page 1 of 43 diabetesdiabetes.diabetesjournals.org/content/diabetes/early/2014/05/30/db14... ·...

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The adaptor protein APPL2 inhibits insulin-stimulated glucose uptake by interacting with

TBC1D1 in skeletal muscle

#Kenneth KY Cheng

1,2, #Weidong Zhu

2, Bin Chen

2, Yu Wang

1,3, Donghai Wu

4, Gary Sweeney

5,

Baile Wang1,2, Karen SL Lam

1,2 and Aimin Xu

1,2,3

1 State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong,

2 Department of Medicine, The University of Hong Kong,

3Department of Pharmacology & Pharmacy, The University of Hong Kong,

4The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and

Health, Chinese Academy of Sciences,

5Department of Biology, York University, Toronto, Canada.

Correspondence to Aimin Xu or Kenneth KY Cheng, Department of Medicine, The University

of Hong Kong, L8, 21 Sassoon Road, Hong Kong, Tel: (852) 2819 9754, Fax: (852) 2816 2095,

E-mail: [email protected], [email protected]

#KKY Cheng and W Zhu equally contribute in this study.

Running title: APPL2 controls insulin-induced glucose uptake.

Word count: 3998 words

Figures: 8 (5 supplementary figures)

Tables: 2 supplementary tables

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Diabetes Publish Ahead of Print, published online May 30, 2014

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Abstract

Insulin stimulates glucose uptake by promoting the trafficking of GLUT4 to the plasma

membrane in muscle cells, and impairment of this insulin action contributes to hyperglycemia in

type 2 diabetes. The adaptor protein APPL1 potentiates insulin-stimulated Akt activation and

downstream actions. However, the physiological functions of APPL2, a close homologue of

APPL1, in regulating glucose metabolism remain elusive. Here we showed that insulin-evoked

plasma membrane recruitment of GLUT4 and glucose uptake were impaired by APPL2

overexpression, but enhanced by APPL2 knockdown. Likewise, conditional deletion of APPL2

in skeletal muscles enhanced insulin sensitivity, leading to an improvement of glucose tolerance.

We identified the Rab-GTPase-activating protein TBC1D1 as an interacting partner of APPL2.

Insulin stimulated TBC1D1 phosphorylation on serine-235, leading to enhanced interaction with

the BAR domain of APPL2, which in turn suppressed insulin-evoked TBC1D1 phosphorylation

on threonine-596 in cultured myotubes and skeletal muscle. Substitution of serine-235 with

alanine diminished APPL2-mediated inhibition on insulin-dependent TBC1D1 phosphorylation

on threonine-596 and the suppressive effects of TBC1D1 on insulin-induced glucose uptake and

GLUT4 translocation to the plasma membrane in cultured myotubes. Therefore, the APPL2-

TBC1D1 interaction serves as a key step to fine-tune insulin-stimulated glucose uptake by

regulating the membrane recruitment of GLUT4 in skeletal muscle.

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Introduction

Insulin maintains glucose homeostasis by facilitating the uptake of postprandial glucose into

adipose tissues and skeletal muscle, the latter of which accounts for approximately 75% of total

glucose disposal (1). The binding of insulin to its receptors elicits tyrosine phosphorylation of

insulin receptor substrates, which in turn activates phosphatidylinositol 3-kinase (PI3K), leading

to the membrane recruitment and activation of Akt. Activated Akt subsequently induces the

translocation of glucose transporter 4 (GLUT4) from intracellular vesicular compartments to the

plasma membrane for glucose uptake. In type 2 diabetic patients, the ability of insulin to

stimulate glucose uptake is significantly 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 muscle causes insulin resistance and diabetes (4), whereas overexpression of

GLUT4 alleviates hyperglycemia and insulin resistance in db/db diabetic mice (5). GLUT4

translocation is tightly controlled by insulin signaling cascades and a series of small GTPase

proteins (6). Recent studies have demonstrated that the Rab-GTPase-activating proteins (GAP),

TBC (Tre-2/Bub2/Cdc16) domain family, member 1 (TBC1D1) and member 4 (TBC1D4, also

known as AS160), may integrate insulin signaling and Rab-GTPase activity, thereby regulating

GLUT4 trafficking and glucose uptake (6). However, the detailed intracellular signaling events

that confer insulin-elicited glucose uptake are still not fully characterized.

Mounting evidence suggests that the adaptor protein APPL1, which consists of 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

multiple insulin-responsive tissues (7). Genetic disruption of APPL1 causes insulin resistance

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and defective glucose-stimulated insulin secretion, leading to glucose intolerance in mice (8). In

contrast, transgenic overexpression of APPL1 prevents obesity-induced deleterious effects on

glucose homeostasis and endothelial and cardiac functions (8-10). Hepatic overexpression of

APPL1 improves hyperglycemia, glucose intolerance and insulin sensitivity in db/db diabetic

mice, whereas hepatic silencing of APPL1 causes insulin resistance and hyperglycemia in lean

mice (11). In pancreatic β-cells, APPL1 promotes glucose-stimulated insulin secretion by

upregulating expression of soluble N-ethylmaleimide-sensitive factor attachment protein receptor

proteins in an Akt dependent manner (8). At the molecular level, the interaction between APPL1

and Akt prevents Akt from binding to the pseudokinase tribble-3 (TRB3), thereby promoting Akt

to the plasma membrane for further activation in hepatocytes, endothelial cells and pancreatic β-

cells (8, 9, 11, 12). Furthermore, APPL1 potentiates the insulin-sensitizing effects of adiponectin

on promotion of glucose uptake by direct interaction with the two adiponectin receptors (13).

APPL2 is a close homolog of APPL1 abundantly expressed in skeletal muscle. These two

adaptor proteins share 52% identity and 72% similarity in amino acid sequence, and the same

domain organization (7). APPL1 and APPL2 appear to play a similar role in mediating growth

factors-induced cell proliferation in fibroblasts and apoptosis in zebrafish (14, 15). On the other

hand, a recent in vitro study demonstrated that these two proteins possess distinct or even

opposite functions in regulating glucose and lipid metabolism (16). Structural analysis revealed

that APPL2 incorporates two homodimers, whereas APPL1 incorporates only one homodimer in

the asymmetric unit (17-19). While APPL1 binds to the adiponectin receptors and increases

adiponectin-induced glucose uptake and fatty acid oxidation, APPL2 inhibits adiponectin actions

in muscle cells (13, 16).

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Although the insulin-sensitizing effects of APPL1 are well characterized, little is known

about the physiological role of APPL2 in insulin signaling and glucose metabolism. In this study,

we provide both in vitro and in vivo evidence that APPL2 is a negative regulator of insulin-

stimulated glucose transport in skeletal muscle. Furthermore, we have identified TBC1D1 as an

interacting partner and a downstream effector that mediates the suppressive effect of APPL2 on

insulin-elicited plasma membrane translocation of GLUT4.

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Research Design and Methods

Materials. Rabbit monoclonal antibodies against total Akt, glyceraldehyde 3-phosphate

dehydrogenase (GAPDH), β-actin and insulin receptor-β (IRβ), rabbit polyclonal antibody

against TBC1D1 (#5929) and mouse monoclonal antibodies against phospho-tyrosine and

GLUT4 were from Cell Signaling Technology. Rabbit anti-HA polyclonal and mouse

monoclonal antibodies against FLAG and c-Myc were from Sigma, and a rabbit polyclonal

antibody against TBC1D1 (ab56191) was from Abcam. Rabbit polyclonal 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

obtained from Antibody and Immunoassay Services, The University of Hong Kong (HKU).

Human recombinant insulin was from Novo Nordisk. The PI3K inhibitor LY294002 and

cytochalasin B were from Sigma. 2-deoxy-[3H]-glucose and [

14C]-mannitol were from Perkin

Elmer.

Animal studies. To generate APPL2 transgenic (APPL2 Tg) mice, human APPL2 cDNA was

cloned into a transgenic vector under the control of cytomegalovirus (CMV) immediate early-β-

actin promoter (9). The transgenic mice were generated as described previously (9), and were

screened by PCR analysis using genotyping primers as listed in Supplementary Table 1.

APPL2 knockout (KO) mice were generated by Shanghai Nanfang Center for Model Organisms.

The targeting construct containing loxP sites flanking the exon 5 of the APPL2 gene and the

FRT-flanked selection cassette upstream of the loxP sites was electroporated into embryonic

stem (ES) cells, followed by selection of positive ES clones, microinjection and chimera

identification as described previously (9). To generate muscle-specific APPL2 KO mice and its

wild-type (WT) littermates, APPL2floxed

mice were crossed with transgenic mice expressing Cre

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recombinase under the control of muscle creatine kinase (MCK) promoter (Jackson Laboratory),

and their genotypes were identified by PCR analysis using the primers listed in Supplementary

Table 1.

Both APPL2 Tg and APPL2 KO mice were backcrossed onto a C57BL/6 genetic background for

at least seven generations and housed in a room with temperature (23±1

oC) and light (12:12 hour

light-dark cycle) control. 4-week-old male APPL2 Tg mice, muscle-specific APPL2 KO and

their WT littermates were fed with a standard chow (STC) (Purina mills) composing of 20 kcal%

protein, 10 kcal% fat and 70 kcal% combined simple carbohydrates. Glucose tolerance test (GTT)

and insulin tolerance test (ITT) were performed in 16 hour- and 6 hour-fasted animals as

previously described (8). All animal experimental protocols were approved by the Animal Ethics

Committee of HKU.

RNAi preparation and transfection. The sequences of RNAi duplex oligos against APPL1,

APPL2 and scrambled control (Invitrogen) were listed in Supplementary Table 1. These oligos

were transfected into C2C12 or L6 cells by electroporation according to manufacturer’s manual

(Bio-Rad).

Mutagenesis, generation and purification of adenoviruses. The adenoviruses encoding

APPL1 and luciferase were generated in our previous study (11). To construct adenoviral vectors

for overexpression of APPL2 or TBC1D1, cDNA encoding human APPL2 or human TBC1D1

were cloned into pAdeasy-1 adenoviral backbone vector (Stratagene) as described previously

(11). PCR-based site-directed mutagenesis was performed to introduce S235A, S237A and

T596D mutations in human TBC1D1 using the mutagenic primers as previously described (12).

C2C12 and L6 myotubes were infected with various adenoviruses at multiplicity of infection of

50.

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Analysis of glucose uptake and GLUT4 translocation in muscle cells and isolated skeletal

muscles. Glucose uptake assays were performed in C2C12 and L6 myotubes using 2-deoxy-

[3H]-glucose as tracer as described in our previous study (20).

For ex vivo glucose uptake assay, mice fasted for 4 hours were anesthetized. Isolated extensor

digitorum longus (EDL) or soleus muscles were stimulated with or without insulin (60 µU/ml),

followed by measurement of 2-deoxy- [3H]-glucose uptake (21).

For determination of GLUT4 translocation, an antibody-coupled densitometric assay was

employed to measure the content of surface Myc-GLUT4 in L6 myotubes stably expressing

Myc-tagged GLUT4 as described in previous studies (20, 22).

Immunoprecipitation and mass spectrometry. C2C12 myotubes or human embryonic kidney

(HEK) 293 cells were subjected to immunoprecipitation as described in our previous study (12).

The immunocomplexes were eluted and subjected to immunoblotting analysis with different

antibodies as specified in each figure legend or mass spectrometry analysis for identification of

interacting partners of APPL2 as previously described (23).

Statistical analysis. All the experiments were performed routinely with four to six repeats in

each group. Data were presented as means ± standard error. Statistical significance was

determined by student t test or two-way ANOVA (for the experiments that involved 2 factors)

followed by Bonferroni post hoc tests. In all statistical comparisons, a p-value < 0.05 was used to

indicate a significant difference.

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Results

APPL1 and APPL2 exert opposite effects on insulin-stimulated glucose uptake in muscle

cells

To compare the effects of APPL1 and APPL2 on glucose uptake and metabolism, we

employed the adenoviral gene delivery system for overexpression of these two adaptor proteins

in L6 myotubes. The APPL1 and APPL2 protein levels in cells with ectopic expression of both

APPL1 and APPL2 were increased by approximately 3 folds relative to its endogenous levels in

L6 myotubes (Figure 1A). Consistent with a previous study (24), ectopic overexpression of

APPL1 enhanced insulin-stimulated glucose uptake and GLUT4 translocation to the plasma

membrane when compared to cells with ectopic expression of luciferase controls. On the

contrary, overexpression of APPL2 inhibited such insulin actions (Figure 1B-C). A similar result

was also evidenced in C2C12 myotubes (data not shown)

To verify the above in vitro findings in cultured cells, we evaluated the impacts of APPL1 and

APPL2 overexpression on insulin-stimulated glucose uptake in skeletal muscle in mice.

Transgenic mice with overexpression of FLAG-tagged human APPL1 driven by CMV-β-actin

promoter were generated in our previous study (9, 10). We used a similar strategy for generation

of transgenic mice with overexpression of human APPL2 (Supplementary Figure 1A), which

was confirmed by PCR (Supplementary Figure 1B) and immunoblotting analysis

(Supplementary Figure 1C). The expression levels of APPL2 in EDL and SOL muscles of the

transgenic mice are elevated approximately 4-6 folds compared to WT controls (Supplementary

Figure 1C). Consistently, ex vivo studies in isolated muscles showed that transgenic expression

of APPL2 suppressed, while APPL1 enhanced, insulin-stimulated glucose uptake in EDL muscle

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when compared to their WT controls (Figure 1D). A similar result was also observed in soleus

muscle (data not shown).

APPL2 Tg mice exhibited a trend of increase in both fasting glucose and insulin levels

(Supplementary Figure 1D-E), and also displayed a modest but significant impairment in both

glucose tolerance and insulin sensitivity as compared to its WT littermates (Supplementary

Figure 1F-G). Noticeably, overexpression of APPL2 had no effect on protein abundance of

GLUT4 in EDL muscles (Supplementary Figure 2).

Transfection of L6 myotubes with the duplex RNAi against rat APPL1 and APPL2 led to a

reduction of APPL1 and APPL2 expression by 76% and 81% respectively, when compared to

scrambled controls (Figure 1E). Notably, knockdown of APPL2 expression potentiated, while

suppression of APPL1 expression inhibited, insulin-stimulated glucose uptake and plasma

membrane recruitment of GLUT4 (Figure 1F-G). Likewise, the potentiating effects of APPL1

and the inhibitory effects of APPL2 on insulin-evoked glucose uptake were also observed in

C2C12 myotubes (data not shown).

APPL2 is a key regulator of glucose homeostasis in mice

To test whether deletion of APPL2 in skeletal muscles protects mice from glucose

intolerance, we generated muscle-specific APPL2 KO mice by crossing APPL2floxed

mice with

transgenic mice expressing Cre recombinase under control of MCK promoter, which resulted in

the disruption of the APPL2 gene at exon 5 (Figure 2A). Immunoblotting analysis confirmed the

dramatic reduction of APPL2 protein in EDL and soleus muscles but not in the brain of APPL2

KO mice (Figure 2B). The residual expression of APPL2 in EDL and soleus muscles in APPL2

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KO mice is perhaps due to its ubiquitous expression in other non-myocyte cells (16). Genetic

ablation of APPL2 in muscles had no obvious effects on food intake, body weight, fasting

glucose and insulin levels (Supplementary Table 2). GTT revealed that APPL2 KO mice

exhibited a significant improvement of glucose tolerance in response to glucose challenge when

compared to WT littermates (Figure 2C-D). Serum insulin levels during the GTT were similar

between the two groups of mice (Figure 2E). Insulin sensitivity, as determined by ITT, was also

obviously enhanced by APPL2 deletion (Figure 2F). Ex vivo studies demonstrated that insulin-

stimulated glucose uptake in EDL muscles of APPL2 KO mice was significantly increased

compared to WT littermates (Figure 2G). A similar result was also observed in soleus muscles

(data not shown). Insulin-elicited phosphorylation of Akt and IRβ in EDL muscle was

comparable between two groups of animals (Figure 2H).

TBC1D1 is an interacting partner and downstream effector of APPL2

To identify the proximal downstream effectors of APPL2, we established a stable cell line

expressing FLAG-tagged human APPL2 for affinity pull-down purification of its potential

interacting partners in HEK293 cells. Tandem mass spectrometry-based analysis identified

several putative interacting partners of APPL2, including heat shock protein (HSP)70, HSP90,

APPL1, centaurin-delta-1, TBC1D1 and son of sevenless homolog-1. Among these APPL2-

interacting proteins, TBC1D1, a member of the TBC1 Rab-GTPase family proteins abundantly

expressed in skeletal muscles, is an important regulator of insulin signaling and glucose

metabolism (25, 26). Notably, our co-immunoprecipitation analysis showed that TBC1D1 bound

to APPL2 but not APPL1 (Figure 3A). The specificity of immunoprecipitation for TBC1D1 was

confirmed by the fact that RNAi-mediated knockdown of TBC1D1 expression in C2C12

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myotubes also leads to a substantial decrease in immunoprecipated TBC1D1 (Supplementary

Figure 3). On the other hand, APPL2 did not bind to TBC1D4 (Figure 3B), a paralog of TBC1D1

that is also involved in the regulation of glucose transport in muscle cells and adipocytes (6). The

APPL2-TBC1D1 interaction was enhanced by insulin stimulation in EDL muscle of C57 mice

(Figure 3C) and C2C12 myotubes (Figure 3D), and such an enhancement was largely blocked by

the PI3K inhibitor LY294002 (Figure 3D).

To determine which domain of APPL2 is responsible for its TBC1D1 binding, we next

generated a series of vectors that express different domains of APPL2 (Figure 4A). Co-

immunoprecipitation analysis revealed that TBC1D1 interacted with the BAR domain, but not

the PH or PTB domain of APPL2 (Figure 4B). The pull-down assay further confirmed the direct

interaction between TBC1D1 and the BAR domain of APPL2 (Supplementary Figure 4). To test

whether the BAR domain mediates the suppressive effect of APPL2 on insulin-stimulated

glucose uptake, we transduced L6 myotubes with adenovirus encoding the BAR domain or the

PH-PTB domain or luciferase as control. Similar to full-length APPL2 (Figure 1B-C), ectopic

overexpression of the BAR domain, but not the PH-PTB mutant, inhibited insulin-stimulated

glucose uptake and GLUT4 translocation to the plasma membrane (Figure 4C and D). Taken

together, these findings suggest that the BAR domain of APPL2 exerts an inhibitory effect on

insulin-evoked glucose uptake by interacting with TBC1D1.

Phosphorylation of TBC1D1 at serine-235 is required for its interaction with APPL2 and

the inhibitory effects of APPL2 on insulin-dependent glucose uptake

To further delineate how the APPL2-TBC1D1 interaction regulates insulin-stimulated glucose

uptake, we next generated a series of truncated mutants of TBC1D1 (Figure 5A) to map the

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minimal domain mediating its binding to APPL2. Co-immunoprecipitation analysis revealed that

all the mutants of TBC1D1 containing the linker region between the two PTB domains (amino

acids 165-279) were able to interact with APPL2, whereas those mutants without the linker

region lost their APPL2-binding property (Figure 5B). Furthermore, the linker region alone was

sufficient to bind with APPL2 (Figure 5B).

TBC1D1 is a hyperphosphorylated protein and insulin induces its phosphorylation via

Akt activation (6). Since insulin prompted the APPL2-TBC1D1 interaction in a PI3K-dependent

manner, we searched for phospho-Akt substrate (PAS) site within the linker region that may be

crucial for its association with APPL2. Notably, a previous study showed that Ser-229 of mouse

TBC1D1 (equivalent to Ser-235 on human TBC1D1) within this region is potentially

phosphorylated by Akt (27). We confirmed that insulin stimulated TBC1D1 phosphorylation at

Ser-235 in a time-dependent manner in both EDL muscle of C57 mice and C2C12 myotubes

(Figure 5C-D), and such an insulin action was largely abolished by the PI3K inhibitor LY294002

(Figure 5D). Substitution of Ser-235 with non-phosphorylatable alanine (S235A) markedly

attenuated insulin-stimulated interaction between APPL2 and TBC1D1, whereas mutation of

Ser-237 to alanine had no obvious effect (Figure 5E).

We next compared the effects of TBC1D1 and its mutant S235A on insulin-stimulated

glucose uptake in L6 myotubes. Consistent with a previous study (27), we found that adenovirus-

mediated expression of TBC1D1 did not affect insulin-evoked Akt phosphorylation (data not

shown), but led to a significant inhibition of insulin-stimulated glucose uptake compared to cells

expressing luciferase control (Figure 5F). The inhibitory effects of TBC1D1 on insulin-evoked

glucose uptake were abrogated by S235A but not S237A mutation (Figure 5F).

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Several previous studies have shown that insulin-induced Akt activation leads to direct

TBC1D1 phosphorylation on Thr-596 (equivalent to Thr-590 in mouse TBC1D1) (6, 26-28),

which in turn promotes the transport of GLUT4 to plasma membrane by activating Rab-GTPases

(6, 26-28). To investigate the effects of APPL2 on insulin-elicited phosphorylation of TBC1D1,

we injected APPL2 Tg and APPL2 KO mice intraperitoneally with insulin. Immunoblotting

analysis demonstrated that insulin-stimulated TBC1D1 phosphorylation on Thr-596 in EDL and

soleus muscles was enhanced by APPL2 deletion, but was diminished by APPL2 overexpression

(Figure 6). On the other hand, APPL2 overexpression or deletion had no obvious effect on

expression levels of total TBC1D1 or TBC1D1 phosphorylation on Ser-235 (Figure 6).

To further investigate the interplay between APPL2 and TBC1D1 in regulating insulin-

stimulated glucose uptake, we co-overexpressed APPL2 and TBC1D1 or its S235A mutant in L6

myotubes by adenoviral gene transfer system (Figure 7A). Similar to the findings in APPL2 Tg

mice, insulin-stimulated TBC1D1 phosphorylation on Thr-596 was significantly impaired by

overexpression of APPL2 as compared to cells overexpressing luciferase controls (Figure 7A-B).

However, the suppressive effect of APPL2 overexpression on insulin-stimulated phosphorylation

of Thr-596 was abolished in cells expressing the TBC1D1-S235A mutant (Figure 7A-B).

Noticeably, overexpression of APPL2 only caused a modest suppressive effect on insulin-elicited

Akt phosphorylation but had no effect on IRβ phosphorylation in L6 myotubes expressing either

TBC1D1 or its S235A mutant (Figure 7C-D). As expected, the inhibitory effect of TBC1D1

overexpression on insulin-stimulated glucose uptake was further aggravated by overexpression

of APPL2 (Figure 7E). However, this suppressive effect of APPL2 was lost in cells expressing

the TBC1D1-S235A mutant (Figure 7E).

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To further investigate whether APPL2 suppresses insulin-elicited glucose uptake by

modulating TBC1D1 phosphorylation on Thr-596, Thr-596 of TBC1D1 was mutated to aspartic

acid (T596D) to mimic its phosphorylation. The level of adenovirus-mediated expression of the

TBC1D1-T590D mutant was comparable to wild-type TBC1D1 in L6 myotubes (Figure 8A).

However, the suppressive effects of APPL2 overexpression on insulin-induced glucose uptake

was only observed in L6 myotubes with ectopic expression of wild-type TBC1D1, but not in L6

cells expressing the TBC1D1-T590D mutant (Figure 8B).

Discussion

In this study, we provide both in vivo and in vitro evidence 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

overexpression of APPL2, but is enhanced by suppression of APPL2 expression.

Despite of the high similarity in domain organization and amino acid sequences between

APPL1 and APPL2, we demonstrate that these two adaptor proteins exert opposite effects on

insulin-stimulated glucose uptake in skeletal muscle. APPL1 exerts its insulin-sensitizing effects

by competing with TRB3 for binding to Akt, thereby promoting the translocation of Akt to

plasma membrane for further activation (11, 16, 24). On the other hand, our present study shows

that APPL2 suppresses insulin-dependent glucose uptake at a step downstream of Akt. Unlike

APPL1, APPL2 does not interact with Akt or the regulatory subunit p85 and catalytic subunit

p110 of PI3K (7, 29, 30). Indeed, overexpression of APPL2 only results in a modest decrease in

insulin-elicited Akt phosphorylation in myotubes, whereas targeted deletion of APPL2 in skeletal

muscle has no obvious effect on Akt phosphorylation. Such a modest effect of APPL2 on Akt

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activity in cultured cells is perhaps due to its heterodimerization with APPL1, which in turn

prevents the binding of APPL1 to Akt (16). We demonstrates that APPL2, but not APPL1,

interacts with TBC1D1, a downstream substrate of Akt that is critically involved in GLUT4

vesicle trafficking. Therefore, the opposite effects of APPL1 and APPL2 on insulin-stimulated

glucose uptake are attributed to the differential binding of these two adaptor proteins to Akt and

TBC1D1. In line with our findings, APPL1 and APPL2 have been shown to bind to different

types of Rab-GTPases involved in membrane trafficking (17, 19). The binding of APPL1 and

APPL2 to different sets of intracellular signaling molecules may be due to their difference in

oligomerization, surface charges and/or subcellular localization (17, 18, 31, 32).

TBC1D1 and TBC1D4, both of which are Rab-GAP proteins sharing ~47% sequence

identity and a similar domain organization, are the important regulators of both insulin- and

contraction-induced trafficking of GLUT4 vesicles (6, 33, 34). Upon insulin stimulation,

activated Akt induces phosphorylation of both TBC1D1 and TBC1D4 at multiple sites flanking

the second PTB domain (26-28, 35, 36). Substitution of these phosphorylation sites with non-

phosphorylatable alanine in TBC1D1 and TBC1D4 abolishes insulin-induced GLUT4

translocation to the plasma membrane in both adipocytes and muscle cells (25-27, 36-38).

Although the precise mechanisms by which TBC1D1 and TBC1D4 regulate GLUT4 vesicle

trafficking remain unclear, it has been proposed that non-phosphorylated TBC1D4 in the basal

state binds to GLUT4-containing vesicles to maintain its substrates Rab-GTPases in their

inactive GDP-loaded form, thereby trapping GLUT4 inside cells (27, 35-38). Insulin-evoked

TBC1D1 phosphorylation on Thr-649, possibly via interaction with 14-3-3 proteins, inhibits its

GAP activity, which in turn allows GTP loading and activation of Rab-GTPases required for

docking and fusion of GLUT4-containing vesicles to plasma membrane (36-38). A recent study

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demonstrated that mice with TBC1D4-Thr649Ala knockin mutation, in which Thr-649 was

mutated to non-phosphorylatable alanine, display impaired glucose disposal and insulin

sensitivity due to reduced GLUT4 trafficking to cell surface in skeletal muscles (39). Likewise,

insulin-elicited phosphorylation of TBC1D1 at Thr-596, a site equivalent to Thr-649 of TBC1D4,

is also obligatory for GLUT4 trafficking possibly by inactivation of the GAP activity (26-28). In

the present study, we find that the inhibitory effects of APPL2 overexpression on glucose uptake

in muscle cells and skeletal muscle are associated with impaired phosphorylation of TBC1D1 at

Thr-596 in response to insulin stimulation. Furthermore, mutation of Thr-596 of TBC1D1 to

aspartic acid reverses the inhibitory effect of APPL2 on insulin-stimulated glucose uptake,

suggesting that the APPL2-TBC1D1 interaction prevents Akt-mediated phosphorylation of

TBC1D1 at Thr-596, thereby impairing insulin-evoked GLUT4 translation to plasma membrane.

Although a previous study has identified Ser-235, a highly conserved amino acid located

within the linker region of TBC1D1, as a likely PAS site responsive to insulin stimulation (27),

its physiological relevance has never been explored. In this study, we further confirm that

insulin induces TBC1D1 phosphorylation on Ser-235 phosphorylation in a time-dependent

manner using an anti-phospho-Ser-235 antibody, and this phosphorylation is suppressed by

pharmacological inhibition of PI3K. Furthermore, we find that mutation of Ser-235 to alanine

not only abolishes APPL2-TBC1D1 interaction, but also abrogates the suppressive effects of

APPL2 on insulin-induced TBC1D1 phosphorylation on Thr-596 and glucose uptake in muscle

cells. Based on these findings, it is likely that Akt-dependent phosphorylation of Ser-235 at N-

terminal TBC1D1 triggers its interaction with APPL2, which in turn prevents phosphorylation of

Thr-596 by either conformational changes or direct blockage of Akt access to Thr-596 and its

surrounding motif. Notably, Ser-235 is absent in TBC1D4, which may explain in part the

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preferential interaction of APPL2 with TBC1D1, but not TBC1D4. On the other hand, mutation

of Ser-237 to alanine has no effect on the APPL2-TBC1D1 interaction as well as on the

suppressive effect of TBC1D1 on insulin-stimulated glucose uptake. Interestingly, Ser-237 is

phosphorylated by AMPK in response to contraction but not by insulin stimulation (40-42),

suggesting that APPL2 may only regulate glucose uptake via the TBC1D1 signaling pathway in

response to insulin, but not contraction in skeletal muscle.

In summary, our present study identifies APPL2 as a negative regulator of insulin-

stimulated GLUT4 translocation to the plasma membrane by interacting with and modulating

phosphorylation of TBC1D1 (Supplementary Figure 5). Insulin stimulation induces

phosphorylation of both Ser-235 and Thr-596 of TBC1D1. Phosphorylation of TBC1D1 at Ser-

235 triggers its interaction with APPL2, which in turn blocks further phosphorylation of Thr-596

required for plasma membrane targeting of GLUT4. Such a feedback regulatory loop may serve

as an important mechanism for insulin to fine-tune glucose homeostasis in mammals.

Author contributions

K.K.Y.C. and W.Z. researched data and wrote the manuscript. B.C. and B.W. researched data.

Y.W. contributed to research data and discussion. D.W. researched data and edited manuscript.

G.S. researched data and reviewed the manuscript. K.S.L.L. supervised the study and edited the

manuscript. A.X. designed and supervised the study and wrote the manuscript. K.K.Y.C. is the

guarantor of this work and, as such, had full access to all the data in the study and takes

responsibility for the integrity of the data and the accuracy of the data analysis.

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Acknowledgment

This work was supported by General Research Fund Grants HKU 783010M, HKU matching

funds for State Key Laboratory of Pharmaceutical Biotechnology, and the National Science

Foundation of China (81270881), and the National Basic Research Program of China

(2011CB504004 and 2010CB945500)

Conflict of interest

All the authors declare that there are no conflicts of interest.

References

1. Bogan JS. Regulation of glucose transporter translocation in health and diabetes. Annu

Rev Biochem 2012; 81: p. 507-532.

2. Ryder JW, Yang J, Galuska D, Rincon J, Bjornholm M, Krook A, Lund S, Pedersen O,

Wallberg-Henriksson H, Zierath JR, and Holman GD. Use of a novel impermeable

biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface

GLUT4 content in skeletal muscle from type 2 diabetic patients. Diabetes 2000; 49: p.

647-654.

3. Garvey WT, Maianu L, Zhu JH, Brechtel-Hook G, Wallace P, and Baron AD. Evidence

for defects in the trafficking and translocation of GLUT4 glucose transporters in skeletal

muscle as a cause of human insulin resistance. J Clin Invest 1998; 101: p. 2377-2386.

4. Zisman A, Peroni OD, Abel ED, Michael MD, Mauvais-Jarvis F, Lowell BB,

Wojtaszewski JF, Hirshman MF, Virkamaki A, Goodyear LJ, Kahn CR, and Kahn BB.

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin

resistance and glucose intolerance. Nat Med 2000; 6: p. 924-928.

5. Gibbs EM, Stock JL, McCoid SC, Stukenbrok HA, Pessin JE, Stevenson RW, Milici AJ,

and McNeish JD. Glycemic improvement in diabetic db/db mice by overexpression of the

human insulin-regulatable glucose transporter (GLUT4). J Clin Invest 1995; 95: p. 1512-

1518.

6. Sakamoto K and Holman GD. Emerging role for AS160/TBC1D4 and TBC1D1 in the

regulation of GLUT4 traffic. Am J Physiol Endocrinol Metab 2008; 295: p. E29-37.

7. Cheng KK, Lam KS, Wang B, and Xu A. Signaling mechanisms underlying the insulin-

sensitizing effects of adiponectin. Best Pract Res Clin Endocrinol Metab 2014; 28: p. 3-

13.

8. Cheng KK, Lam KS, Wu D, Wang Y, Sweeney G, Hoo RL, Zhang J, and Xu A. APPL1

potentiates insulin secretion in pancreatic beta cells by enhancing protein kinase Akt-

of 43 Diabetes

20

dependent expression of SNARE proteins in mice. Proc Natl Acad Sci U S A 2012; 109:

p. 8919-8924.

9. Wang Y, Cheng KK, Lam KS, Wu D, Wang Y, Huang Y, Vanhoutte PM, Sweeney G, Li

Y, and Xu A. APPL1 counteracts obesity-induced vascular insulin resistance and

endothelial dysfunction by modulating the endothelial production of nitric oxide and

endothelin-1 in mice. Diabetes 2011; 60: p. 3044-3054.

10. Park M, Wu D, Park T, Choi CS, Li RK, Cheng KK, Xu A, and Sweeney G. APPL1

transgenic mice are protected from high-fat diet-induced cardiac dysfunction. Am J

Physiol Endocrinol Metab 2013; 305: p. E795-804.

11. Cheng KK, Iglesias MA, Lam KS, Wang Y, Sweeney G, Zhu W, Vanhoutte PM,

Kraegen EW, and Xu A. APPL1 potentiates insulin-mediated inhibition of hepatic

glucose production and alleviates diabetes via Akt activation in mice. Cell Metab 2009; 9:

p. 417-427.

12. Cheng KK, Lam KS, Wang Y, Wu D, Zhang M, Wang B, Li X, Hoo RL, Huang Z,

Sweeney G, and Xu A. TRAF6-mediated ubiquitination of APPL1 enhances hepatic

actions of insulin by promoting the membrane translocation of Akt. Biochem J 2013; 455:

p. 207-216.

13. Mao X, Kikani CK, Riojas RA, Langlais P, Wang L, Ramos FJ, Fang Q, Christ-Roberts

CY, Hong JY, Kim RY, Liu F, and Dong LQ. APPL1 binds to adiponectin receptors and

mediates adiponectin signalling and function. Nat Cell Biol 2006; 8: p. 516-523.

14. Schenck A, Goto-Silva L, Collinet C, Rhinn M, Giner A, Habermann B, Brand M, and

Zerial M. The endosomal protein Appl1 mediates Akt substrate specificity and cell

survival in vertebrate development. Cell 2008; 133: p. 486-497.

15. Miaczynska M, Christoforidis S, Giner A, Shevchenko A, Uttenweiler-Joseph S,

Habermann B, Wilm M, Parton RG, and Zerial M. APPL proteins link Rab5 to nuclear

signal transduction via an endosomal compartment. Cell 2004; 116: p. 445-456.

16. Wang C, Xin X, Xiang R, Ramos FJ, Liu M, Lee HJ, Chen H, Mao X, Kikani CK, Liu F,

and Dong LQ. Yin-Yang regulation of adiponectin signaling by APPL isoforms in

muscle cells. J Biol Chem 2009; 284: p. 31608-31615.

17. King GJ, Stockli J, Hu SH, Winnen B, Duprez WG, Meoli CC, Junutula JR, Jarrott RJ,

James DE, Whitten AE, and Martin JL. Membrane curvature protein exhibits interdomain

flexibility and binds a small GTPase. J Biol Chem 2012; 287: p. 40996-41006.

18. Li J, Mao X, Dong LQ, Liu F, and Tong L. Crystal structures of the BAR-PH and PTB

domains of human APPL1. Structure 2007; 15: p. 525-533.

19. Zhu G, Chen J, Liu J, Brunzelle JS, Huang B, Wakeham N, Terzyan S, Li X, Rao Z, Li G,

and Zhang XC. Structure of the APPL1 BAR-PH domain and characterization of its

interaction with Rab5. EMBO J 2007; 26: p. 3484-3493.

20. Vu V, Bui P, Eguchi M, Xu A, and Sweeney G. Globular adiponectin induces

LKB1/AMPK-dependent glucose uptake via actin cytoskeleton remodeling. J Mol

Endocrinol 2013; 51: p. 155-165.

21. Zhang X, Xu A, Chung SK, Cresser JH, Sweeney G, Wong RL, Lin A, and Lam KS.

Selective inactivation of c-Jun NH2-terminal kinase in adipose tissue protects against

diet-induced obesity and improves insulin sensitivity in both liver and skeletal muscle in

mice. Diabetes 2011; 60: p. 486-495.

of 43Diabetes

21

22. Wang Q, Khayat Z, Kishi K, Ebina Y, and Klip A. GLUT4 translocation by insulin in

intact muscle cells: detection by a fast and quantitative assay. FEBS Lett 1998; 427: p.

193-197.

23. Wang Y, Xu LY, Lam KS, Lu G, Cooper GJ, and Xu A. Proteomic characterization of

human serum proteins associated with the fat-derived hormone adiponectin. Proteomics

2006; 6: p. 3862-3870.

24. Cleasby ME, Lau Q, Polkinghorne E, Patel SA, Leslie SJ, Turner N, Cooney GJ, Xu A,

and Kraegen EW. The adaptor protein APPL1 increases glycogen accumulation in rat

skeletal muscle through activation of the PI3-kinase signalling pathway. J Endocrinol

2011; 210: p. 81-92.

25. Taylor EB, An D, Kramer HF, Yu H, Fujii NL, Roeckl KS, Bowles N, Hirshman MF,

Xie J, Feener EP, and Goodyear LJ. Discovery of TBC1D1 as an insulin-, AICAR-, and

contraction-stimulated signaling nexus in mouse skeletal muscle. J Biol Chem 2008; 283:

p. 9787-9796.

26. Chen S, Murphy J, Toth R, Campbell DG, Morrice NA, and Mackintosh C.

Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK

activators. Biochem J 2008; 409: p. 449-459.

27. Peck GR, Chavez JA, Roach WG, Budnik BA, Lane WS, Karlsson HK, Zierath JR, and

Lienhard GE. Insulin-stimulated phosphorylation of the Rab GTPase-activating protein

TBC1D1 regulates GLUT4 translocation. J Biol Chem 2009; 284: p. 30016-30023.

28. Roach WG, Chavez JA, Miinea CP, and Lienhard GE. Substrate specificity and effect on

GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1. Biochem J 2007;

403: p. 353-358.

29. Yang L, Lin HK, Altuwaijri S, Xie S, Wang L, and Chang C. APPL suppresses androgen

receptor transactivation via potentiating Akt activity. J Biol Chem 2003; 278: p. 16820-

16827.

30. Nechamen CA, Thomas RM, and Dias JA. APPL1, APPL2, Akt2 and FOXO1a interact

with FSHR in a potential signaling complex. Mol Cell Endocrinol 2007; 260-262: p. 93-

99.

31. Chial HJ, Wu R, Ustach CV, McPhail LC, Mobley WC, and Chen YQ. Membrane

targeting by APPL1 and APPL2: dynamic scaffolds that oligomerize and bind

phosphoinositides. Traffic 2008; 9: p. 215-229.

32. Erdmann KS, Mao Y, McCrea HJ, Zoncu R, Lee S, Paradise S, Modregger J,

Biemesderfer D, Toomre D, and De Camilli P. A role of the Lowe syndrome protein

OCRL in early steps of the endocytic pathway. Dev Cell 2007; 13: p. 377-390.

33. Szekeres F, Chadt A, Tom RZ, Deshmukh AS, Chibalin AV, Bjornholm M, Al-Hasani H,

and Zierath JR. The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle

glucose metabolism. Am J Physiol Endocrinol Metab 2012; 303: p. E524-533.

34. Lansey MN, Walker NN, Hargett SR, Stevens JR, and Keller SR. Deletion of Rab GAP

AS160 modifies glucose uptake and GLUT4 translocation in primary skeletal muscles

and adipocytes and impairs glucose homeostasis. Am J Physiol Endocrinol Metab 2012;

303: p. E1273-1286.

35. Geraghty KM, Chen S, Harthill JE, Ibrahim AF, Toth R, Morrice NA, Vandermoere F,

Moorhead GB, Hardie DG, and MacKintosh C. Regulation of multisite phosphorylation

and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR. Biochem J

2007; 407: p. 231-241.

of 43 Diabetes

22

36. Sano H, Kane S, Sano E, Miinea CP, Asara JM, Lane WS, Garner CW, and Lienhard GE.

Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4

translocation. J Biol Chem 2003; 278: p. 14599-14602.

37. Thong FS, Bilan PJ, and Klip A. The Rab GTPase-activating protein AS160 integrates

Akt, protein kinase C, and AMP-activated protein kinase signals regulating GLUT4

traffic. Diabetes 2007; 56: p. 414-423.

38. Zeigerer A, McBrayer MK, and McGraw TE. Insulin stimulation of GLUT4 exocytosis,

but not its inhibition of endocytosis, is dependent on RabGAP AS160. Mol Biol Cell

2004; 15: p. 4406-4415.

39. Chen S, Wasserman DH, MacKintosh C, and Sakamoto K. Mice with AS160/TBC1D4-

Thr649Ala knockin mutation are glucose intolerant with reduced insulin sensitivity and

altered GLUT4 trafficking. Cell Metab 2011; 13: p. 68-79.

40. Vichaiwong K, Purohit S, An D, Toyoda T, Jessen N, Hirshman MF, and Goodyear LJ.

Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle.

Biochem J 2010; 431: p. 311-320.

41. Pehmoller C, Treebak JT, Birk JB, Chen S, Mackintosh C, Hardie DG, Richter EA, and

Wojtaszewski JF. Genetic disruption of AMPK signaling abolishes both contraction- and

insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal

muscle. Am J Physiol Endocrinol Metab 2009; 297: p. E665-675.

42. Frosig C, Pehmoller C, Birk JB, Richter EA, and Wojtaszewski JF. Exercise-induced

TBC1D1 Ser237 phosphorylation and 14-3-3 protein binding capacity in human skeletal

muscle. J Physiol 2010; 588: p. 4539-4548.

Figure Legend

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 hours, followed by serum

starvation for 6 hours. (A) The starved cells were subjected to immunoblotting using a rabbit

polyclonal antibody against APPL1 or APPL2, or a rabbit monoclonal against β-actin. (B)

Insulin-stimulated glucose uptake was measured in the infected cells using 2-deoxy-[3H]-glucose.

(C) Insulin-stimulated GLUT4 translocation was measured in L6 myotubes stably expressing

Myc-tagged GLUT4 infected with indicated adenoviruses using colorimetric assay. The

reference values for 2-deoxy-[3H]-glucose uptake and membrane bound Myc-GLUT4 are

4.82±0.41 pmol/min/mg protein and 4.12±0.35 fmol/mg protein in non-insulin-treated cells

expressing luciferase control, respectively. Data is expressed as fold changes relative to basal

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levels in luciferase-expressing cells. (D) Insulin-stimulated glucose uptake was measured in EDL

muscles isolated from the 20-week-old APPL1 transgenic (APPL1 Tg), APPL2 transgenic

(APPL2 Tg) mice and their wild-type (WT) controls using 2-deoxy-[3H]-glucose and normalized

with [14

C]-mannitol as described in Methods. (E-F) L6 myotubes were transfected with RNAi

against APPL1, APPL2 or scrambled control for 24 hours, followed by serum starvation for 6

hours. 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 myotubes stable cell line expressing Myc-tagged GLUT4 transfected with RNAi as

indicated. The reference values for glucose uptake and membrane bound Myc-GLUT4 are


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), student t test.

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 extensor

digitorum longus (EDL) muscles and brain isolated from 8-week-old heterozygous (hetero) and

homozygous muscle-specific APPL2 KO mice and its wild-type (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 with STC. (D) Area

under the curve (AUC) of the GTT in panel C. Data is expressed as fold change relative to WT

controls. (E) Serum insulin levels during GTT in panel C. (F) ITT in 22-week-old APPL2 KO

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mice and WT controls. 0.5 U/Kg of insulin was intraperitoneally injected. (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 Methods. (H) EDL muscles from fasted

C57BL/6 mice injected with or without insulin (0.5 U/Kg) for 15 minutes were subjected to

immunoprecipitation (IP) using a rabbit anti-IRβ monoclonal antibody, followed by

immunoblotting using a mouse anti-phospho-tyrosine (pY) monoclonal or rabbit anti-IRβ

monoclonal antibody. Total tissue lysates were subjected to immunoblotting using a rabbit anti-

phospho-Akt (Ser 473) (pAkt) polyclonal antibody or rabbit anti-total Akt (Akt) monoclonal

antibody. The bar chart on the right panel represents fold changes of phosphorylation of Akt

versus total Akt or tyrosine phosphorylation of IRβ versus total IRβ relative to the basal levels in

WT controls as quantified by densitometry. *p<0.05 (n=5), student t test.

Figure 3. APPL2 binds to TBC1D1 in an insulin- and PI3K-dependent manner. (A) C2C12

myotubes were collected and subjected to immunoprecipitation (IP) using a rabbit anti-TBC1D1

polyclonal antibody (Abcam) or mouse IgG as control, followed by immunoblotting using

indicated antibodies. (B) C2C12 infected with adenovirus encoding FLAG-tagged APPL2

(FLAG-APPL2) and HA-tagged TBC1D1 (HA-TBC1D1) or HA-tagged TBC1D4 (HA-TBC1D4)

were subjected to IP using a mouse anti-FLAG monoclonal antibody, followed by

immunoblotting using indicated antibodies. (C) C57 mice were fasted overnight, followed by

intraperitoneal injection of insulin (0.5U/kg) for indicated time points. EDL muscles were

isolated and subjected to IP using a rabbit anti-APPL2 polyclonal antibody, followed by

immunoblotting using a rabbit polyclonal antibody against APPL2 or TBC1D1 (Abcam). (D)

C2C12 myotubes expressing FLAG-APPL2 and HA-TBC1D1 were serum starved for 12 hours,

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followed by pre-incubation with or without the PI3K inhibitor LY294002 (LY, 50 µm) for 30

minutes. The cells treated with or without insulin (10 nM) for 15 minutes were subjected to IP

using a mouse anti-FLAG monoclonal antibody, followed by immunoblotting with a mouse anti-

FLAG monoclonal or rabbit anti-HA polyclonal antibody as indicated. All experiments were

repeated at least three times and representative images are shown.

Figure 4. The BAR domain of APPL2 interacts with TBC1D1 and mediates the suppressive

effects of APPL2 on insulin-stimulated glucose uptake. (A) Schematic diagram of FLAG-

tagged wild-type (WT) APPL2 and its truncated mutants containing different domain(s) used for

IP assays. (B) HEK293 cells were co-transfected with plasmids encoding HA-tagged TBC1D1

and FLAG-WT-APPL2 or FLAG-APPL2 mutants (BAR-PH, PH-PTB, BAR and PH) or an

empty vector as negative control (-ve) for 48 hours, followed by IP with a mouse anti-FLAG

monoclonal antibody and immunoblotting using a mouse anti-FLAG monoclonal or rabbit anti-

HA polyclonal antibody as indicated. (C) L6 myotubes infected with various truncated mutants

of APPL2 or luciferase (Luci) control were subjected to glucose uptake assay as described in

Methods. (D) L6 myotubes stably expressing Myc-tagged GLUT4 were infected with indicated

adenoviruses, followed by serum starvation for 6 hours. Insulin-stimulated GLUT4 translocation

to plasma membrane was measured using the antibody-coupled densitometric assay as described

in Methods. The reference values for glucose uptake and membrane bound Myc-GLUT4 are


expressing luciferase control, respectively. Data is expressed as fold changes relative to basal

levels in cells expressing luciferase. *p<0.05 (n=5), student t test.

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Figure 5. Effect of serine 235 phosphorylation of TBC1D1 on its APPL2 binding and

suppressive action on insulin-dependent glucose uptake. (A) Schematic presentation of HA-

tagged wild-type (WT) TBC1D1 and its truncated mutants (Mut 1-5) used for IP assays. (B)

HEK293 cells transfected with FLAG-tagged APPL2 or HA-WT-TBC1D1 or HA-TBC1D1

mutants (Mut-1: 374-1168, 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 U/kg) for 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 molecular

weight of ~160 kDa in cells expressing non-phosphorylatable TBC1D1 S235A mutant (data not

shown). (D) C2C12 myotubes infected with adenovirus encoding HA-WT-TBC1D1 were pre-

treated with or without the PI3K inhibitor LY294002 and then stimulated with insulin (10 nM)

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 (serine 235 is mutated to alanine) or S237A (serine 237 is mutated to alanine) and

FLAG-APPL2 were stimulated with insulin (10 nM) for 10 minutes, followed by IP with a

mouse anti-FLAG monoclonal antibody and immunoblotting to detect HA-tagged TBC1D1. (F)

L6 myotubes infected with various recombinant adenoviruses as indicated were subjected to

immunoblotting using a rabbit anti-TBC1D1 polyclonal (Abcam) or a rabbit anti-β-actin

monoclonal antibody (left panel) and insulin-stimulated glucose uptake assay (right panel) as

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described in Methods. The reference value for glucose uptake is 4.2±0.32 pmol/min/mg protein

in non-insulin-treated cells expressing luciferase control, and the result is expressed as fold

changes relative to non-insulin-treated cells with expressing luciferase control. *p<0.05 (n=5),

student t test. CBD: calmodulin-binding domain; PTB: phosphotyrosine binding domain; LK:

linker region; GAP: GTPase-activating protein.

Figure 6. APPL2 suppresses insulin-elicited phosphorylation of TBC1D1 at Thr-590 in

skeletal muscles. (A-B) 12-week-old male APPL2 knockout (KO) mice, APPL2 Tg mice and

their respective wild-type (WT) littermates were fasted overnight, followed by intraperitoneal

injection with or without insulin (0.5 U/Kg) for 10 minutes. EDL and soleus muscles were

isolated and subjected to 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 bar charts

on the right panels are the relative abundance of phosphorylated TBC1D1 at Thr-596 or at Ser-

235 versus total TBC1D1 as determined by densitometric analysis. The specificity of the rabbit

anti-phospho-TBC1D1 (Thr-596) polyclonal antibody was validated by immunoblotting showing

the disappearance of a specific band with molecular weight of ~160 kDa in cells expressing non-

phosphorylatable TBC1D1 T596A mutant (data not shown). The results are expressed as fold

changes relative to non-insulin-treated WT controls. *p<0.05 (n=4), student t test. not significant

(N.S.)

Figure 7. The APPL2-TBC1D1 interaction modulates insulin-elicited phosphorylation of

TBC1D1 at threonine 596 and the suppressive effect of APPL2 overexpression on insulin-

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dependent glucose uptake. L6 myotubes were infected with adenovirus encoding HA-tagged

wild-type (WT)-TBC1D1, TBC1D1-S235A mutant, luciferase (Luci) or APPL2 for 24 hours,

followed by serum starvation for 12 hours. (A) The cells were treated with or without insulin (10

nM) for 10 minutes, and the cell lysates were either subjected to immunoblotting with various

antibodies as indicated, or IP using a rabbit anti-IRβ monoclonal antibody. The

immunoprecipitated IRβ was subjected to immunoblotting analysis for tyrosine phosphorylation

using a mouse anti-tyrosine monoclonal antibody. (B-D) The bar charts are the relative

abundance of phosphorylated TBC1D1 at Thr-596 versus total TBC1D1 (B), phosphorylated Akt

at Ser-473 versus total Akt (C) and tyrosine phosphorylation of IRβ versus total IRβ (D). (E) The

starved cells infected with indicated adenoviruses were subjected to glucose uptake assay as

described in Methods. The reference value for glucose uptake is 4.87±0.25 pmol/min/mg protein

in non-insulin-treated cells expressing luciferase control. The results are expressed as fold

changes relative to non-insulin-treated luciferase controls (E) or non-insulin-treated luciferase

control plus WT-TBC1D1 (B-D) as indicated. In panel E, comparisons were made with two-way

ANOVA followed by Bonferroni post hoc tests. *p<0.05, #p<0.01 (n=5). Not significant (N.S.).

Figure 8. A phosphomimetic mutation of TBC1D1 at Thr-596 abolishes the inhibitory

effects of APPL2 on insulin-stimulated glucose uptake. L6 myotubes were infected with

adenovirus encoding HA-tagged wild-type (WT)-TBC1D1, TBC1D1-T596D mutant, luciferase

(Luci) or FLAG-tagged APPL2 as specified for 24 hours, followed by serum starvation for 6

hours and subsequent stimulation with insulin for 10 minutes. The cells were subjected to

immunoblotting (A) using a rabbit polyclonal antibody against HA or APPL2 or a rabbit

monoclonal antibody against β-actin, or insulin-stimulated glucose uptake assay (B) as in Figure

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1. The reference value for glucose uptake is 4.03±0.42 pmol/min/mg protein in non-insulin-

treated cells expressing luciferase control. Comparisons were made with two-way ANOVA

followed by Bonferroni post hoc tests.*p<0.05, #p<0.01 (n=6), not significant (N.S.).

of 43 Diabetes

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of 43Diabetes

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of 43 Diabetes

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0.5

1

1.5

Luci BAR PH-PTB

0

0.51

1.5

Mem

bran

eG

LUT4

(fol

d

Luci BAR PH-PTB

of 43 Diabetes

Figure 5

HATotallysate

TBC1D1:BA

Mut‐1

1 153

96 373

301

721

994

800

1168

279

201

WT

GAPGAP

LKLKPTBPTB PTBPTB CBDCBD GAPGAP

CBDCBD lysate

FLAG

HA

IP:FLAG

Mut 1

Mut‐2

Mut‐3

Mut‐4

Mut‐5

GAPGAP

PTBPTB

PTBPTB

CBDCBD

PTBPTB PTBPTBLKLK

LKLK

LKLK

2 5 *al)

IP:FLAG

E

TBC1D1:

FLAG

HA

D

0 15 30 30Insulin:pTBC1D1

(S235)

HA

LY: - - - +

pTBC1D1(S235)

TBC1D1

Insulin: 0 10 20 minC

IP:TBC1D1

00.51

1.52

2.5 *

Glu

cose

upt

ake

(fold

cha

nge

over

bas

aTBC1D1

β-actin

TBC1D1

F

TBC1D1

(

of 43Diabetes

Figure 6

EDL SoleusA

0

2

4

6

WT KO WT KO

Fold

cha

nge

(ove

r bas

al)

WT KO WT KO

*

* N.S.N.S.pTBC1D1

(Thr-596)

WT KOEDL

WT KOSoleus

pTBC1D1(Ser-235)

Insulin - + - + - + - +

IP:TBC1D1

WT KO WT KO

pTBC1D1(Thr-596)/TBC1D1

pTBC1D1(Ser-235)/TBC1D1

EDL SoleusWT KO WT KO

EDL Soleus

3

4 *

*N.S. N.S.e al

)

TBC1D1

WT Tg

EDL

WT Tg

SoleusB

0

1

2

3

WT Tg WT Tg WT Tg WT TgEDL Soleus EDL Soleus

Fold

cha

ng(o

ver b

asa

- + - +

IP:TBC1D1

WT Tg WT Tg

TBC1D1

pTBC1D1(Thr-596)pTBC1D1(Ser-235)

Insulin - + - +

pTBC1D1(Thr-596)/TBC1D1

pTBC1D1(Ser-235)/TBC1D1

EDL Soleus EDL Soleus

of 43 Diabetes

Insulin:APPL2

_ + _ +Luci APPL2WT-TBC1D1

_ + _ +Luci APPL2TBC1D1-S235A

A

3

4

B

BC1D

1er

bas

al) * N.S.

C

6

8

basa

l) * *

Figure 7

pTBC1D1(Thr -596)

HA

APPL2

pAkt

Akt0

2

1

pTB

C1D

1/H

A-T

(fold

cha

nge

ove

0

2

4

6

pAkt

/Akt

(fold

cha

nge

over

pYWT-

TBC1D1TBC1D1-S235A

WT-TBC1D1

TBC1D1-S235A

E

2

2.5

ake

r bas

al)

N.S.*

*#

pIP:IRβ

IRβ

D

7.5

10

IRβ

over

bas

al) N.S. N.S.

0

0.5

1

1.5

Glu

cose

upt

a(fo

ld c

hang

e ov

e

0

2.5

5

pIRβ

/(fo

ld c

hang

e o

WT- TBC1D1WT-

TBC1D1TBC1D1-S235A

TBC1D1 -S235A

of 43Diabetes

B #

Figure 8

A B

1

2

3

Glu

cose

upt

ake

d ch

ange

ove

r bas

al)

HA

APPL2

WT-TBC1D1

TBC1D1-T596D

*

N.S.*

#

0(foldAPPL2

β-actin

WT-TBC1D1

TBC1D1-T596D

of 43 Diabetes

Online Appendix

1

Supplementary Figure 1. Transgenic expression of APPL2 causes insulin resistance and

glucose intolerance. (A) Schematic diagram of the transgenic construct. Shown is cDNA

encoding FLAG-tagged full-length human APPL2 under the control of cytomegalovirus (CMV)-

β-actin promoter. (B) PCR genotyping using genomic DNA from APPL2 transgenic (Tg) mice

and its wild-type (WT) littermate or the transgene construct as positive control (+ve), distilled

water as negative control (-ve). M, DNA marker; bp, base pair. (C) Extensor digitorum longus

(EDL) and soleus muscles isolated from 8-week-old APPL2 Tg mice and its WT littermates were

subjected to immunoblotting using a rabbit anti-APPL2 polyclonal or rabbit anti-β-actin

monoclonal antibody. (D) Glucose and (E) insulin levels in 24-week-old mice fasted for 16 hours.

(F) GTT in 24-week-old mice. The overnight-fasted mice were intraperitoneally injected with

2g/kg of D-glucose. (G) ITT in 22-week-old mice. The mice fasted for 6 hours were

intraperitoneally injected with insulin (0.5 U/kg). (H) Food intake and (I) body weight in 20-

week-old mice. *p<0.05 vs WT (n=6 each group), student t test. Representative images were

shown.

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Online Appendix

2

Supplementary Figure 2. Effect of APPL1 and APPL2 overexpression on protein

expressions of GLUT4 in skeletal muscle. EDL muscles isolated from 16-week-old APPL1

and APPL2 transgenic (Tg) mice and their wild-type (WT) littermates were subjected to

immunoblotting using a mouse anti-GLUT4 monoclonal or rabbit anti-GAPDH monoclonal

antibody as indicated. The bar chart on the right panel is the relative abundance of GLUT4

normalized against GAPDH as determined by densitometric analysis. (n=4), student t test. Not

significant (N.S).

Supplementary Figure 3. Confirmation of the specificity of the anti-TBC1D1 antibody used

for immunoprecipitation. C2C12 cells were transfected with RNAi against scrambled control

or mouse TBC1D1 for 48 hours. The transfected cells were collected and subjected to

immunoprecipitation (IP) using a rabbit anti-TBC1D1 polyclonal antibody from Abcam

(ab56191), followed by immunoblotting with a rabbit polyclonal anti-TBC1D1 antibody from

Cell Signaling Technology (#5929) or rabbit anti-β-actin monoclonal antibody. Representative

images were shown.

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Online Appendix

3

Supplementary Figure 4. The BAR domain of APPL2 interacts with TBC1D1. His-tagged

recombinant proteins of wild-type (WT) APPL1 or WT-APPL2 or APPL2’s truncated mutants

(the BAR-PH and PH-PTB domains) were purified from E.Coli and analyzed by SDS-PAGE

stained with commassie brilliant blue (upper panel). One microgram of the purified recombinant

proteins were immobilized on Ni-NTA agarose and then incubated with cell lysates extracted

from HEK293 cells with expression of HA-tagged TBC1D1, followed by washing for 3 times

with a lysis buffer (50 mM TrisCl, 150 mM NaCl, 0.1% Triton X-100, protease inhibitor cocktail,

pH 8.0). The pull-down complex was eluted by boiling with 1XSDS loading buffer for 10

minutes and then analyzed by immunoblotting using a rabbit anti-HA polyclonal antibody. All

experiments were performed at least 3 times and representative images were shown.

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Online Appendix

4

Supplementary Figure 5. APPL2 negatively regulates insulin-stimulated glucose uptake by

interacting with TBC1D1 in skeletal muscle cells. Upon insulin stimulation, activation of the

signaling cascade leads to phosphorylation of Akt by its upstream kinases (including PDK1 and

mTOR2), which in turn induces phosphorylation of TBC1D1 at threonine 596 (Thr-596).

Phosphorylation of TBC1D1 is thought to inhibit its GAP activity, leading to formation of GTP-

loaded Rab substrate and subsequent membrane recruitment of GLUT4 and glucose uptake. On

the other hand, activation of Akt phoshorylates TBC1D1 at serine 235 (Ser-235), resulting the

binding of APPL2 to TBC1D1. The interaction diminishes the phosphorylation of TBC1D1 at

Thr-596, resulting inhibition of GLUT4 translocation and glucose uptake, thereby maintaining

glucose homeostasis. Abbreviations: insulin receptor (IR), insulin receptor substrate (IRS), 3-

phosphoinositide-dependent protein kinase-1 (PDK1), phosphatidylinositol 3-kinase (PI3K),

mammalian target of rapamycin complex (mTOR2), phosphatidylinositol (3,4,5)-triphosphate

(PIP3), Rab-GTPase-activating protein (GAP), guanosine triphosphate (GTP), guanosine-5’-

diphosphate (GDP).

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Online Appendix

5

Genotyping Primers:

Primer name Sequences of oligonucleotides

APPL2 transgene Fw: 5’ TAG AGC CTC TGC TAA CCA TGT TCA TG 3’

Rv: 5’ TCC GAC TTC GGT CTT CAC CT 3’

APPL2 floxed allele Fw: 5’ CAC ACA GGA GCG TCT GTG GTG GTC 3’

Rv: 5’ CCT CCC TCT GTT GAA CCA GGA ACG 3’

MCK-Cre transgene Fw: 5’ GTG AAA CAG CAT TGC TGT CAC TT 3’

Rv: 5’ TAA GTC TGA ACC CGG TCT GC 3’

RNAi duplex oligos

Gene name Sequences of RNAi

Scrambled control 5’ UAG CGA CUA AAC ACA UCA AUU 3’

5’ AAU UGA UGU GUU UAG UCG CUA 3’

Mouse APPL1 5’ GAG UGG AUC UGU ACA AUA A 3’

5’ UUA UUG UAC AGA UCC ACU C 3’

Mouse APPL2 5’ CAG ACA CUA UGG UUC UCC CUG UUA U 3’

5’ AUA ACA GGG AGA ACC AUA GUG UCU G 3’

Mouse TBC1D1 5’ GAU CAG AGG UCA UAU UUA A 3’

5’ UUA AAU AUG ACC UCU GAU C 3’

Rat APPL1 5’ GAG UGG AUC UGU ACA AUA A 3’

5’ UUA UUG UAC AGA UCC ACU C 3’

Rat APPL2 5’ CCC UCA CAG ACU ACA CCA A 3’

5’ UUG GUG UAG UCU GUG AGG G 3’

Supplementary Table 1: The oligonucleotides used for genotyping of genetically modified

mouse models and RNAi transfection.

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Online Appendix

6

WT APPL2 KO

Fasting glucose (mmol/L) 4.41±0.25 4.0±0.30

Fasting insulin (ng/ml) 0.19±0.049 0.18±0.059

Food intake (Kcal/day) 15.04±0.21 15.11±0.49

Body weight (gram) 31.25±0.41 30.97±0.76

Supplementary Table 2. Basic metabolic parameters of 20-week-old male muscle-specific

APPL2 KO mice and WT littermates fed with standard chow. Mice were fasted for 16 hours

before measurement of glucose and insulin levels. (n=6 each group).

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