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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
Page 1 of 43 Diabetes
Diabetes Publish Ahead of Print, published online May 30, 2014
2
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.
Page 2 of 43Diabetes
3
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
Page 3 of 43 Diabetes
4
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).
Page 4 of 43Diabetes
5
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.
Page 5 of 43 Diabetes
6
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
Page 6 of 43Diabetes
7
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.
Page 7 of 43 Diabetes
8
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.
Page 8 of 43Diabetes
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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
Page 9 of 43 Diabetes
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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
Page 10 of 43Diabetes
11
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
Page 11 of 43 Diabetes
12
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
Page 12 of 43Diabetes
13
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).
Page 13 of 43 Diabetes
14
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).
Page 14 of 43Diabetes
15
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
Page 15 of 43 Diabetes
16
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
Page 16 of 43Diabetes
17
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
Page 17 of 43 Diabetes
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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.
Page 18 of 43Diabetes
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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.
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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
Page 22 of 43Diabetes
23
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
4.53±0.31 pmol/min/mg protein and 3.8±0.42 fmol/mg protein in non-insulin-treated cells
transfected with RNAi against scrambled control, respectively. All experiments were repeated at
least three times and representative images are shown. *p<0.05 (n=6), 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
Page 23 of 43 Diabetes
24
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,
Page 24 of 43Diabetes
25
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
5.2±0.49 pmol/min/mg protein and 4.70±0.54 fmol/mg protein in non-insulin-treated cells
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.
Page 25 of 43 Diabetes
26
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
Page 26 of 43Diabetes
27
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-
Page 27 of 43 Diabetes
28
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
Page 28 of 43Diabetes
29
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.).
Page 29 of 43 Diabetes
2
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Page 30 of 43Diabetes
AWT
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Page 31 of 43 Diabetes
BA Totallysate IP
Figure 3
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Page 32 of 43Diabetes
A B
Figure 4
HA
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Page 33 of 43 Diabetes
Figure 5
HATotallysate
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Mut‐1
1 153
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301
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WT
GAPGAP
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Page 34 of 43Diabetes
Figure 6
EDL SoleusA
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Fold
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nge
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r bas
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*
* N.S.N.S.pTBC1D1
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WT KOEDL
WT KOSoleus
pTBC1D1(Ser-235)
Insulin - + - + - + - +
IP:TBC1D1
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pTBC1D1(Thr-596)/TBC1D1
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EDL Soleus EDL Soleus
Page 35 of 43 Diabetes
Insulin:APPL2
_ + _ +Luci APPL2WT-TBC1D1
_ + _ +Luci APPL2TBC1D1-S235A
A
3
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Figure 7
pTBC1D1(Thr -596)
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ove
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Page 36 of 43Diabetes
B #
Figure 8
A B
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#
0(foldAPPL2
β-actin
WT-TBC1D1
TBC1D1-T596D
Page 37 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.
Page 38 of 43Diabetes
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.
Page 39 of 43 Diabetes
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.
Page 40 of 43Diabetes
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).
Page 41 of 43 Diabetes
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.
Page 42 of 43Diabetes
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).
Page 43 of 43 Diabetes