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 Protein APPL2Inhibits Insulin-StimulatedGlucose Uptake by InteractingWith TBC1D1 in Skeletal MuscleDiabetes 2014;63:3748–3758 | DOI: 10.2337/db14-0337

Insulin stimulates glucose uptake by promoting thetrafficking of GLUT4 to the plasma membrane in musclecells, and impairment of this insulin action contributes tohyperglycemia in type 2 diabetes. The adaptor proteinAPPL1 potentiates insulin-stimulated Akt activation anddownstream actions. However, the physiological func-tions of APPL2, a close homolog of APPL1, in regulatingglucose metabolism remain elusive. We show thatinsulin-evoked plasma membrane recruitment of GLUT4and glucose uptake are impaired by APPL2 overexpres-sion but enhanced by APPL2 knockdown. Likewise,conditional deletion of APPL2 in skeletal musclesenhances insulin sensitivity, leading to an improvementin glucose tolerance. We identified the Rab-GTPase–activating protein TBC1D1 as an interacting partner ofAPPL2. Insulin stimulates TBC1D1 phosphorylation onserine 235, leading to enhanced interaction with theBAR domain of APPL2, which in turn suppresses insulin-evoked TBC1D1 phosphorylation on threonine 596 incultured myotubes and skeletal muscle. Substitution ofserine 235 with alanine diminishes APPL2-mediated in-hibition on insulin-dependent TBC1D1 phosphorylationon threonine 596 and the suppressive effects of TBC1D1on insulin-induced glucose uptake and GLUT4 transloca-tion to the plasmamembrane in cultured myotubes. There-fore, the APPL2–TBC1D1 interaction is a key step to finetune insulin-stimulated glucose uptake by regulating themembrane recruitment of GLUT4 in skeletal muscle.

Insulin maintains glucose homeostasis by facilitating theuptake of postprandial glucose into adipose tissues andskeletal muscle, the latter of which accounts for ;75% oftotal glucose disposal (1). The binding of insulin to its recep-tors elicits tyrosine phosphorylation of insulin receptor sub-strates, which in turn activates phosphatidylinositol 3-kinase(PI3K), leading to the membrane recruitment and activationof Akt. Activated Akt subsequently induces the translocationof GLUT4 from intracellular vesicular compartments to theplasma membrane for glucose uptake. In type 2 diabeticpatients, the ability of insulin to stimulate glucose uptake issignificantly impaired, owing in part to the defective insulin-dependent recruitment of GLUT4 to the plasma membrane(2,3). In rodents, genetic ablation of GLUT4 in skeletal musclecauses insulin resistance and diabetes (4), whereas overex-pression of GLUT4 alleviates hyperglycemia and insulin re-sistance in db/db diabetic mice (5). GLUT4 translocation istightly controlled by insulin signaling cascades and a se-ries of small GTPase proteins (6). Studies have demon-strated that the Rab-GTPase–activating proteins (GAPs)Tre-2/Bub2/Cdc16 domain family, member 1 (TBC1D1)and member 4 (TBC1D4 [also known as AS160]) mayintegrate insulin signaling and Rab-GTPase activity,thereby regulating GLUT4 trafficking and glucose up-take (6). However, the detailed intracellular signalingevents that confer insulin-elicited glucose uptake arestill not fully characterized.

1State Key Laboratory of Pharmaceutical Biotechnology, The University of HongKong, Hong Kong2Department of Medicine, The University of Hong Kong, Hong Kong3Department of Pharmacology & Pharmacy, The University of Hong Kong, HongKong4The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicineand Health, Chinese Academy of Sciences, Guangzhou, China5Department of Biology, York University, Toronto, Ontario, Canada

Corresponding author: Aimin Xu, amxu@hku.hk, or Kenneth K.Y. Cheng,dorncky@hku.hk.

Received 25 February 2014 and accepted 23 May 2014.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0337/-/DC1.

K.K.Y.C. and W.Z. contributed equally to this study.

© 2014 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

3748 Diabetes Volume 63, November 2014

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Mounting evidence suggests that the adaptor proteinAPPL1, which comprises an NH2-terminal Bin/amphiphysin/Rvs (BAR) domain, a central pleckstrin homology (PH)domain, and a COOH-terminal phosphotyrosine-binding(PTB) domain, is an insulin-sensitizing molecule in multipleinsulin-responsive tissues (7). Genetic disruption of APPL1causes insulin resistance and defective glucose-stimulatedinsulin secretion, leading to glucose intolerance in mice(8). In contrast, transgenic overexpression of APPL1 pre-vents obesity-induced deleterious effects on glucose homeo-stasis and endothelial and cardiac functions (8–10). Hepaticoverexpression of APPL1 improves hyperglycemia, glucoseintolerance, and insulin sensitivity in db/db diabetic mice,whereas hepatic silencing of APPL1 causes insulin resistanceand hyperglycemia in lean mice (11). In pancreatic b-cells,APPL1 promotes glucose-stimulated insulin secretion byupregulating the expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteinsin an Akt-dependent manner (8). At the molecular level,the interaction between APPL1 and Akt prevents Aktfrom binding to the pseudokinase tribble 3 (TRB3),thereby promoting Akt to the plasma membrane for fur-ther activation in hepatocytes, endothelial cells, and pan-creatic b-cells (8,9,11,12). Furthermore, APPL1 potentiatesthe insulin-sensitizing effects of adiponectin on promotionof glucose uptake by direct interaction with the two adipo-nectin receptors (13).

APPL2 is a close homolog of APPL1 abundantly expressedin skeletal muscle. These two adaptor proteins share 52%identity and 72% similarity in amino acid sequence and thesame domain organization (7). APPL1 and APPL2 appear toplay a similar role in mediating growth factor–induced cellproliferation in fibroblasts and apoptosis in zebrafish (14,15).On the other hand, an in vitro study demonstrated that thesetwo proteins possess distinct or even opposite functions inregulating glucose and lipid metabolism (16). Structural anal-ysis revealed that APPL2 incorporates two homodimers,whereas APPL1 incorporates only one homodimer in theasymmetric unit (17–19). Although APPL1 binds to theadiponectin receptors and increases adiponectin-inducedglucose uptake and fatty acid oxidation, APPL2 inhibitsadiponectin actions in muscle cells (13,16).

Although the insulin-sensitizing effects of APPL1 arewell characterized, little is known about the physiologicalrole of APPL2 in insulin signaling and glucose metabolism.In this study, we provide both in vitro and in vivo evidencethat APPL2 is a negative regulator of insulin-stimulatedglucose transport in skeletal muscle. Furthermore, weidentify TBC1D1 as an interacting partner and a down-stream effector that mediates the suppressive effect ofAPPL2 on insulin-elicited plasma membrane translocationof GLUT4.

RESEARCH DESIGN AND METHODS

MaterialsRabbit monoclonal antibodies against total Akt, GAPDH,b-actin and insulin receptor-b (IRb), rabbit polyclonal

antibody against TBC1D1 (#5929), and mouse monoclo-nal antibodies against phosphotyrosine and GLUT4 werefrom Cell Signaling Technology. Rabbit anti-HA polyclonaland mouse monoclonal antibodies against FLAG andc-Myc were from Sigma, and a rabbit polyclonal antibodyagainst TBC1D1 (ab56191) was from Abcam. Rabbitpolyclonal antibodies against APPL1, APPL2, phospho-TBC1D1 (serine [Ser] 235 and threonine [Thr] 596),phospho-Akt (Ser-473), and recombinant proteins of full-length and truncated APPL1 and APPL2 were obtainedfrom Antibody and Immunoassay Services, The Universityof Hong Kong (HKU). Human recombinant insulin wasfrom Novo Nordisk. The PI3K inhibitor LY294002 andcytochalasin B were from Sigma, and 2-deoxy-[3H]-glucoseand [14C]-mannitol were from PerkinElmer.

Animal StudiesTo generate APPL2 transgenic (APPL2 Tg) mice, humanAPPL2 cDNA was cloned into a transgenic vector underthe control of cytomegalovirus immediate early b-actinpromoter (9). The transgenic mice were generated asdescribed previously (9) and were screened by PCR anal-ysis with genotyping primers as listed in SupplementaryTable 1.

APPL2 knockout (KO) mice were generated by Shang-hai Nanfang Center for Model Organisms. The targetingconstruct containing loxP sites flanking exon 5 of theAPPL2 gene and the FRT-flanked selection cassette up-stream of the loxP sites was electroporated into embryonicstem cells, followed by selection of positive embryonic stemclones, microinjection, and chimera identification as de-scribed previously (9). To generate muscle-specific APPL2KO mice and their wild-type (WT) littermates, APPL2floxed

mice were crossed with transgenic mice expressing Crerecombinase under the control of muscle creatine kinasepromoter (The Jackson Laboratory), and their genotypeswere identified by PCR analysis using the primers listedin Supplementary Table 1.

Both APPL2 Tg and APPL2 KO mice were backcrossedonto a C57BL/6 genetic background for at least sevengenerations and housed in a room with temperature(23 6 1°C) and light (12-h light-dark cycle) control.Four-week-old male APPL2 Tg mice, muscle-specificAPPL2 KO, and their WT littermates were fed with astandard chow (Purina Mills) comprising 20% kcal fromprotein, 10% kcal from fat, and 70% kcal from combinedsimple carbohydrates. Glucose tolerance test (GTT) andinsulin tolerance test (ITT) were performed in 16-h– and6-h–fasted animals as previously described (8). All animalexperimental protocols were approved by the AnimalEthics Committee of HKU.

RNA Interference Preparation and TransfectionThe sequences of RNA interference (RNAi) duplex oligosagainst APPL1, APPL2, and scrambled control (Invitrogen)are listed in Supplementary Table 1. These oligos weretransfected into C2C12 or L6 cells by electroporationaccording to the manufacturer’s protocol (Bio-Rad).

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Mutagenesis, Generation, and Purification ofAdenovirusesThe adenoviruses encoding APPL1 and luciferase were gen-erated in our previous study (11). To construct adenoviralvectors for overexpression of APPL2 or TBC1D1, cDNAencoding human APPL2 or human TBC1D1 were clonedinto pAdeasy-1 adenoviral backbone vector (Stratagene) asdescribed previously (11). PCR-based site-directed muta-genesis was performed to introduce S235A, S237A, andT596D mutations in human TBC1D1 by using the muta-genic primers as previously described (12). C2C12 and L6myotubes were infected with various adenoviruses ata multiplicity of infection of 50.

Analysis of Glucose Uptake and GLUT4 Translocationin Muscle Cells and Isolated Skeletal MusclesGlucose uptake assays were performed in C2C12 and L6myotubes using 2-deoxy-[3H]-glucose as tracer as de-scribed in our previous study (20). For ex vivo glucoseuptake assay, mice fasted for 4 h were anesthetized. Iso-lated extensor digitorum longus (EDL) or soleus muscleswere stimulated without or with insulin 60 mU/mL fol-lowed by measurement of 2-deoxy-[3H]-glucose uptake (21).For determination of GLUT4 translocation, an antibody-coupled densitometric assay was used to measure thecontent of surface Myc-GLUT4 in L6 myotubes stablyexpressing Myc-tagged GLUT4 as described in previousstudies (20,22).

Immunoprecipitation and Mass SpectrometryC2C12 myotubes or human embryonic kidney (HEK) 293cells were subjected to immunoprecipitation as describedin our previous study (12). The immunocomplexes wereeluted and subjected to immunoblotting analysis withvarious antibodies as specified in each figure legend orby mass spectrometry analysis for identification of inter-acting partners of APPL2 as previously described (23).

Statistical AnalysisAll experiments were performed routinely, with four tosix repeats in each group. Data are presented as mean 6SE. Statistical significance was determined by Studentt test or two-way ANOVA (for the experiments that in-volved two factors) followed by Bonferroni post hoc tests.In all statistical comparisons, P , 0.05 indicated a signif-icant difference.

RESULTS

APPL1 and APPL2 Exert Opposite Effects on Insulin-Stimulated Glucose Uptake in Muscle CellsTo compare the effects of APPL1 and APPL2 on glucoseuptake and metabolism, we used the adenoviral genedelivery system for overexpression of these two adaptorproteins in L6 myotubes. The APPL1 and APPL2 proteinlevels in cells with ectopic expression of both APPL1 andAPPL2 were increased by approximately threefold relativeto their endogenous levels in L6 myotubes (Fig. 1A). Con-sistent with a previous study (24), ectopic overexpressionof APPL1 enhanced insulin-stimulated glucose uptake and

GLUT4 translocation to the plasma membrane comparedwith cells with ectopic expression of luciferase controls.On the contrary, overexpression of APPL2 inhibited suchinsulin actions (Fig. 1B and C). A similar result was alsofound in C2C12 myotubes (data not shown).

To verify these in vitro findings in cultured cells, weevaluated the impact of APPL1 and APPL2 overexpressionon insulin-stimulated glucose uptake in skeletal muscle inmice. Transgenic mice with overexpression of FLAG-tagged human APPL1 driven by cytomegalovirus-b-actinpromoter were generated in our previous study (9,10). Weused a similar strategy to generate transgenic mice withoverexpression of human APPL2 (Supplementary Fig. 1A),which was confirmed by PCR (Supplementary Fig. 1B) andimmunoblotting (Supplementary Fig. 1C). The expressionlevels of APPL2 in EDL and soleus muscles of the trans-genic mice were elevated approximately four- to sixfoldcompared with WT controls (Supplementary Fig. 1C).Consistently, ex vivo studies in isolated muscles showedthat transgenic expression of APPL2 suppressed, whereasAPPL1 enhanced, insulin-stimulated glucose uptake inEDL muscle compared with WT controls (Fig. 1D). A sim-ilar result was also observed in soleus muscle (data notshown).

APPL2 Tg mice exhibited a trend of increased fastingglucose and insulin levels (Supplementary Fig. 1D and E)and displayed a modest, but significant impairment inboth glucose tolerance and insulin sensitivity comparedwith WT littermates (Supplementary Fig. 1F and G). Ofnote, overexpression of APPL2 had no effect on proteinabundance of GLUT4 in EDL muscles (Supplementary Fig.2).

Transfection of L6 myotubes with the duplex RNAiagainst rat APPL1 and APPL2 led to a reduction of APPL1and APPL2 expression by 76% and 81%, respectively,compared with scrambled controls (Fig. 1E). Of note,knockdown of APPL2 expression potentiated, whereas sup-pression of APPL1 expression inhibited, insulin-stimulatedglucose uptake and plasma membrane recruitment ofGLUT4 (Fig. 1F and G). Likewise, the potentiating effectsof APPL1 and the inhibitory effects of APPL2 on insulin-evoked glucose uptake were observed in C2C12 myotubes(data not shown).

APPL2 Is a Key Regulator of Glucose Homeostasis inMiceTo test whether deletion of APPL2 in skeletal musclesprotects mice from glucose intolerance, we generatedmuscle-specific APPL2 KO mice by crossing APPL2floxed

mice with transgenic mice expressing Cre recombinaseunder the control of muscle creatine kinase promoter,which resulted in the disruption of the APPL2 gene atexon 5 (Fig. 2A). Immunoblotting analysis confirmedthe dramatic reduction of APPL2 protein in EDL andsoleus muscles but not in the brain of APPL2 KO mice(Fig. 2B). The residual expression of APPL2 in EDL andsoleus muscles in APPL2 KO mice is perhaps due to its

3750 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

ubiquitous expression in other nonmyocyte cells (16). Ge-netic ablation of APPL2 in muscles had no obvious effectson food intake, body weight, and fasting glucose and in-sulin levels (Supplementary Table 2). GTT revealed thatAPPL2 KO mice exhibited a significant improvement ofglucose tolerance in response to glucose challenge com-pared with WT littermates (Fig. 2C and D). Serum insulinlevels during the GTT were similar between the two groupsof mice (Fig. 2E). Insulin sensitivity, as determined by ITT,was also enhanced by APPL2 deletion (Fig. 2F). Ex vivostudies demonstrated that insulin-stimulated glucose up-take in EDL muscles of APPL2 KO mice was significantlyincreased compared with WT littermates (Fig. 2G). A sim-ilar result was observed in soleus muscles (data not shown).Insulin-elicited phosphorylation of Akt and IRb in EDLmuscle was comparable between the two groups (Fig. 2H).

TBC1D1 Is an Interacting Partner and DownstreamEffector of APPL2To identify the proximal downstream effectors of APPL2,we established a stable cell line expressing FLAG-taggedhuman APPL2 for affinity pull-down purification of itspotential interacting partners in HEK293 cells. Tandem

mass spectrometry identified several putative interactingpartners of APPL2, including heat shock protein (HSP)70, HSP90, APPL1, centaurin delta 1, TBC1D1, and son ofsevenless homolog 1. Among these APPL2-interactingproteins, TBC1D1, a member of the TBC1 Rab-GTPasefamily of proteins abundantly expressed in skeletalmuscles, is an important regulator of insulin signalingand glucose metabolism (25,26). Of note, our coimmuno-precipitation analysis showed that TBC1D1 binds toAPPL2 but not to APPL1 (Fig. 3A). The specificity of im-munoprecipitation for TBC1D1 was confirmed by RNAi-mediated knockdown of TBC1D1 expression in C2C12myotubes, leading to a substantial decrease in immunopre-cipated TBC1D1 (Supplementary Fig. 3). On the otherhand, APPL2 did not bind to TBC1D4 (Fig. 3B), a paralogof TBC1D1 that is also involved in the regulation of glucosetransport in muscle cells and adipocytes (6). The APPL2–TBC1D1 interaction was enhanced by insulin stimulationin EDL muscle of C57 mice (Fig. 3C) and C2C12 myotubes(Fig. 3D), and such an enhancement was largely blocked bythe PI3K inhibitor LY294002 (Fig. 3D).

To determine which domain of APPL2 is responsiblefor TBC1D1 binding, we generated a series of vectors that

Figure 1—Opposite actions of APPL1 and APPL2 on insulin-stimulated glucose uptake and GLUT4 translocation in myotubes and skeletalmuscles. L6 myotubes were infected with adenovirus-encoding luciferase (Luci), APPL1, or APPL2 for 24 h followed by serum starvation for6 h. A: The starved cells were subjected to immunoblotting using a rabbit polyclonal antibody against APPL1 or APPL2 or a rabbitmonoclonal 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 GLUT4infected with the indicated adenoviruses. The reference values for 2-deoxy-[3H]-glucose uptake and membrane-bound Myc-GLUT4 are4.82 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 EDLmuscles 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, orscrambled control for 24 h followed by serum starvation for 6 h. The starved cells were subjected to immunoblotting (E) or measurementof insulin-stimulated glucose uptake (F ). G: Insulin-stimulated GLUT4 plasma membrane recruitment in the L6 myotube stable cell lineexpressing 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 againstscrambled control, respectively. All experiments were repeated at least three times, and representative images are shown. *P < 0.05 (n = 6)by Student t test.

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express various domains of APPL2 (Fig. 4A). Coimmu-noprecipitation analysis revealed that TBC1D1 inter-acted with the BAR domain but not with the PH orPTB domain of APPL2 (Fig. 4B). The pull-down assayfurther confirmed the direct interaction betweenTBC1D1 and the BAR domain of APPL2 (SupplementaryFig. 4). To test whether the BAR domain mediates thesuppressive effect of APPL2 on insulin-stimulated glucoseuptake, we transduced L6 myotubes with adenovirusencoding the BAR domain or the PH-PTB domain orluciferase as control. Similar to full-length APPL2 (Fig.1B and C), ectopic overexpression of the BAR domain butnot the PH-PTB mutant inhibited insulin-stimulated glu-cose uptake and GLUT4 translocation to the plasmamembrane (Fig. 4C and D). Taken together, these find-ings suggest that the BAR domain of APPL2 exerts aninhibitory effect on insulin-evoked glucose uptake byinteracting with TBC1D1.

Phosphorylation of TBC1D1 at Ser-235 Is Required forIts Interaction With APPL2 and the Inhibitory Effects ofAPPL2 on Insulin-Dependent Glucose UptakeTo further delineate how the APPL2–TBC1D1 interactionregulates insulin-stimulated glucose uptake, we generateda series of truncated mutants of TBC1D1 (Fig. 5A) to mapthe minimal domain mediating its binding to APPL2.Coimmunoprecipitation analysis revealed that all themutants of TBC1D1 containing the linker region betweenthe two PTB domains (amino acids 165–279) were able tointeract with APPL2, whereas those mutants without thelinker region lost their APPL2-binding property (Fig. 5B).Furthermore, the linker region alone was sufficient tobind with APPL2 (Fig. 5B).

TBC1D1 is a hyperphosphorylated protein, and insulininduces its phosphorylation through Akt activation (6).Because insulin prompted the APPL2–TBC1D1 interac-tion in a PI3K-dependent manner, we searched for the

Figure 2—Generation and metabolic characterization of muscle-specific APPL2 KO mice. A: Strategy for generating muscle-specificAPPL2 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-oldheterozygous (hetero) and homozygous muscle-specific APPL2 KO mice and WT littermates were subjected to immunoblotting usinga rabbit anti-APPL2 polyclonal or rabbit anti-GAPDH monoclonal antibody. C: GTT in 20-week-old APPL2 KO mice and WT controls fedstandard chow. D: Area under the curve (AUC) of the GTT in panel C. Data are fold change relative to WT controls. E: Serum insulin levelsduring 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: Exvivo 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 RESEARCHDESIGN ANDMETHODS. H: EDL muscles from fasted C57BL/6 mice injected without or with insulin 0.5 units/kgfor 15 min were subjected to immunoprecipitation (IP) using a rabbit anti-IRb monoclonal antibody followed by immunoblotting usinga 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 inthe right panel represents fold changes of phosphorylation of Akt vs. total Akt or tyrosine phosphorylation of IRb vs. total IRb relative to thebasal levels in WT controls as quantified by densitometry. *P < 0.05 (n = 5) by Student t test.

3752 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

phospho-Akt substrate site within the linker region thatmay be crucial for its association with APPL2. Of note,a previous study showed that Ser-229 of mouse TBC1D1(equivalent to Ser-235 on human TBC1D1) within thisregion is potentially phosphorylated by Akt (27). We con-firmed that insulin stimulated TBC1D1 phosphorylationat Ser-235 in a time-dependent manner in both EDL mus-cle of C57 mice and C2C12 myotubes (Fig. 5C and D), andsuch an insulin action was largely abolished by the PI3Kinhibitor LY294002 (Fig. 5D). Substitution of Ser-235with nonphosphorylatable alanine (S235A) markedly at-tenuated insulin-stimulated interaction between APPL2and TBC1D1, whereas mutation of Ser-237 to alaninehad no obvious effect (Fig. 5E).

We next compared the effects of TBC1D1 and itsmutant S235A on insulin-stimulated glucose uptake in L6myotubes. Consistent with a previous study (27), wefound that adenovirus-mediated expression of TBC1D1did not affect insulin-evoked Akt phosphorylation (datanot shown) but led to a significant inhibition of insulin-stimulated glucose uptake compared with cells expressing

luciferase control (Fig. 5F). The inhibitory effects ofTBC1D1 on insulin-evoked glucose uptake were abrogatedby the S235A but not S237A mutation (Fig. 5F).

Several previous studies have shown that insulin-induced Akt activation leads to direct TBC1D1 phos-phorylation on Thr-596 (equivalent to Thr-590 in mouseTBC1D1) (6,26–28), which in turn promotes the trans-port of GLUT4 to the plasma membrane by activatingRab-GTPases (6,26–28). To investigate the effects ofAPPL2 on insulin-elicited phosphorylation of TBC1D1,we injected APPL2 Tg and APPL2 KO mice intraper-itoneally with insulin. Immunoblotting demonstratedthat insulin-stimulated TBC1D1 phosphorylation onThr-596 in EDL and soleus muscles was enhanced byAPPL2 deletion but diminished by APPL2 overexpression(Fig. 6). On the other hand, APPL2 overexpression ordeletion had no obvious effect on expression levels oftotal TBC1D1 or TBC1D1 phosphorylation on Ser-235(Fig. 6).

Figure 3—APPL2 binds to TBC1D1 in an insulin- and PI3K-dependentmanner. A: C2C12 myotubes were collected and subjected to im-munoprecipitation (IP) using a rabbit anti-TBC1D1 polyclonal anti-body (Abcam) or rabbit IgG as control followed by immunoblottingwith the indicated antibodies. B: C2C12 infected with adenovirusencoding FLAG-tagged APPL2 (FLAG-APPL2) and HA-taggedTBC1D1 (HA-TBC1D1) or HA-tagged TBC1D4 (HA-TBC1D4) weresubjected to IP using a mouse anti-FLAG monoclonal antibodyfollowed by immunoblotting with the indicated antibodies. C: C57mice were fasted overnight followed by insulin 0.5 units/kg i.p. forthe indicated time points. EDL muscles were isolated and subjectedto IP using a rabbit anti-APPL2 polyclonal antibody followed byimmunoblotting using a rabbit polyclonal antibody against APPL2or TBC1D1 (Abcam). D: C2C12 myotubes expressing FLAG-APPL2and HA-TBC1D1 were serum starved for 12 h followed by preincu-bation with or without the PI3K inhibitor LY294002 (LY 50 mmol/L)for 30 min. The cells treated without or with insulin 10 nmol/L for 15min were subjected to IP using a mouse anti-FLAG monoclonalantibody followed by immunoblotting with a mouse anti-FLAGmonoclonal or rabbit anti-HA polyclonal antibody as indicated. Allexperiments were repeated at least three times, and representativeimages are shown.

Figure 4—The BAR domain of APPL2 interacts with TBC1D1 andmediates the suppressive effects of APPL2 on insulin-stimulatedglucose uptake. A: Schematic diagram of FLAG-tagged WT APPL2and its truncated mutants containing various domains used for im-munoprecipitation (IP) assays. B: HEK293 cells were cotransfectedwith plasmids encoding HA-tagged TBC1D1 and FLAG-WT-APPL2or FLAG-APPL2 mutants (BAR-PH, PH-PTB, BAR, and PH) or anempty vector as negative control (-ve) for 48 h followed by IP witha mouse anti-FLAG monoclonal antibody and immunoblotting usinga mouse anti-FLAG monoclonal or rabbit anti-HA polyclonal anti-body as indicated. C: L6 myotubes infected with various truncatedmutants of APPL2 or luciferase (Luci) control were subjected toglucose uptake assay as described in RESEARCH DESIGN AND METHODS.D: L6 myotubes stably expressing Myc-tagged GLUT4 wereinfected with the indicated adenoviruses, followed by serum star-vation for 6 h. Insulin-stimulated GLUT4 translocation to plasmamembrane was measured using the antibody-coupled densitomet-ric assay as described in RESEARCH DESIGN ANDMETHODS. The referencevalues for glucose uptake and membrane-bound Myc-GLUT4 are5.2 6 0.49 pmol/min/mg protein and 4.70 6 0.54 fmol/mg proteinin noninsulin-treated cells expressing luciferase control, respec-tively. Data are fold changes relative to basal levels in cells express-ing luciferase. *P < 0.05 (n = 5) by Student t test.

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To further investigate the interplay between APPL2and TBC1D1 in regulating insulin-stimulated glucoseuptake, we co-overexpressed APPL2 and TBC1D1 or itsS235A mutant in L6 myotubes by adenoviral gene trans-fer system (Fig. 7A). Similar to the findings in APPL2 Tgmice, insulin-stimulated TBC1D1 phosphorylation onThr-596 was significantly impaired by overexpression ofAPPL2 compared with cells overexpressing luciferase con-trols (Fig. 7A and B). However, the suppressive effect ofAPPL2 overexpression on insulin-stimulated phosphory-lation of Thr-596 was abolished in cells expressing theTBC1D1-S235A mutant (Fig. 7A and B). Of note, over-expression of APPL2 caused only a modest suppressiveeffect on insulin-elicited Akt phosphorylation and hadno effect on IRb phosphorylation in L6 myotubes express-ing either TBC1D1 or its S235A mutant (Fig. 7C and D).

As expected, the inhibitory effect of TBC1D1 overexpres-sion on insulin-stimulated glucose uptake was further ag-gravated by overexpression of APPL2 (Fig. 7E). However,this suppressive effect of APPL2 was lost in cells express-ing the TBC1D1-S235A mutant (Fig. 7E).

To further investigate whether APPL2 suppressesinsulin-elicited glucose uptake by modulating TBC1D1phosphorylation on Thr-596, Thr-596 of TBC1D1 wasmutated to aspartic acid (T596D) to mimic its phosphor-ylation. The level of adenovirus-mediated expressionof the TBC1D1-T590D mutant was comparable to WTTBC1D1 in L6 myotubes (Fig. 8A). However, the suppres-sive effects of APPL2 overexpression on insulin-inducedglucose uptake was observed in only L6 myotubes withectopic expression of WT TBC1D1, not in L6 cells express-ing the TBC1D1-T590D mutant (Fig. 8B).

Figure 5—Effect of Ser-235 phosphorylation of TBC1D1 on its APPL2-binding and -suppressive action on insulin-dependent glucoseuptake. 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 byimmunoblotting using antibodies as indicated. C: EDL muscles isolated from C57 mice injected without or with insulin 0.5 units/kg for theindicated time points were subjected to IP with a rabbit anti-TBC1D1 polyclonal antibody (Abcam) followed by immunoblotting usinga rabbit polyclonal antibody against phospho-TBC1D1 (Ser-235) or total TBC1D1 (Cell Signaling Inc.) as specified. The specificity of therabbit phospho-TBC1D1 (Ser-235) polyclonal antibody was confirmed by immunoblotting, showing the disappearance of a specific bandwith a molecular weight of ;160 kDa in cells expressing nonphosphorylatable TBC1D1 S235A mutant (data not shown). D: C2C12myotubes infected with adenovirus encoding HA-WT-TBC1D1 were pretreated with or without the PI3K inhibitor LY294002 and thenstimulated with insulin 10 nmol/L for various time periods followed by immunoblotting with a rabbit polyclonal antibody against HA orphospho-TBC1D1 (Ser-235). E: C2C12 myotubes infected with HA-WT-TBC1D1 or its mutant S235A (Ser-235 is mutated to alanine) orS237A (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 monoclonalantibody (left panel) and insulin-stimulated glucose uptake assay (right panel) as described in RESEARCH DESIGN AND METHODS. The referencevalue for glucose uptake is 4.2 6 0.32 pmol/min/mg protein in noninsulin-treated cells expressing luciferase control. Data are fold changesrelative to noninsulin-treated cells expressing luciferase control. *P < 0.05 (n = 5) by Student t test. CBD, calmodulin-binding domain; LK,linker region.

3754 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

DISCUSSION

In this study, we provide both in vivo and in vitroevidence showing that APPL2 negatively regulates insulin-stimulated glucose transport by interacting with TBC1D1.In both skeletal muscle and cultured muscle cells, insulin-induced glucose uptake is diminished by overexpressionof APPL2 but is enhanced by suppression of APPL2expression.

Despite of the high similarity in domain organizationand amino acid sequences between APPL1 and APPL2, wedemonstrate that these two adaptor proteins exertopposite effects on insulin-stimulated glucose uptake inskeletal muscle. APPL1 exerts its insulin-sensitizingeffects by competing with TRB3 for binding to Akt,thereby promoting the translocation of Akt to the plasmamembrane for further activation (11,16,24). On the otherhand, the present study shows that APPL2 suppressesinsulin-dependent glucose uptake at a step downstreamof Akt. Unlike APPL1, APPL2 does not interact withAkt or the regulatory subunit p85 and catalytic subunitp110 of PI3K (7,29,30). Indeed, overexpression of APPL2results in only a modest decrease in insulin-elicited Aktphosphorylation in myotubes, whereas targeted deletionof APPL2 in skeletal muscle has no obvious effect on Aktphosphorylation. Such a modest effect of APPL2 on Aktactivity in cultured cells is perhaps a result of its

heterodimerization with APPL1, which in turn preventsthe binding of APPL1 to Akt (16). We demonstrate thatAPPL2 but not APPL1 interacts with TBC1D1, a down-stream substrate of Akt that is critically involved inGLUT4 vesicle trafficking. Therefore, the opposite effectsof APPL1 and APPL2 on insulin-stimulated glucose uptakeare attributed to the differential binding of these twoadaptor proteins to Akt and TBC1D1. In line with thesefindings, APPL1 and APPL2 have been shown to bind tovarious types of Rab-GTPases involved in membrane traf-ficking (17,19). The binding of APPL1 and APPL2 to var-ious sets of intracellular signaling molecules may be dueto their differences in oligomerization, surface charges,and/or subcellular localization (17,18,31,32).

TBC1D1 and TBC1D4, both of which are Rab-GAPproteins sharing ;47% sequence identity and a similardomain organization, are the important regulators of bothinsulin- and contraction-induced trafficking of GLUT4vesicles (6,33,34). Upon insulin stimulation, activated Aktinduces phosphorylation of both TBC1D1 and TBC1D4at multiple sites flanking the second PTB domain(26–28,35,36). Substitution of these phosphorylationsites with nonphosphorylatable alanine in TBC1D1 andTBC1D4 abolishes insulin-induced GLUT4 translocationto the plasma membrane in both adipocytes and musclecells (25–27,36–38). Although the precise mechanisms by

Figure 6—APPL2 suppresses insulin-elicited phosphorylation of TBC1D1 at Thr-590 in skeletal muscle. A and B: Twelve-week-old maleAPPL2 KO mice, APPL2 Tg mice, and their respective WT littermates were fasted overnight followed by intraperitoneal injection without orwith 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 SignalingInc.), phospho-TBC1D1 (Ser-235), or phospho-TBC1D1 (Thr-596) as indicated. The charts in the right panels are the relative abundance ofphosphorylated TBC1D1 at Thr-596 or Ser-235 vs. total TBC1D1 as determined by densitometric analysis. The specificity of the rabbitantiphospho-TBC1D1 (Thr-596) polyclonal antibody was validated by immunoblotting, showing the disappearance of a specific band witha molecular weight of ;160 kDa in cells expressing nonphosphorylatable TBC1D1 T596A mutant (data not shown). Data are fold changesrelative to noninsulin-treated WT controls. *P < 0.05 (n = 4) by Student t test. N.S., not significant.

diabetes.diabetesjournals.org Cheng and Associates 3755

which TBC1D1 and TBC1D4 regulate GLUT4 vesicletrafficking remain unclear, it has been proposed thatnonphosphorylated TBC1D4 in the basal state binds toGLUT4-containing vesicles to maintain its substrate Rab-GTPases in their inactive guanosine diphosphate–loadedform, thereby trapping GLUT4 inside cells (27,35–38).Insulin-evoked TBC1D4 phosphorylation on Thr649, pos-sibly through interaction with 14-3-3 proteins, inhibits itsGAP activity, which in turn allows guanosine triphosphateloading and activation of Rab-GTPases required for dock-ing and fusion of GLUT4-containing vesicles to the plasmamembrane (36–38). A previous study demonstrated thatmice with TBC1D4-Thr649Ala knockin mutation, in whichThr649 was mutated to nonphosphorylatable alanine, dis-play impaired glucose disposal and insulin sensitivity asa result of reduced GLUT4 trafficking to the cell surfacein skeletal muscles (39). Likewise, insulin-elicited phos-phorylation of TBC1D1 at Thr-596, a site equivalent toThr-649 of TBC1D4, is obligatory for GLUT4 traffickingpossibly by inactivation of GAP activity (26–28). In thecurrent study, we found that the inhibitory effects ofAPPL2 overexpression on glucose uptake in muscle cells

and skeletal muscle are associated with impaired phos-phorylation of TBC1D1 at Thr-596 in response to insulinstimulation. Furthermore, mutation of Thr-596 of TBC1D1to aspartic acid reverses the inhibitory effect of APPL2on insulin-stimulated glucose uptake, suggesting thatthe APPL2–TBC1D1 interaction prevents Akt-mediatedphosphorylation of TBC1D1 at Thr-596, thereby im-pairing insulin-evoked GLUT4 translation to the plasmamembrane.

Although a previous study has identified Ser-235, ahighly conserved amino acid located within the linkerregion of TBC1D1, as a likely phospho-Akt substrate siteresponsive to insulin stimulation (27), its physiologicalrelevance has never been explored. In the current study,we further confirm that insulin induces TBC1D1 phosphor-ylation on Ser-235 in a time-dependent manner by usingan antiphospho-Ser-235 antibody, and this phosphoryla-tion is suppressed by pharmacological inhibition of PI3K.Furthermore, we found that mutation of Ser-235 to ala-nine not only abolishes the APPL2–TBC1D1 interactionbut also abrogates the suppressive effects of APPL2 oninsulin-induced TBC1D1 phosphorylation on Thr-596

Figure 7—The APPL2-TBC1D1 interaction modulates insulin-elicited phosphorylation of TBC1D1 at Thr-596 and the suppressive effect ofAPPL2 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 treatedwithout or with insulin 10 nmol/L for 10 min, and the cell lysates were subjected to either immunoblotting with various antibodies asindicated or immunoprecipitation (IP) using a rabbit anti-IRbmonoclonal antibody. The IP IRb was subjected to immunoblotting analysis fortyrosine phosphorylation using a mouse antityrosine monoclonal antibody. B–D: The charts show the relative abundance of phosphorylatedTBC1D1 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 luciferasecontrol. 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.

3756 APPL2 Controls Insulin-Induced Glucose Uptake Diabetes Volume 63, November 2014

and glucose uptake in muscle cells. On the basis of thesefindings, Akt-dependent phosphorylation of Ser-235 at N-terminal TBC1D1 likely triggers the interaction betweenTBC1D1 and APPL2, which in turn prevents phosphory-lation of Thr-596 of TBC1D1 by either conformationalchanges or direct blockage of Akt access to Thr-596 andits surrounding motif. Of note, Ser-235 is absent inTBC1D4, which may partly explain the preferential inter-action of APPL2 with TBC1D1 but not with TBC1D4. Onthe other hand, mutation of Ser-237 to alanine has noeffect on the APPL2–TBC1D1 interaction as well as on thesuppressive effect of TBC1D1 on insulin-stimulated glu-cose uptake. Of note, Ser-237 is phosphorylated by AMPKin response to contraction but not by insulin stimulation(40–42), suggesting that APPL2 may only regulate glucoseuptake through the TBC1D1 signaling pathway in re-sponse to insulin and not contraction in skeletal muscle.

In summary, the present study identifies APPL2 asa negative regulator of insulin-stimulated GLUT4 trans-location to the plasma membrane by interacting with andmodulating phosphorylation of TBC1D1 (SupplementaryFig. 5). Insulin stimulation induces phosphorylation ofboth Ser-235 and Thr-596 of TBC1D1. Phosphorylationof TBC1D1 at Ser-235 triggers its interaction with APPL2,which in turn blocks further phosphorylation of Thr-596required for plasma membrane targeting of GLUT4. Sucha feedback regulatory loop may be an important mechanismfor insulin to fine tune glucose homeostasis in mammals.

Funding. This work was supported by General Research Fund Grant HKU783010M, HKU matching funds for State Key Laboratory of Pharmaceutical

Biotechnology, the National Science Foundation of China (81270881),and the National Basic Research Program of China (2011CB504004 and2010CB945500).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. K.K.Y.C. and W.Z. contributed to researching dataand 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 andreviewing the manuscript. K.S.L.L. contributed to supervising the study andediting the manuscript. A.X. contributed to the design and supervision of thestudy and writing of the manuscript. K.K.Y.C. is the guarantor of this work and, assuch, had full access to all the data in the study and takes responsibility for theintegrity of the data and the accuracy of the data analysis.

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