ORIGINAL ARTICLE

Journal of
974
Cellular
Physiology
Metformin Induces Rab4
Through AMPK and Modulates
GLUT4 Translocation in Skeletal
Muscle Cells
JUNG OK LEE,1 SOO KYUNG LEE,1 JIN HEE JUNG,1 JI HAE KIM,1 GA YOUNG YOU,1 SU JIN KIM,1
SUN HWA PARK,1 KYUNG-OK UHM,2 AND HYEON SOO KIM1*
1
Department of Anatomy, Korea University College of Medicine, Seoul, Korea
2
Department of Alzheimer’s Disease Research, National Institute for Geriatrics and Gerontology, Morioka, Aichi, Japan

Metformin is a major oral anti-diabetic drug and is known as an insulin sensitizer. However, the mechanism by which metformin acts is
unclear. In this study, we found that AICAR, an AMPK activator, and metformin increased the expression of Rab4 mRNA and protein levels
in skeletal muscle C2C12 cells. The promoter activity of Rab4 was increased by metformin in an AMPK-dependent manner. Metformin
stimulated the phosphorylation of AS160, Akt substrate, and Rab GTPase activating protein (GAP), and also increased the
phosphorylation of PKC-zeta, which is a critical molecule for glucose uptake. Knockdown of AMPK blocked the metformin-induced
phosphorylation of AS160/PKC-zeta. In addition, a colorimetric absorbance assay showed that insulin-induced translocation of GLUT4
was suppressed in Rab4 knockdown cells. Moreover, Rab4 interacted with PKC-zeta but not with GLUT4. The C-terminal-deleted Rab4
mutant, Rab4DCT, showed diffuse sub-cellular localization, while wild-type Rab4 localized exclusively to the perinuclear membrane.
Unlike Rab4DCT, wild-type Rab4 co-localized with PKC-zeta. Together, these results demonstrate that metformin induces Rab4
expression via AMPK-AS160-PKC-zeta and modulates insulin-mediated GLUT4 translocation.
J. Cell. Physiol. 226: 974–981, 2011. ß 2010 Wiley-Liss, Inc.

Metformin is an oral anti-diabetic drug of the biguanide class.
Metformin originates from the French lilac, Galega officinalis, a
plant known to reduce the symptoms of diabetic mellitus
(Witters, 2001). There are many effects of metformin on insulin
sensitivity in muscle and liver, such as a decrease in hepatic
glucose production, an increase in peripheral glucose
utilization, and positive effects on insulin receptor expression
and tyrosine kinase activity (Meuillet et al., 1999; Kumar and
Dey, 2002; Gunton et al., 2003). Metformin also ameliorates
insulin resistance by inducing glucose transporter4 (GLUT4)
translocation (Sarabia et al., 1992; Fischer et al., 1995; Yang and
Holman, 2006). It is still unclear, however, how metformin
achieves GLUT4 translocation.
AMP-activated protein kinase (AMPK) is an enzyme that
plays a role in cellular energy homeostasis. AMPK, a
heterotrimeric complex comprised a catalytic subunit and two
regulatory subunits, is activated when cellular energy is
depleted (Hardie and Carling, 1997). Upon activation by
allosteric binding of AMP or phosphorylation at Thr172 of the
catalytic subunit by AMPK kinase, AMPK accelerates ATP-
generating catabolic pathways, including glucose and fatty acid
oxidation (Makinde et al., 1997; Ai et al., 2002; Zong et al.,
2002), while simultaneously reducing ATP-consuming anabolic
pathways (cholesterol, fatty acid, and triacylglycerol synthesis;
Henin et al., 1995). In addition to roles in energy homeostasis,
AMPK has also been shown to regulate insulin signaling (Fisher
et al., 2002; Towler and Hardie, 2007). Insulin is a glucose-
regulating hormone. When blood glucose is high, insulin
facilitates the uptake of glucose into cells via translocation of
GLUT4. It has been shown that exercise (Cormont and Le
Marchand-Brustel, 2001) and AMPK (Kaddai et al., 2008)
increase GLUT4 translocation. Moreover, metformin acts by
stimulating peripheral AMPK, leading to reduced insulin
resistance in muscle (Imamura et al., 2003). These facts confirm
the existence of a metformin–AMPK–GLUT4 axis; however,
the molecular link by which metformin acts has not been
established.
The Rab family of proteins is a member of the Ras superfamily
of monomeric G proteins. To date, approximately 70 types of
Rabs have been identified in humans. Rab GTPases regulate
many steps in membrane traffic, including vesicle formation,
movement, and membrane fusion. These processes make up
the route through which cell surface proteins are trafficked
from the Golgi to the plasma membrane and are recycled.
Protein recycling serves as a means of regulating the number of
a certain type of protein molecules on the surface (Huang et al.,
2001; Ishikura et al., 2008). Insulin-responsive tissues express
several Rab isozymes, including Rab4, Rab5, Rab8, and Rab10
(Sano et al., 2007; Ishikura and Klip, 2008). Of these isozymes,
only Rab4 has been shown to play a role in mediating insulin
action within cells (Shibata et al., 1997). Specifically, the level of
expression of Rab4 affects insulin-mediated GLUT4
translocation (Vollenweider et al., 1997; Kaddai et al., 2009).
Together, these facts suggest that Rab4 plays a key role in
insulin-mediated cellular processes, including GLUT4 vesicle
trafficking.
In the current study, we determined the effects of metformin
on Rab4 expression to gain an understanding of the role of
The authors confirm that there are no conflicts of interest.
Contract grant sponsor: Korea University College of Medicine.
Contract grant sponsor: Korea Science and Engineering
Foundation (KOSEF);
Contract grant number: R01-2008-000-11180-0.
*Correspondence to: Hyeon Soo Kim, 126-1, Anam-dong 5-ga,
Seongbuk-gu, Seoul 136-701, Korea. E-mail:
anatomykim@korea.ac.kr
Received 19 April 2010; Accepted 19 August 2010
Published online in Wiley Online Library
(wileyonlinelibrary.com), 20 September 2010.
DOI: 10.1002/jcp.22410

ß 2 0 1 0 W I L E Y - L I S S , I N C .
 METFORMIN REGULATES GLUT4 VIA AMPK
metformin in GLUT4 transporter regulation. We have shown
that metformin induces Rab4 expression via the AMPK pathway
and demonstrated that the activities of Akt substrate160
(AS160) and PKC-zeta are involved in metformin-induced Rab4
regulation. These findings provide novel insight into the manner
in which metformin contributes to GLUT4-regulating functions
in skeletal muscle cells.
Materials and Methods
Reagents
Anti-phospho-acetyl-CoA carboxylase (ACC; Ser79), anti-
phospho-AMPK (Thr172), anti-AMPK, anti-Rab4, and anti-ACC
antibodies were purchased from Cell Signaling Technology (New
England Biolabs, Beverly, MA). Anti-phospho-PKCz (Thr560)
antibody was purchased from Epitomics, Inc. (Burlingame, CA).
Anti-PKCz and anti-GFP antibodies, and GAPDH were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-
phospho-AS160 (Thr642) and anti-AS160 antibodies were
purchased from Millipore–Upstate (Billerica, MA). Anti-GLUT4
antibody was purchased from Abcam (Cambridge, UK). Anti-rabbit
Cy3-labeled IgG secondary antibodies were purchased from
Invitrogen–Molecular Probes (Carlsbad, CA). Horseradish
peroxidase-conjugated secondary antibodies were purchased from
Assay Designs and Stressgen (Ann Arbor, MI). Metformin, insulin,
compound C, and 5-aminoimidazole-4-carboxy-amide-1-D-
ribofuranoside (AICAR) were purchased from Calbiochem (San
Diego, CA).
Cell culture
Mouse myoblast C2C12 cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM; GibcoTM, Auckland, NZ)
supplemented with 10% fetal bovine serum (FBS) and antibiotics at
378C in an incubator with 5% CO2. Cells were grown in culture
medium consisting of 500 ml of DMEM containing 0.584 g/L of L-
glutamate and 4.5 g/L of glucose, and mixed with 500 ml of F-12
medium containing 0.146 g/L of L-glutamate, 1.8 g/L of glucose,
100 mg/ml of gentamicin, 2.5 g/L of sodium carbonate, and 10%
heat-inactivated FBS. L6-GLUT4myc-tagged myoblasts were
maintained in cell monolayers in a-MEM supplemented with 10%
FBS, 50 U/ml of penicillin, and 50 mg/ml of streptomycin in a
5% CO2 humidified atmosphere at 378C. Two days after the
myoblasts achieved confluence, differentiation to myotubes was
induced by incubating the cells for 6–7 days in a-MEM
supplemented with 2% FBS, which was changed every 2 days
(Wijesekara et al., 2006). The monolayer myotubes were then
serum-starved in DMEM for 2 h and used in the following
experiments.
Immunoblot analysis
Cells were grown on six-well plates and serum-starved for 36 h
prior to treatment with the indicated agents. Following treatment
of the cells, the media were aspirated and the cells were washed
twice in ice-cold PBS and lysed in 100 ml of lysis buffer. The samples
were then briefly sonicated, heated for 5 min at 958C, and
centrifuged for 5 min. The supernatants were electrophoresed on
SDS–PAGE (8%) gels and transferred to polyvinylidene difluoride
membranes. The blots were incubated overnight at room
temperature with primary antibodies, and then washed six times in
Tris-buffered saline/0.1% Tween-20 prior to a 1-h incubation with
horseradish peroxidase-conjugated secondary antibodies at room
temperature. The blots were then visualized via ECL (Amersham
Biosciences, Buckinghamshire, UK). In some cases, the blots were
stripped and reprobed using other antibodies.
Immunoprecipitation
The amount of proteins from L6-GLUT4myc-tagged myoblasts was
determined by the Bradford method. Cellular protein (100 mg) was
JOURNAL OF CELLULAR PHYSIOLOGY
975
mixed with 1 mg of anti-PKCz antibody and incubated overnight at
48C. Then, 10 ml of protein A sepharose (Amersham, Uppsala,
Sweden) was added to the samples and incubated for another 3 h at
48C. After incubation, the samples were thrice washed with wash
buffer (25 mM HEPES, 5 mM EDTA, 1% Triton X-100, 50 mM NaF,
150 mM NaCl, 10 mM phenylmethylsulfonyl fluoride, 1 mM
leupeptin, 1 mM pepstatin, and 1 mM aprotinin A [pH 7.2]). The
washed samples were resuspended in SDS sample buffer (125 mM
Tris–HCl [pH 6.8], 20% [v/v] glycerol, 4% [w/v] SDS, 100 mM
dithiothreitol, and 0.1% [w/v] bromophenol blue), and heated at
1008C for 5 min prior to electrophoresis.
Silencing AMPK and Rab4
L6-GLUT4myc-tagged myoblasts were seeded in six-well plates and
allowed to grow to 70% confluence for 24 h. Transient
transfections were performed with transfection reagent
(Lipofectamine 2000; Invitrogen, Carlsbad, CA), according to the
manufacturer’s protocol. Both AMPKa2 (NM_001013367;
Dharmacon, Inc., Chicago, IL) and non-targeted control siRNAs
were designed. Five microliters of siRNA and 5 ml of transfection
reagent (Lipofectamine 2000) were each diluted with 95 ml of
reduced serum media (Opti-MEM; Invitrogen, CA), then mixed.
The mixtures were allowed to incubate for 30 min at room
temperature, and then added dropwise to each culture well
containing 800 ml of reduced serum media (final siRNA
concentration, 100 nM). Four hours after transfection, the medium
was replaced with fresh complete medium. The cells were
cultivated for 24 h and lysed, and the expression of AMPKa2
protein was assayed with Western blotting.
Immunofluorescence
L6-GLUT4myc-tagged myoblasts were split onto collagen-coated
glass cover slips. After 24 h, the cells were serum-starved for 2 h
and stimulated with 100 nM insulin for 20 min at 378C. The cells
were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at
48C. The cells were then incubated for 3 h with primary p-PKCz
and p-AS160 antibodies in buffer (5% BSA in PBS) at 48C, followed
by incubation with Cy3-conjugated secondary antibody at a dilution
of 1:250 for 1 h at room temperature. The cells were observed
using a Zeiss LSM510 confocal fluorescence microscope.
Reverse transcription-polymerase chain reaction (RT-PCR)
First-strand cDNA synthesis was performed using 1 mg of total
RNA isolated from C2C12 at 558C for 20 min using the
Thermoscript II one-step RT-PCR Kit (Invitrogen, Paisley, UK).
Amplification of cDNA was carried out in the same tube using the
Gene Amp System 9700 thermocycler (Applied Biosystems,
Warrington, UK). Heating to 948C for 5 min inactivated the
reverse transcriptase. The following PCR conditions were used: 27
cycles of 30 sec at 948C; 30 sec at 568C; and 30 sec at 728C,
followed by 7 min at 728C. The number of PCR cycles used was
optimized to ensure amplification during the exponential phase.
Ten microliters of samples from each RT-PCR product were
removed and analyzed by agarose gel electrophoresis. Bands were
stained with ethidium bromide and visualized under ultraviolet
light. Band intensity quantification was determined by a gel
documentation system (Gene Genius, Syngene, UK). The following
primers were used: Rab4-sense (50 -GTG CCT GCA CCA ACC
TTA AT-30 ) and Rab4-antisense (5-CTT GAA GTG GCC TTG
ACT CC-3); and GAPDH-sense (5-
ATTTGGTCGTATTGGGCGCCTGGTCACC-3) and GAPDH
antisense (5-GAAGATGGTGATGGGATTTC-3).
Immunodetection of GLUT4myc
Cell surface GLUT4myc was quantified using antibody-coupled
colorimetric absorbance assays, as previously described
(Wijesekara et al., 2006). Briefly, following stimulation with insulin
or metformin, L6-GLUT4myc-tagged myoblasts were exposed to
976 LEE ET AL.
polyclonal anti-myc antibody (1:100) for 60 min, fixed with 4% PFA
for 10 min, and incubated with peroxidase-conjugated goat anti-
rabbit IgG (1:1,000) for 1 h. Cells were then washed six times, and
1 ml of OPD reagent (0.4 mg/ml of o-phenylenediamine
dihydrochloride) was added for 30 min. The optical absorbance of
the supernatant was measured at 492 nm.
Fluorescence microscopy of FITC-conjugated AMPK siRNA and
Rab4 siRNA
AMPK-siRNA was conjugated with fluorescein isothiocyanate
(FITC-siRNA; Shanghai GenePharma Co., Ltd, Shanghai, China) and
transfected to cells seeded on chamber slides. Twenty-four hours
after transfection, the cells were washed in 1 PBS and fixed for
30 min in 4% PFA at 48C. After an additional washing in PBS, the
nuclei were counterstained with 1 mg/ml of Hoechst staining
(Sigma, St. Louis, MO) for 20 min at room temperature in the dark.
The slides were covered with fluorescent mounting medium
(Dako, Carpinteria, CA). Fluorescence microscopy was performed
using a Zeiss Axioplan microscope (Carl Zeiss, Göttingen,
Germany) at 490 nm excitation (FITC-siRNA, 525 nm emission)
and 536 nm excitation (propidium iodide, 617 nm emission). Digital
image analysis was performed using Open Lab software
(Improvision, Heidelberg, Germany).
Plasmid construction of Rab4 promoter and GFP-tagged wild-
type Rab4 and GFP-tagged Rab4DCT mutant
A 1,082-bp genomic fragment (from 1278 to þ573) containing
the Rab4 promoter was cloned into an empty pGL3-basic vector
(pGL3; Promega, Madison, WI) between the kpn1 and Sac1 sites
(Promega). Genomic DNA from C2C12 subjects was amplified by
PCR (forward primer, 50 -GG GGT ACC CC GGC TAG ATC AGG
CTA GCC ACG-30 ; reverse primer 50 -GG AGC TCG GTA AGT
CTC GGA CAT GGC GGT-30 ). Kpn1 and Sac1 digested products
were ligated into a linearized pGL3-basic vector containing the P1
promoter (pGL3-Rab4). A positive control construct was made by
cloning a pCMV5 promoter into the pGL3-basic vector (pGL3-
pCMV). Mouse Rab4a cDNA is D86563.1. The cDNAs for Rab4
(141–798) and Rab4DCT (141–789) were amplified by PCR and
subcloned into the HindIII/BamH/site in the pEGFP-C1 vector
(Clontech, Mountain View, CA) encoded for fusion with the green
fluorescent protein (GFP). All constructs were verified by direct
sequencing.
Luciferase assay
After transfection, cells were harvested and the extracts were
prepared using reporter lysis buffer (Promega). Cell lysates were
analyzed for luciferase activity using a dual luciferase assay kit
(Promega) and an illuminometer (VICTORTM3; PerkinElmer,
Waltham, MA). Each extract was assayed three times.
Data analysis
Data are expressed as the mean  SEM. One-way ANOVA was
used, followed by the Holm–Sidak multiple-range test for between-
group comparisons. P-values <0.05 were considered statistically
significant.
Results
AICAR and metformin induce Rab4 RNA and protein
levels in C2C12 cells
To gain insight into the role of AMPK on GLUT4 translocation,
we evaluated the effect of AICAR, an AMPK activator, on the
expression of Rab4, a member of the Ras superfamily of
monomeric G proteins. Rab regulates many steps involving
membrane traffic, including vesicle formation, movement, and
fusion. The administration of AICAR induced a time-dependent
increase in the level of expression of Rab4 messenger RNA in
C2C12 cells (Fig. 1A). Protein levels of Rab4 were also
JOURNAL OF CELLULAR PHYSIOLOGY
Fig. 1. AICAR and metformin induces Rab4 mRNA and protein
levels in C2C12 cells. A,C: Total RNA was prepared for these cells
after AICAR or metformin treatment, and RT-PCR was conducted
using specific primers. The PCR product was then gel-run in 2%
agarose and visualized in UV. GAPDH mRNA was employed as a
positive control. B,D: C2C12 cells were stimulated for the indicated
times with 1 mM AICAR or 10 mM metformin. The cell lysates (20 mg)
were analyzed via Western blotting for anti-Rab4 antibody. Blotting
with anti-GAPDH antibody was conducted as a protein loading
control. The results shown are representative of three independent
experiments.
increased by treatment of C2C12 cells with AICAR (Fig. 1B). To
provide physiologic relevance of Rab4 expression, we assessed
the effects of metformin, an AMPK activator, and one of the
most widely used oral hypoglycemic agents, on Rab4
expression. To this end, C2C12 cells were treated with
metformin before measuring the levels of Rab4 mRNA. The
levels of Rab4 mRNA were increased by treatment of C2C12
cells with metformin (Fig. 1C). The levels of Rab4 protein were
increased by the treatment of C2C12 cells with metformin
(Fig. 1D). Together, these results demonstrate that AMPK
regulates the expression of Rab4 in skeletal muscle cells.
Metformin stimulates Rab4 promoter in an AMPK-
dependent manner
To evaluate whether or not AMPK regulates Rab4
transcription, we examined the effects of AICAR and
metformin on the promoter activity of Rab4. AICAR stimulated
the promoter activity of Rab4 in a time-dependent manner
(Fig. 2A). Compound C, an AMPK inhibitor, blocked
metformin-induced Rab4 promoter activation (Fig. 2B),
suggesting the involvement of AMPK in metformin-induced
Rab4 induction. In addition, the administration of both AICAR
and metformin induced a time-dependent increase in AMPK
phosphorylation in C2C12 cells (Fig. 2C,D). The level of
phosphorylation of Thr172, which is in the active site of the
AMPK-alpha subunit and essential for enzyme activity, reached
a maximum level 12 h after treatment, then decreased to basal
levels by 24 h. Consistent with the increase in AMPK activity,
the phosphorylation of ACC-Ser79, the best-characterized
phosphorylation site by AMPK, increased after metformin
administration. These results demonstrate that metformin has
a stimulatory role with respect to the Rab4 promoter in an
AMPK-dependent manner.
Metformin stimulates the phosphorylation of AS160
To further characterize the signal pathway of metformin, we
examined the effects of metformin on the activity of AS160, a
protein with a Rab-GTPase activating protein (Rab-GAP)
domain that is involved in the regulation of GLUT4
translocation. We observed that AICAR initiated an increase in
AS160 phosphorylation in C2C12 cells when used at a
 METFORMIN REGULATES GLUT4 VIA AMPK
Fig. 2. Metformin stimulates Rab4 promoter in an AMPK-
dependent manner. A: HeLa cells were transiently transfected with
Rab4 promoter reporter plasmid. Cells were stimulated for the
indicated times with 1 mM AICAR. The promoter activity was then
measured. Results are the mean W SEM from three experiments.
M
P < 0.05. B: Promoter analysis of Rab4 in HeLa. Cells were transiently
transfected with Rab4 promoter reporter plasmid. HeLa cells were
pre-treated with compound C (5 mM) for 30 min, then exposed to
10 mM metformin for 12 h. The promoter activity was then
measured. The results are the mean W SEM from three experiments.
M
P < 0.05. C: C2C12 cells were stimulated for the indicated times with
1 mM AICAR. The cell lysates (20 mg) were analyzed via Western
blotting for anti-phospho-AMPK/ACC antibodies. Blotting with anti-
AMPK/ACC antibodies was conducted as a protein loading control. D:
C2C12 cells were stimulated for the indicated times with 10 mM
metformin. The cell lysates (20 mg) were analyzed via Western
blotting for anti-phospho-AMPK/ACC antibodies. Blotting with anti-
AMPK/ACC antibodies served as a protein loading control. The
results that are shown are representative of three independent
experiments.
concentration of 1 mM, with a maximal increase occurring at
12 h (Fig. 3A). Metformin also influenced the phosphorylation of
AS160 in C2C12 cells at a concentration of 10 mM (Fig. 3B).
Compound C, an AMPK inhibitor, blocked the metformin-
induced AS160 phosphorylation (Fig. 3C). To verify the roles of
AMPK in the metformin-mediated signaling pathway, we
assessed the effects of AMPK knockdown on AS160
phosphorylation at the cellular level. Knockdown of AMPK
suppressed the phosphorylation of AS160, suggesting that
AMPK plays a role upstream of AS160 (Fig. 3D). These results
demonstrate that metformin has a stimulatory role in AS160
phosphorylation in an AMPK-dependent manner.
Metformin activates the PKCz pathway
It has been reported that PKCz acts as a downstream effector of
PI3 kinase and contributes to the activation of GLUT4
translocation (Standaert et al., 1997). In an effort to understand
the signaling pathways involved in metformin-mediated GLUT4
translocation, we investigated the effects of metformin on
PKCz. AICAR activated PKCz in a time-dependent manner in
C2C12 cells (Fig. 4A). The phosphorylation of PKCz reached a
maximum level at 12 h. This pattern was also observed in
C2C12 cells upon metformin treatment (Fig. 4B). The
phosphorylation of PKCz was inhibited in cells pre-treated with
compound C (Fig. 4C). To corroborate the role of AMPK in the
metformin-mediated signaling pathway, we assessed the effect
of AMPK knockdown in L6-GLUT4myc-tagged myoblasts.
Metformin-mediated phosphorylation of PKCz was blocked by
AMPK knockdown (Fig. 4D). To confirm this effect at the level
of a single cell, we constructed FITC-conjugated AMPK siRNA.
JOURNAL OF CELLULAR PHYSIOLOGY
977
Fig. 3. Metformin stimulates the phosphorylation of AS160. A:
C2C12 cells were stimulated for the indicated times with 1 mM
AICAR. The cell lysates (20 mg) were analyzed via Western blotting
for anti-phospho-AS160 antibody. Blotting with anti-AS160 antibody
served as a protein loading control. B: C2C12 cells were stimulated for
the indicated times with 10 mM metformin. The cell lysates (20 mg)
were analyzed via Western blotting for anti-phospho-AS160
antibody. Blotting with anti-AS160 antibody served as a protein
loading control. C: C2C12 cells were pre-treated with compound C
(5 mM) for 30 min, then exposed to 10 mM metformin for 12 h. The cell
lysates (20 mg) were analyzed via Western blotting for anti-phospho-
AS160 antibody. Blotting with anti-AS160 antibody served as a
protein loading control. D: C2C12 cells were transiently transfected
with AMPK siRNA. The cell lysates (20 mg) were analyzed via
Western blotting for anti-phospho-AS160 antibody. Blotting with
anti-GAPDH antibody served as a protein loading control. The results
shown are representative of three independent experiments. AS160
indicates Akt substrate of 160 kDa.
The phosphorylation of PKCz (red signal) was decreased in
FITC-conjugated AMPK siRNA-transfected cells (Fig. 4E).
These results suggest that AMPK may play a critical role in
metformin-induced Rab4 induction.
Insulin stimulates GLUT4 translocation through Rab4
The stimulation of glucose uptake by insulin in skeletal muscle
cells is mediated mainly by translocation of the specialized
glucose transporter, GLUT4, from the cytoplasm to the plasma
membrane. To verify the roles of Rab4 in insulin-mediated
GLUT4 signaling, we assessed the effects of Rab4 knockdown
on GLUT4 translocation. First, we showed that Rab4 siRNA
reduced the expression Rab4, while non-target siRNA did not
show that effect (Fig. 5A). Since immunofluorescent
microscopy did not provide accurate quantitative analysis of
GLUT4 translocation, we used HRP-coupled secondary
antibody coupled with a colorimetric assay to accurately
measure cell surface GLUT4myc. There was a decrease in the
translocation of GLUT4 to the plasma membranes in Rab4
knockdown cells (Fig. 5B) using day 6 differentiated L6-
GLUT4myc-tagged myoblasts (Fig. 5B, squares). To
characterize the molecular mechanism of Rab4, we evaluated
the insulin effect on the expression of Rab4. The administration
of insulin did not influence the level of expression of
Rab4mRNA in C2C12 cells (Fig. 5C). To determine the
hierarchy between AMPK and Rab4, we determined the level of
expression of Rab4 in AMPK knockdown cells. The levels of
Rab4 protein were downregulated in cells pre-treated with
AMPK siRNA (Fig. 5D). Furthermore, glucose uptake in L6
myotubes was clearly increased in cells treated with AICAR or
metformin (Fig. 5E). These results suggest that Rab4 may affect
insulin-induced GLUT4 translocation downstream of AMPK.
978 LEE ET AL.
Fig. 4. Metformin activates the PKCz pathway. A: C2C12 cells were
stimulated for the indicated times with 1 mM AICAR. The cell lysates
(20 mg) were analyzed via Western blotting for anti-phospho-PKCz
antibody. Blotting with anti-PKCz antibody was conducted as a
protein loading control. B: C2C12 cells were stimulated for the
indicated times with 10 mM metformin. The cell lysates (20 mg) were
analyzed via Western blotting for anti-phospho-PKCz antibody.
Blotting with anti-PKCz antibody served as a protein loading control.
These results represent one of three independent experiments. C: L6-
GLUT4myc-tagged myoblasts were pre-treated with compound C
(5 mM) for 30 min, then exposed to 10 mM metformin for 12 h. The cell
lysates (20 mg) were analyzed via Western blotting for anti-phospho-
PKCz antibody. Blotting with anti-PKCz antibody served as a protein
loading control. D: L6-GLUT4myc-tagged myoblasts were transiently
transfected with AMPK siRNA. The cells were stimulated with
metformin. The cell lysates (20 mg) were analyzed via Western
blotting for anti-phospho-PKCz antibody. Blotting with anti-PKCz
antibody served as a protein loading control. E: L6-GLUT4myc-tagged
myoblasts were transiently transfected with FITC-conjugated AMPK
siRNA. Cells were stimulated for the indicated times with 10 mM
metformin. Cytologic images of phosphorylated PKCz were obtained
with confocal microscopy. The results shown are representative of
three independent experiments.
JOURNAL OF CELLULAR PHYSIOLOGY
Rab4 interacts with PKC-zeta, but not GLUT4
Rab4 was localized to the early endosome compartment and
found in GLUT4-containing vesicles in rat adipocytes and
skeletal muscle. In addition, Rab proteins appear to function in
the regulated assembly of multi-protein complexes required for
specific intracellular trafficking events (Zerial and McBride,
2001). PKCz is involved in insulin regulation of glucose uptake
and binds to and activates Rab5 (Huang et al., 2001). To
corroborate the role of Rab4 in the insulin-mediated signaling
pathway, we assessed the interaction of Rab4 and PKCz. As
shown in Figure 6, PKCz interacted with Rab4 and GLUT4 in L6
cells, indicating the importance of Rab4 in the insulin-mediated
signaling pathway. Furthermore, Rab4 and PKCz co-localized to
the perinuclear membranes (Fig. 6B). To determine the effect of
PKCz on GLUT4 translocation, we assessed the interaction of
GLUT4 and PKCz; GLUT4 interacted with PKCz but not with
Rab4 (Fig. 6C). These results indicate that protein–protein
interactions have are important in the translocation of GLUT4.
Effect of mutant Rab4 on insulin-induced GLUT4
translocation
Rab proteins are membrane proteins that anchor to
membranes via prenyl groups on two cysteine residues within
the C-terminals of Rab proteins. GLUT4 translocation has been
shown to be inhibited by microinjection of a synthetic peptide
corresponding to the C-terminal domain of Rab4 (Shibata et al.,
1996), and expression of prenylation-deficient Rab4 proteins
(Cormont et al., 2001). To confirm the effect of the Rab4 C-
terminal on GLUT4 translocation, we determined the effect of
Rab4DCT-GFP on translocation of GLUT4 in L6 myoblasts
expressing Myc-tagged GLUT4. The Rab4 C-terminal deletion
mutant did not localize to the perinuclear membrane (Fig. 7A).
Moreover, insulin-mediated GLUT4 translocation was
inhibited in cells transiently expressing the Rab4 C-terminal-
deleted mutant, but not by the Rab4 wild type (Fig. 7B).
Together, these results indicate that Rab4 protein plays a
crucial role in the insulin-mediated GLUT4 signaling pathway.
Discussion
The principal finding of this study was that AMPK is involved in
GLUT4 translocation. Specifically, we demonstrated that
AMPK is instrumental in metformin-mediated GLUT4
translocation.
The primary assertion of this study was that AMPK mediates
GLUT4 effects through the Rab4 pathway. The anti-diabetic
role of AMPK has previously been evaluated in conjunction with
as insulin signal modulator (Fischer et al., 1995; Towler and
Hardie, 2007) and GLUT4 (Kurth-Kraczek et al., 1999;
Koistinen et al., 2003; Holmes et al., 2005; Yamaguchi et al.,
2005). The GLUT4-modulating properties of AMPK appear to
be responsible for its role as an insulin signal modulator and may
also contribute to its observed anti-diabetic effects. The
contribution of AMPK to the anti-diabetic role has raised
questions as to which of the activities of AMPK may be relevant
to its metabolic role. The mechanisms of the AMPK-induced
anti-diabetic effects have previously been suggested (Winder
and Hardie, 1999; Barnes and Zierath, 2005). In the present
study, we have established that AMPK induces Rab4 expression.
Additionally, we have demonstrated that metformin activates
Rab4 promoter activity through the AMPK pathway. A
relationship between metformin and glucose regulation has
been suggested (Ciaraldi et al., 2002). Collectively, our results
indicate that AMPK has a crucial role in metformin-mediated
Rab4 expression.
The objective of the present study was to ascertain whether
or not Rab4 is directly regulated by metformin, and if so, to
determine which molecules are involved in this process. Our
 METFORMIN REGULATES GLUT4 VIA AMPK
Fig. 5. Insulin stimulates GLUT4 translocation through Rab4. A:
Confluent monolayers of myotubes were transiently transfected with
either Rab4 siRNA or non-target siRNA (50 nM, 24 h). The levels of
Rab4 were analyzed via Western blotting for anti-Rab4 antibody. B:
Confluent monolayers of myotubes were transiently transfected with
either Rab4 siRNA or non-target siRNA prior to insulin treatment
(100 nM, 10 min). Cell surface GLUT4myc levels on intact cells were
determined, as described in the Materials and Methods Section. The
results are the mean W SEM from three experiments. MP < 0.05. C:
Total RNA was prepared for these cells after AICAR treatment and
RT-PCR was conducted using specific primers. The PCR product was
then gel-run in 2% agarose and visualized with UV. GAPDH mRNA
was used as a positive control. D: L6 cells were transiently transfected
with AMPK siRNA. The cell lysates (20 mg) were analyzed via
Western blotting for anti-Rab4/AMPK antibodies. Blotting with anti-
GAPDH antibody served as a protein loading control. The results
shown are representative of three independent experiments. E:
Confluent monolayers of myotubes were treated for 20 min with
AIACR, metformin, and insulin. Cell surface GLUT4myc levels on
intact cells were determined as described in the Materials and
Methods Section. The results are the mean W SEM from three
experiments. MP < 0.05.
JOURNAL OF CELLULAR PHYSIOLOGY
979
Fig. 6. PKCz interacts with Rab4 and GLUT4. A: Rab4-GFP
overexpression cell lysates (400 mg) were immunoprecipitated with
anti-GFP antibody. The immunoprecipitates were analyzed via
Western blotting for anti-GFP and PKCz antibodies. B: L6 cells
transiently transfected with Rab4-GFP were immunostained for
PKCz. Images were obtained with confocal microscopy. C: Rab4-GFP
overexpressed cell lysates (400 mg) were immunoprecipitated with
anti-GLUT4 antibody. The immunoprecipitates were analyzed via
Western blotting for anti-GFP and PKCz antibodies. The results
shown are representative of three independent experiments. TCL
indicates total cell lysates.
data have revealed a novel role for AS160 downstream of Akt
activation by metformin. The identification of an AMPK–
AS160–Rab4 axis in metformin-treated skeletal muscle cells led
us to hypothesize that Rab4 has a role in AMPK-mediated
modulation of insulin signaling. Indeed, this is the first report of a
link between AMPK and Rab4 in skeletal muscle cells. It is thus
tempting to speculate that AMPK-mediated regulation of Rab4
plays a role in insulin signaling because both metformin and
AMPK have been suggested to counteract impaired glucose
metabolism. In addition to an anti-diabetic role, we further
demonstrated that metformin has a metabolic effect by
activating Rab4. Indeed, we showed that a metformin
concentration of 10 mM affects AMPK phosphorylation,
indicating that somewhat higher concentrations of metformin
are required to effectively investigate its effect in a cell culture
system, even though the relevant physiologic concentration is
low by comparison. There are two possible reasons for this
disparity. First, the in vitro and in vivo environments are
different. Second, artifacts of the immortalized cell line may be
responsible. Although the mechanism by which AMPK
influences insulin signaling is unknown, the findings in this report
suggest that metformin may induce Rab4 expression through
the AMPK pathway as part of the process of metformin-
mediated insulin signal modulation.
The Rab family of small GTP-binding proteins is involved in
each step of vesicular transport, which includes the formation
and movement of vesicles, as well as docking and fusion of
vesicles with acceptor membranes, and exocytosis and
endocytosis (Cormont and Le Marchand-Brustel, 2001; Kaddai
et al., 2008). Different Rab proteins can participate in these
processes depending on the specific functions of the protein
involved and its unique subcellular localization. For example,
Rab7 and Rab9 are located in late endosomes, participating in
late endosomes Golgi transport, while Rab11 is observed in
recycling endosomes and participates in this process (Zerial and
McBride, 2001). Rab5 is located in the plasma membrane and
early endosomes (Zerial and McBride, 2001). Rab5 mediates
980 LEE ET AL.
Fig. 7. Rab4-C-terminal domain is involved in insulin-induced
translocation of GLUT4 to the cell surface. A: L6 cells were transiently
transfected with wild-type Rab4-GFP or Rab4DCT-GFP mutant.
Images were obtained with confocal microscopy. B: L6-GLUTmyc-
tagged cells transfected with either wild-type Rab4-GFP or Rab4DCT-
GFP mutant were serum-starved for 4 h. The cells were then treated
with 100 nM insulin for 20 min. Cell surface GLUT4myc levels on intact
cells were determined as described in the Materials and Methods
Section. The results are the mean W SEM from three experiments.
M E23.
P < 0.05.
clathrin-coated vesicle-mediated transport from the plasma
membrane to the early endosomes, as well as the endocytotic
process of GLUT4 internalization (Cormont and Le Marchand-
Brustel, 2001), whereas Rab4 is localized to the early/recycling
endosomes, where it contributes to cargo sorting and recycling
(Vollenweider et al., 1997). GLUT4 translocation was inhibited
by microinjection of a synthetic peptide corresponding to the
C-terminal domain of Rab4 (Shibata et al., 1996), and by
expression of prenylation-deficient Rab4 proteins (Cormont
et al., 2001). Rab proteins are intrinsically soluble proteins, the
activity of which is dependent upon the cytoplasmic leaflet of
cellular membranes. This association is mediated by a lipid
modification of the C-terminal, which is termed prenylation.
Prenylation consists of the covalent attachment via a thioether
linkage of a C15 (farnesyl) or C20 (geranylgeranyl) isoprenoid
group to a C-terminal cysteine residue in the context of a
prenylation motif. Prenylated proteins play crucial roles in vital
cellular processes such as signal transduction and intracellular
trafficking pathways (Knight et al., 2000). In the present study,
overexpression of the Rab4DCT mutant decreased GLUT4
translocation in response to insulin. In addition, wild-type Rab4
co-localized with PKCz, but the Rab4DCT mutant did not.
Together, these results demonstrate that the C-terminal
domain of Rab4 has an important role in GLUT4 translocation.
The insulin-signaling pathway is required for multi-protein
complexes. Moreover, insulin stimulates the interaction
JOURNAL OF CELLULAR PHYSIOLOGY
between Rab4 and a motor protein kinesin downstream of
aPKC activation (Fiory et al., 2004). We observed an interaction
between Rab4 and GLUT4 and PKCz based on
immunoprecipitation. The interaction between Rab4 and PKCz
was confirmed in Rab4-GFP-overexpressed L6 cells. This
interaction is presumably required for the movement of
GLUT4 vesicles to the plasma membrane. Taken together,
Rab4 appears to be involved in the movement of vesicles along
the microtubule network and the fusion of the vesicles with the
plasma membranes.
In conclusion, the present study supports the hypothesis that
metformin induces expression of Rab4 through the AMPK–
AS160–PKCz pathways and modulates insulin-mediated
GLUT4 translocation in skeletal muscle cells.
Acknowledgments
This study was supported by a grant from the Korea University
College of Medicine and Korea Science and Engineering
Foundation (KOSEF, R01-2008-000-11180-0).
Literature Cited
Ai H, Ihlemann J, Hellsten Y, Lauritzen HP, Hardie DG, Galbo H, Ploug T. 2002. Effect of fiber
type and nutritional state on AICAR- and contraction-stimulated glucose transport in rat
muscle. Am J Physiol Endocrinol Metab 282:E1291–E1300.
Barnes BR, Zierath JR. 2005. Role of AMP-activated protein kinase in the control of glucose
homeostasis. Curr Mol Med 5:341–348.
Ciaraldi TP, Kong AP, Chu NV, Kim DD, Baxi S, Loviscach M, Plodkowski R, Reitz R, Caulfield
M, Mudaliar S, Henry RR. 2002. Regulation of glucose transport and insulin signaling by
troglitazone or metformin in adipose tissue of type 2 diabetic subjects. Diabetes 51:30–36.
Cormont M, Le Marchand-Brustel Y. 2001. The role of small G-proteins in the regulation of
glucose transport (review). Mol Membr Biol 18:213–220.
Cormont M, Gautier N, Ilc K, le Marchand-Brustel Y. 2001. Expression of a prenylation-
deficient Rab4 inhibits the GLUT4 translocation induced by active phosphatidylinositol 3-
kinase and protein kinase B. Biochem J 356:143–149.
Fiory F, Oriente F, Miele C, Romano C, Trencia A, Alberobello AT, Esposito I, Valentino R,
Beguinot F, Formisano P. 2004. Protein kinase C-zeta and protein kinase B regulate distinct
steps of insulin endocytosis and intracellular sorting. J Biol Chem 279:11137–11145.
Fischer Y, Thomas J, Rosen P, Kammermeier H. 1995. Action of metformin on glucose
transport and glucose transporter GLUT1 and GLUT4 in heart muscle cells from healthy
and diabetic rats. Endocrinology 136:412–420.
Fisher JS, Gao J, Han DH, Holloszy JO, Nolte LA. 2002. Activation of AMP kinase enhances
sensitivity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab 282:E18–
Gunton JE, Delhanty PJ, Takahashi S, Baxter RC. 2003. Metformin rapidly increases insulin
receptor activation in human liver and signals preferentially through insulin-receptor
substrate-2. J Clin Endocrinol Metab 88:1323–1332.
Hardie DG, Carling D. 1997. The AMP-activated protein kinase—Fuel gauge of the
mammalian cell? Eur J Biochem 246:259–273.
Henin N, Vincent MF, Gruber HE, Van den Berghe G. 1995. Inhibition of fatty acid and
cholesterol synthesis by stimulation of AMP-activated protein kinase. FASEB J 9:541–546.
Holmes BF, Sparling DP, Olson AL, Winder WW, Dohm GL. 2005. Regulation of muscle
GLUT4 enhancer factor and myocyte enhancer factor 2 by AMP-activated protein kinase.
Am J Physiol Endocrinol Metab 289:E1071–E1076.
Huang J, Imamura T, Olefsky JM. 2001. Insulin can regulate GLUT4 internalization by signaling
to Rab5 and the motor protein dynein. Proc Natl Acad Sci USA 98:13084–13089.
Imamura T, Huang J, Usui I, Satoh H, Bever J, Olefsky JM. 2003. Insulin-induced GLUT4
translocation involves protein kinase C-lambda-mediated functional coupling between
Rab4 and the motor protein kinesin. Mol Cell Biol 23:4892–4900.
Ishikura S, Klip A. 2008. Muscle cells engage Rab8A and myosin Vb in insulin-dependent
GLUT4 translocation. Am J Physiol Cell Physiol 295:C1016–C1025.
Ishikura S, Koshkina A, Klip A. 2008. Small G proteins in insulin action: Rab and Rho families at
the crossroads of signal transduction and GLUT4 vesicle traffic. Acta Physiol (Oxf) 192:61–
74.
Kaddai V, Le Marchand-Brustel Y, Cormont M. 2008. Rab proteins in endocytosis and Glut4
trafficking. Acta Physiol (Oxf) 192:75–88.
Kaddai V, Gonzalez T, Keslair F, Gremeaux T, Bonnafous S, Gugenheim J, Tran A, Gual P,
Le Marchand-Brustel Y, Cormont M. 2009. Rab4b is a small GTPase involved in the control
of the glucose transporter GLUT4 localization in adipocyte. PLoS ONE 4:e5257.
Knight JB, Cao KT, Gibson GV, Olson AL. 2000. Expression of a prenylation-deficient Rab4
interferes with propagation of insulin signaling through insulin receptor substrate-1.
Endocrinology 141:208–218.
Koistinen HA, Galuska D, Chibalin AV, Yang J, Zierath JR, Holman GD, Wallberg-Henriksson
H. 2003. 5-Amino-imidazole carboxamide riboside increases glucose transport and cell-
surface GLUT4 content in skeletal muscle from subjects with type 2 diabetes. Diabetes
52:1066–1072.
Kumar N, Dey CS. 2002. Metformin enhances insulin signalling in insulin-dependent and -
independent pathways in insulin resistant muscle cells. Br J Pharmacol 137:329–336.
Kurth-Kraczek EJ, Hirshman MF, Goodyear LJ, Winder WW. 1999. 50 AMP-activated protein
kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 48:1667–1671.
Makinde AO, Gamble J, Lopaschuk GD. 1997. Upregulation of 50 -AMP-activated protein
kinase is responsible for the increase in myocardial fatty acid oxidation rates following birth
in the newborn rabbit. Circ Res 80:482–489.
Meuillet EJ, Wiernsperger N, Mania-Farnell B, Hubert P, Cremel G. 1999. Metformin
modulates insulin receptor signaling in normal and cholesterol-treated human hepatoma
cells (HepG2). Eur J Pharmacol 377:241–252.
 METFORMIN REGULATES GLUT4 VIA AMPK
Sano H, Eguez L, Teruel MN, Fukuda M, Chuang TD, Chavez JA, Lienhard GE, McGraw TE.
2007. Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated
translocation of GLUT4 to the adipocyte plasma membrane. Cell Metab 5:293–303.
Sarabia V, Lam L, Burdett E, Leiter LA, Klip A. 1992. Glucose transport in human skeletal
muscle cells in culture. Stimulation by insulin and metformin. J Clin Invest 90:1386–
1395.
Shibata H, Omata W, Suzuki Y, Tanaka S, Kojima I. 1996. A synthetic peptide corresponding
to the Rab4 hypervariable carboxyl-terminal domain inhibits insulin action on glucose
transport in rat adipocytes. J Biol Chem 271:9704–9709.
Shibata H, Omata W, Kojima I. 1997. Insulin stimulates guanine nucleotide exchange on Rab4
via a wortmannin-sensitive signaling pathway in rat adipocytes. J Biol Chem 272:14542–
14546.
Standaert ML, Galloway L, Karnam P, Bandyopadhyay G, Moscat J, Farese RV. 1997. Protein
kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin
stimulation in rat adipocytes. Potential role in glucose transport. J Biol Chem 272:30075–
30082.
Towler MC, Hardie DG. 2007. AMP-activated protein kinase in metabolic control and insulin
signaling. Circ Res 100:328–341.
Vollenweider P, Martin SS, Haruta T, Morris AJ, Nelson JG, Cormont M, Le Marchand-Brustel
Y, Rose DW, Olefsky JM. 1997. The small guanosine triphosphate-binding protein Rab4 is
JOURNAL OF CELLULAR PHYSIOLOGY
981
involved in insulin-induced GLUT4 translocation and actin filament rearrangement in 3T3-
L1 cells. Endocrinology 138:4941–4949.
Wijesekara N, Tung A, Thong F, Klip A. 2006. Muscle cell depolarization induces a gain in
surface GLUT4 via reduced endocytosis independently of AMPK. Am J Physiol Endocrinol
Metab 290:E1276–E1286.
Winder WW, Hardie DG. 1999. AMP-activated protein kinase, a metabolic master switch:
Possible roles in type 2 diabetes. Am J Physiol 277:E1–E10.
Witters LA. 2001. The blooming of the French lilac. J Clin Invest 108:1105–1107.
Yamaguchi S, Katahira H, Ozawa S, Nakamichi Y, Tanaka T, Shimoyama T, Takahashi K,
Yoshimoto K, Imaizumi MO, Nagamatsu S, Ishida H. 2005. Activators of AMP-activated
protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1
adipocytes. Am J Physiol Endocrinol Metab 289:E643–E649.
Yang J, Holman GD. 2006. Long-term metformin treatment stimulates cardiomyocyte
glucose transport through an AMP-activated protein kinase-dependent reduction in
GLUT4 endocytosis. Endocrinology 147:2728–2736.
Zerial M, McBride H. 2001. Rab proteins as membrane organizers. Nat Rev Mol Cell Biol
2:107–117.
Zong H, Ren JM, Young LH, Pypaert M, Mu J, Birnbaum MJ, Shulman GI. 2002. AMP kinase is
required for mitochondrial biogenesis in skeletal muscle in response to chronic energy
deprivation. Proc Natl Acad Sci USA 99:15983–15987.