity, as determined by ITT, precipitation analysis showed that TBC1D1 binds to was also enhanced by APPL2 deletion (Fig. 2F). Ex vivo APPL2 but not to APPL1 (Fig. 3A). The specificity of im- studies demonstrated that insulin-stimulated glucose up- munoprecipitation for TBC1D1 was confirmed by RNAi- take in EDL muscles of APPL2 KO mice was significantly mediated knockdown of TBC1D1 expression in C2C12 increased compared with WT littermates (Fig. 2G). A sim- myotubes, leading to a substantial decrease in immunopre- ilar result was observed in soleus muscles (data not shown). cipated TBC1D1 (Supplementary Fig. 3). On the other Insulin-elicited phosphorylation of Akt and IRb in EDL hand, APPL2 did not bind to TBC1D4 (Fig. 3B), a paralog muscle was comparable between the two groups (Fig. 2H). of TBC1D1 that is also involved in the regulation of glucose transport in muscle cells and adipocytes (6). The APPL2– TBC1D1 Is an Interacting Partner and Downstream TBC1D1 interaction was enhanced by insulin stimulation Effector of APPL2 in EDL muscle of C57 mice (Fig. 3C) and C2C12 myotubes To identify the proximal downstream effectors of APPL2, (Fig. 3D), and such an enhancement was largely blocked by we established a stable cell line expressing FLAG-tagged the PI3K inhibitor LY294002 (Fig. 3D). human APPL2 for affinity pull-down purification of its To determine which domain of APPL2 is responsible potential interacting partners in HEK293 cells. Tandem for TBC1D1 binding, we generated a series of vectors that 3752 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

Figure 2—Generation and metabolic characterization of muscle-specific APPL2 KO mice. A: Strategy for generating muscle-specific APPL2 KO mice. The null allele lacking exon 5 of APPL2 as a result of Cre recombinase, which was driven by muscle creatine kinase (MCK) promoter, mediated recombination between the two LoxP sites. B: Soleus and EDL muscles and brain isolated from 8-week-old heterozygous (hetero) and homozygous muscle-specific APPL2 KO mice and WT littermates were subjected to immunoblotting using a rabbit anti-APPL2 polyclonal or rabbit anti-GAPDH monoclonal antibody. C: GTT in 20-week-old APPL2 KO mice and WT controls fed standard chow. D: Area under the curve (AUC) of the GTT in panel C. Data are fold change relative to WT controls. E: Serum insulin levels during the GTT in panel C. F: ITT in 22-week-old APPL2 KO mice and WT controls; 0.5 units/kg insulin was injected intraperitoneally. G:Ex vivo glucose uptake was assessed in EDL muscles isolated from 20-week-old APPL2 KO mice and WT controls using 2-deoxy-[3H]- glucose as described in RESEARCH DESIGN AND METHODS. H: EDL muscles from fasted C57BL/6 mice injected without or with insulin 0.5 units/kg for 15 min were subjected to immunoprecipitation (IP) using a rabbit anti-IRb monoclonal antibody followed by immunoblotting using a mouse antiphosphotyrosine (pY) monoclonal or rabbit anti-IRb monoclonal antibody. Total tissue lysates were subjected to immuno- blotting using a rabbit antiphospho-Akt (Ser-473) (pAkt) polyclonal antibody or rabbit antitotal Akt (Akt) monoclonal antibody. The chart in the right panel represents fold changes of phosphorylation of Akt vs. total Akt or tyrosine phosphorylation of IRb vs. total IRb relative to the basal levels in WT controls as quantified by densitometry. *P < 0.05 (n = 5) by Student t test.

express various domains of APPL2 (Fig. 4A). Coimmu- Phosphorylation of TBC1D1 at Ser-235 Is Required for noprecipitation analysis revealed that TBC1D1 inter- Its Interaction With APPL2 and the Inhibitory Effects of acted with the BAR domain but not with the PH or APPL2 on Insulin-Dependent Glucose Uptake – PTB domain of APPL2 (Fig. 4B). The pull-down assay To further delineate how the APPL2 TBC1D1 interaction further confirmed the direct interaction between regulates insulin-stimulated glucose uptake, we generated TBC1D1 and the BAR domain of APPL2 (Supplementary a series of truncated mutants of TBC1D1 (Fig. 5A) to map Fig. 4). To test whether the BAR domain mediates the the minimal domain mediating its binding to APPL2. suppressive effect of APPL2 on insulin-stimulated glucose Coimmunoprecipitation analysis revealed that all the uptake, we transduced L6 myotubes with adenovirus mutants of TBC1D1 containing the linker region between encoding the BAR domain or the PH-PTB domain or the two PTB domains (amino acids 165–279) were able to luciferase as control. Similar to full-length APPL2 (Fig. interact with APPL2, whereas those mutants without the 1B and C), ectopic overexpression of the BAR domain but linker region lost their APPL2-binding property (Fig. 5B). not the PH-PTB mutant inhibited insulin-stimulated glu- Furthermore, the linker region alone was sufficient to cose uptake and GLUT4 translocation to the plasma bind with APPL2 (Fig. 5B). membrane (Fig. 4C and D). Taken together, these find- TBC1D1 is a hyperphosphorylated protein, and insulin ings suggest that the BAR domain of APPL2 exerts an induces its phosphorylation through Akt activation (6). inhibitory effect on insulin-evoked glucose uptake by Because insulin prompted the APPL2–TBC1D1 interac- interacting with TBC1D1. tion in a PI3K-dependent manner, we searched for the diabetes.diabetesjournals.org Cheng and Associates 3753

Figure 4—The BAR domain of APPL2 interacts with TBC1D1 and Figure 3—APPL2 binds to TBC1D1 in an insulin- and PI3K-dependent mediates the suppressive effects of APPL2 on insulin-stimulated manner. A: C2C12 myotubes were collected and subjected to im- glucose uptake. A: Schematic diagram of FLAG-tagged WT APPL2 munoprecipitation (IP) using a rabbit anti-TBC1D1 polyclonal anti- and its truncated mutants containing various domains used for im- body (Abcam) or rabbit IgG as control followed by immunoblotting munoprecipitation (IP) assays. B: HEK293 cells were cotransfected with the indicated antibodies. B: C2C12 infected with adenovirus with plasmids encoding HA-tagged TBC1D1 and FLAG-WT-APPL2 encoding FLAG-tagged APPL2 (FLAG-APPL2) and HA-tagged or FLAG-APPL2 mutants (BAR-PH, PH-PTB, BAR, and PH) or an TBC1D1 (HA-TBC1D1) or HA-tagged TBC1D4 (HA-TBC1D4) were empty vector as negative control (-ve) for 48 h followed by IP with subjected to IP using a mouse anti-FLAG monoclonal antibody a mouse anti-FLAG monoclonal antibody and immunoblotting using followed by immunoblotting with the indicated antibodies. C: C57 a mouse anti-FLAG monoclonal or rabbit anti-HA polyclonal anti- mice were fasted overnight followed by insulin 0.5 units/kg i.p. for body as indicated. C: L6 myotubes infected with various truncated the indicated time points. EDL muscles were isolated and subjected mutants of APPL2 or luciferase (Luci) control were subjected to to IP using a rabbit anti-APPL2 polyclonal antibody followed by glucose uptake assay as described in RESEARCH DESIGN AND METHODS. immunoblotting using a rabbit polyclonal antibody against APPL2 D: L6 myotubes stably expressing Myc-tagged GLUT4 were or TBC1D1 (Abcam). D: C2C12 myotubes expressing FLAG-APPL2 infected with the indicated adenoviruses, followed by serum star- and HA-TBC1D1 were serum starved for 12 h followed by preincu- vation for 6 h. Insulin-stimulated GLUT4 translocation to plasma bation with or without the PI3K inhibitor LY294002 (LY 50 mmol/L) membrane was measured using the antibody-coupled densitomet- for 30 min. The cells treated without or with insulin 10 nmol/L for 15 ric assay as described in RESEARCH DESIGN AND METHODS. The reference min were subjected to IP using a mouse anti-FLAG monoclonal values for glucose uptake and membrane-bound Myc-GLUT4 are antibody followed by immunoblotting with a mouse anti-FLAG 5.2 6 0.49 pmol/min/mg protein and 4.70 6 0.54 fmol/mg protein monoclonal or rabbit anti-HA polyclonal antibody as indicated. All in noninsulin-treated cells expressing luciferase control, respec- experiments were repeated at least three times, and representative tively. Data are fold changes relative to basal levels in cells express- images are shown. ing luciferase. *P < 0.05 (n = 5) by Student t test. phospho-Akt substrate site within the linker region that may be crucial for its association with APPL2. Of note, a previous study showed that Ser-229 of mouse TBC1D1 luciferase control (Fig. 5F). The inhibitory effects of (equivalent to Ser-235 on human TBC1D1) within this TBC1D1 on insulin-evoked glucose uptake were abrogated region is potentially phosphorylated by Akt (27). We con- by the S235A but not S237A mutation (Fig. 5F). firmed that insulin stimulated TBC1D1 phosphorylation Several previous studies have shown that insulin- at Ser-235 in a time-dependent manner in both EDL mus- induced Akt activation leads to direct TBC1D1 phos- cle of C57 mice and C2C12 myotubes (Fig. 5C and D), and phorylationonThr-596(equivalenttoThr-590inmouse such an insulin action was largely abolished by the PI3K TBC1D1) (6,26–28), which in turn promotes the trans- inhibitor LY294002 (Fig. 5D). Substitution of Ser-235 port of GLUT4 to the plasma membrane by activating with nonphosphorylatable alanine (S235A) markedly at- Rab-GTPases (6,26–28). To investigate the effects of tenuated insulin-stimulated interaction between APPL2 APPL2 on insulin-elicited phosphorylation of TBC1D1, and TBC1D1, whereas mutation of Ser-237 to alanine we injected APPL2 Tg and APPL2 KO mice intraper- had no obvious effect (Fig. 5E). itoneally with insulin. Immunoblotting demonstrated We next compared the effects of TBC1D1 and its that insulin-stimulated TBC1D1 phosphorylation on mutant S235A on insulin-stimulated glucose uptake in L6 Thr-596 in EDL and soleus muscles was enhanced by myotubes. Consistent with a previous study (27), we APPL2 deletion but diminished by APPL2 overexpression found that adenovirus-mediated expression of TBC1D1 (Fig. 6). On the other hand, APPL2 overexpression or did not affect insulin-evoked Akt phosphorylation (data deletion had no obvious effect on expression levels of not shown) but led to a significant inhibition of insulin- total TBC1D1 or TBC1D1 phosphorylation on Ser-235 stimulated glucose uptake compared with cells expressing (Fig. 6). 3754 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

Figure 5—Effect of Ser-235 phosphorylation of TBC1D1 on its APPL2-binding and -suppressive action on insulin-dependent glucose uptake. A: Schematic presentation of HA-tagged WT TBC1D1 and its truncated mutants (Mut-1–Mut-5) used for immunoprecipitation (IP) assays. B: HEK293 cells transfected with FLAG-tagged APPL2 or HA-WT-TBC1D1 or HA-TBC1D1 mutants (Mut-1: 374–1,168; Mut-2: 1– 378; Mut-3: 165–378; Mut-4: 165–279; Mut-5: 280–378) were subjected to IP using a mouse anti-FLAG monoclonal antibody followed by immunoblotting using antibodies as indicated. C: EDL muscles isolated from C57 mice injected without or with insulin 0.5 units/kg for the indicated time points were subjected to IP with a rabbit anti-TBC1D1 polyclonal antibody (Abcam) followed by immunoblotting using a rabbit polyclonal antibody against phospho-TBC1D1 (Ser-235) or total TBC1D1 (Cell Signaling Inc.) as specified. The specificity of the rabbit phospho-TBC1D1 (Ser-235) polyclonal antibody was confirmed by immunoblotting, showing the disappearance of a specific band with a molecular weight of ;160 kDa in cells expressing nonphosphorylatable TBC1D1 S235A mutant (data not shown). D: C2C12 myotubes infected with adenovirus encoding HA-WT-TBC1D1 were pretreated with or without the PI3K inhibitor LY294002 and then stimulated with insulin 10 nmol/L for various time periods followed by immunoblotting with a rabbit polyclonal antibody against HA or phospho-TBC1D1 (Ser-235). E: C2C12 myotubes infected with HA-WT-TBC1D1 or its mutant S235A (Ser-235 is mutated to alanine) or S237A (Ser-237 is mutated to alanine) and FLAG-APPL2 were stimulated with insulin 10 nmol/L for 10 min followed by IP with a mouse anti- FLAG monoclonal antibody and immunoblotting to detect HA-tagged TBC1D1. F: L6 myotubes infected with various recombinant adeno- viruses as indicated were subjected to immunoblotting using a rabbit anti-TBC1D1 polyclonal (Abcam) or a rabbit anti–b-actin monoclonal antibody (left panel) and insulin-stimulated glucose uptake assay (right panel) as described in RESEARCH DESIGN AND METHODS. The reference value for glucose uptake is 4.2 6 0.32 pmol/min/mg protein in noninsulin-treated cells expressing luciferase control. Data are fold changes relative to noninsulin-treated cells expressing luciferase control. *P < 0.05 (n = 5) by Student t test. CBD, calmodulin-binding domain; LK, linker region.

To further investigate the interplay between APPL2 As expected, the inhibitory effect of TBC1D1 overexpres- and TBC1D1 in regulating insulin-stimulated glucose sion on insulin-stimulated glucose uptake was further ag- uptake, we co-overexpressed APPL2 and TBC1D1 or its gravated by overexpression of APPL2 (Fig. 7E). However, S235A mutant in L6 myotubes by adenoviral gene trans- this suppressive effect of APPL2 was lost in cells express- fer system (Fig. 7A). Similar to the findings in APPL2 Tg ing the TBC1D1-S235A mutant (Fig. 7E). mice, insulin-stimulated TBC1D1 phosphorylation on To further investigate whether APPL2 suppresses Thr-596 was significantly impaired by overexpression of insulin-elicited glucose uptake by modulating TBC1D1 APPL2 compared with cells overexpressing luciferase con- phosphorylation on Thr-596, Thr-596 of TBC1D1 was trols (Fig. 7A and B). However, the suppressive effect of mutated to aspartic acid (T596D) to mimic its phosphor- APPL2 overexpression on insulin-stimulated phosphory- ylation. The level of adenovirus-mediated expression lation of Thr-596 was abolished in cells expressing the of the TBC1D1-T590D mutant was comparable to WT TBC1D1-S235A mutant (Fig. 7A and B). Of note, over- TBC1D1 in L6 myotubes (Fig. 8A). However, the suppres- expression of APPL2 caused only a modest suppressive sive effects of APPL2 overexpression on insulin-induced effect on insulin-elicited Akt phosphorylation and had glucose uptake was observed in only L6 myotubes with no effect on IRb phosphorylation in L6 myotubes express- ectopic expression of WT TBC1D1, not in L6 cells express- ing either TBC1D1 or its S235A mutant (Fig. 7C and D). ing the TBC1D1-T590D mutant (Fig. 8B). diabetes.diabetesjournals.org Cheng and Associates 3755

Figure 6—APPL2 suppresses insulin-elicited phosphorylation of TBC1D1 at Thr-590 in skeletal muscle. A and B: Twelve-week-old male APPL2 KO mice, APPL2 Tg mice, and their respective WT littermates were fasted overnight followed by intraperitoneal injection without or with insulin 0.5 units/kg for 10 min. EDL and soleus muscles were isolated and subjected to immunoprecipitation (IP) with a rabbit anti- TBC1D1 polyclonal antibody (Abcam) followed by immunoblotting using a rabbit polyclonal antibody against total TBC1D1 (Cell Signaling Inc.), phospho-TBC1D1 (Ser-235), or phospho-TBC1D1 (Thr-596) as indicated. The charts in the right panels are the relative abundance of phosphorylated TBC1D1 at Thr-596 or Ser-235 vs. total TBC1D1 as determined by densitometric analysis. The specificity of the rabbit antiphospho-TBC1D1 (Thr-596) polyclonal antibody was validated by immunoblotting, showing the disappearance of a specific band with a molecular weight of ;160 kDa in cells expressing nonphosphorylatable TBC1D1 T596A mutant (data not shown). Data are fold changes relative to noninsulin-treated WT controls. *P < 0.05 (n = 4) by Student t test. N.S., not significant.

DISCUSSION heterodimerization with APPL1, which in turn prevents In this study, we provide both in vivo and in vitro the binding of APPL1 to Akt (16). We demonstrate that evidence showing that APPL2 negatively regulates insulin- APPL2 but not APPL1 interacts with TBC1D1, a down- stimulated glucose transport by interacting with TBC1D1. stream substrate of Akt that is critically involved in In both skeletal muscle and cultured muscle cells, insulin- GLUT4 vesicle trafficking. Therefore, the opposite effects induced glucose uptake is diminished by overexpression of APPL1 and APPL2 on insulin-stimulated glucose uptake of APPL2 but is enhanced by suppression of APPL2 are attributed to the differential binding of these two expression. adaptor proteins to Akt and TBC1D1. In line with these Despite of the high similarity in domain organization findings, APPL1 and APPL2 have been shown to bind to and amino acid sequences between APPL1 and APPL2, we various types of Rab-GTPases involved in membrane traf- demonstrate that these two adaptor proteins exert ficking (17,19). The binding of APPL1 and APPL2 to var- opposite effects on insulin-stimulated glucose uptake in ious sets of intracellular signaling molecules may be due skeletal muscle. APPL1 exerts its insulin-sensitizing to their differences in oligomerization, surface charges, effects by competing with TRB3 for binding to Akt, and/or subcellular localization (17,18,31,32). thereby promoting the translocation of Akt to the plasma TBC1D1 and TBC1D4, both of which are Rab-GAP membrane for further activation (11,16,24). On the other proteins sharing ;47% sequence identity and a similar hand, the present study shows that APPL2 suppresses domain organization, are the important regulators of both insulin-dependent glucose uptake at a step downstream insulin- and contraction-induced trafficking of GLUT4 of Akt. Unlike APPL1, APPL2 does not interact with vesicles (6,33,34). Upon insulin stimulation, activated Akt Akt or the regulatory subunit p85 and catalytic subunit induces phosphorylation of both TBC1D1 and TBC1D4 p110 of PI3K (7,29,30). Indeed, overexpression of APPL2 at multiple sites flanking the second PTB domain results in only a modest decrease in insulin-elicited Akt (26–28,35,36). Substitution of these phosphorylation phosphorylation in myotubes, whereas targeted deletion sites with nonphosphorylatable alanine in TBC1D1 and of APPL2 in skeletal muscle has no obvious effect on Akt TBC1D4 abolishes insulin-induced GLUT4 translocation phosphorylation. Such a modest effect of APPL2 on Akt to the plasma membrane in both adipocytes and muscle activity in cultured cells is perhaps a result of its cells (25–27,36–38). Although the precise mechanisms by 3756 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

Figure 7—The APPL2-TBC1D1 interaction modulates insulin-elicited phosphorylation of TBC1D1 at Thr-596 and the suppressive effect of APPL2 overexpression on insulin-dependent glucose uptake. L6 myotubes were infected with adenovirus encoding HA-tagged WT- TBC1D1, TBC1D1-S235A mutant, luciferase (Luci), or APPL2 for 24 h followed by serum starvation for 12 h. A: The cells were treated without or with insulin 10 nmol/L for 10 min, and the cell lysates were subjected to either immunoblotting with various antibodies as indicated or immunoprecipitation (IP) using a rabbit anti-IRb monoclonal antibody. The IP IRb was subjected to immunoblotting analysis for tyrosine phosphorylation using a mouse antityrosine monoclonal antibody. B–D: The charts show the relative abundance of phosphorylated TBC1D1 at Thr-596 vs. total TBC1D1 (B), phosphorylated Akt at Ser-473 vs. total Akt (C), and tyrosine phosphorylation of IRb vs. total IRb (D). E: The starved cells infected with indicated adenoviruses were subjected to glucose uptake assay as described in RESEARCH DESIGN AND METHODS. The reference value for glucose uptake is 4.87 6 0.25 pmol/min/mg protein in noninsulin-treated cells expressing luciferase control. Data are fold changes relative to noninsulin-treated luciferase controls (E) or noninsulin-treated luciferase control plus WT-TBC1D1 (B–D) as indicated. Comparisons were made with two-way ANOVA followed by Bonferroni post hoc tests (E). *P < 0.05, #P < 0.01 (n = 5). N.S., not significant.

which TBC1D1 and TBC1D4 regulate GLUT4 vesicle and skeletal muscle are associated with impaired phos- trafficking remain unclear, it has been proposed that phorylation of TBC1D1 at Thr-596 in response to insulin nonphosphorylated TBC1D4 in the basal state binds to stimulation. Furthermore, mutation of Thr-596 of TBC1D1 GLUT4-containing vesicles to maintain its substrate Rab- to aspartic acid reverses the inhibitory effect of APPL2 GTPases in their inactive guanosine diphosphate–loaded on insulin-stimulated glucose uptake, suggesting that form, thereby trapping GLUT4 inside cells (27,35–38). the APPL2–TBC1D1 interaction prevents Akt-mediated Insulin-evoked TBC1D4 phosphorylation on Thr649, pos- phosphorylation of TBC1D1 at Thr-596, thereby im- sibly through interaction with 14-3-3 proteins, inhibits its pairing insulin-evoked GLUT4 translation to the plasma GAP activity, which in turn allows guanosine triphosphate membrane. loading and activation of Rab-GTPases required for dock- Although a previous study has identified Ser-235, a ing and fusion of GLUT4-containing vesicles to the plasma highly conserved amino acid located within the linker membrane (36–38). A previous study demonstrated that region of TBC1D1, as a likely phospho-Akt substrate site mice with TBC1D4-Thr649Ala knockin mutation, in which responsive to insulin stimulation (27), its physiological Thr649 was mutated to nonphosphorylatable alanine, dis- relevance has never been explored. In the current study, play impaired glucose disposal and insulin sensitivity as we further confirm that insulin induces TBC1D1 phosphor- a result of reduced GLUT4 trafficking to the cell surface ylation on Ser-235 in a time-dependent manner by using in skeletal muscles (39). Likewise, insulin-elicited phos- an antiphospho-Ser-235 antibody, and this phosphoryla- phorylation of TBC1D1 at Thr-596, a site equivalent to tion is suppressed by pharmacological inhibition of PI3K. Thr-649 of TBC1D4, is obligatory for GLUT4 trafficking Furthermore, we found that mutation of Ser-235 to ala- possibly by inactivation of GAP activity (26–28). In the nine not only abolishes the APPL2–TBC1D1 interaction current study, we found that the inhibitory effects of but also abrogates the suppressive effects of APPL2 on APPL2 overexpression on glucose uptake in muscle cells insulin-induced TBC1D1 phosphorylation on Thr-596 diabetes.diabetesjournals.org Cheng and Associates 3757

Biotechnology, the National Science Foundation of China (81270881), and the National Basic Research Program of China (2011CB504004 and 2010CB945500). Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. K.K.Y.C. and W.Z. contributed to researching data and writing the manuscript. B.C. and B.W. contributed to researching data. Y.W. contributed to researching data and the discussion. D.W. contributed to research- ing data and editing the manuscript. G.S. contributed to researching data and reviewing the manuscript. K.S.L.L. contributed to supervising the study and editing the manuscript. A.X. contributed to the design and supervision of the study and writing of the manuscript. K.K.Y.C. is the guarantor of this work and, as Figure 8—A phosphomimetic mutation of TBC1D1 at Thr-596 abol- such, had full access to all the data in the study and takes responsibility for the ishes the inhibitory effects of APPL2 on insulin-stimulated glucose integrity of the data and the accuracy of the data analysis. uptake. L6 myotubes were infected with adenovirus encoding HA- tagged WT-TBC1D1, TBC1D1-T596D mutant, luciferase (Luci), or References FLAG-tagged APPL2 as specified for 24 h followed by serum star- 1. Bogan JS. Regulation of glucose transporter translocation in health and vation for 6 h and subsequent stimulation with insulin for 10 min. diabetes. Annu Rev Biochem 2012;81:507–532 The cells were subjected to immunoblotting (A) using a rabbit poly- 2. Ryder JW, Yang J, Galuska D, et al. 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