Mol Cell Biochem (2011) 353:305313
DOI 10.1007/s11010-011-0799-0
Apelin stimulates glucose uptake through the PI3K/Akt pathway
and improves insulin resistance in 3T3-L1 adipocytes
Shunming Zhu Fei Sun Weijie Li Yanjie Cao
Chen Wang Yabin Wang Dong Liang Rongqing Zhang
Shenwei Zhang Haichang Wang Feng Cao
Received: 19 November 2010 / Accepted: 18 March 2011 / Published online: 2 April 2011
Springer Science+Business Media, LLC. 2011
Abstract Apelin, a cytokine mainly secreted by adipo-
cytes, is closely related with insulin resistance. The under-
lying molecular mechanisms of how apelin affects insulin
resistance, however, are poorly understood. This study
aimed to investigate the effect of apelin on glucose metab-
olism and insulin resistance in 3T3-L1 adipocytes. After
10 ng/ml TNF-a treatment for 24 h, insulin-stimulated
glucose uptake was reduced by 47% in 3T3-L1 adipocytes.
Apelin treatment improved glucose uptake in a time- and
dose-dependent manner. Treatment of 1,000 nM apelin for
60 min maximally augmented glucose uptake in insulin-
resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incu-
bation also increased adipocytes insulin-stimulated glucose
uptake, and PI3K/Akt pathway were involved in these
effects. In addition, immunocytochemistry staining and
western blotting analysis indicated that apelin could increase
glucose transporter 4 translocation from the cytoplasm to the
plasma membrane. Apelin also increased the anti-inflam-
matory adipokine adiponectin mRNA expression while
reducing that of pro-inflammatory adipokine interleukin-6
in insulin-resistant 3T3-L1 adipocytes. These results suggest
that apelin stimulates glucose uptake through the PI3K/
Akt pathway, promotes GLUT4 translocation from the
S. Zhu  W. Li  Y. Cao  C. Wang  Y. Wang  D. Liang 
R. Zhang  S. Zhang  H. Wang (&)  F. Cao (&)
Department of Cardiology, Xijing Hospital, Fourth Military
Medical University, Xian 710032, Shaanxi, China
e-mail: wanghc@fmmu.edu.cn
F. Cao
e-mail: wind8828@gmail.com
F. Sun
Department of Otolaryngology, Xijing Hospital, Fourth Military
Medical University, Xian 710032, Shaanxi, China
cytoplasm to the plasma membrane, and modulates inflam-
matory responses in insulin-resistant 3T3-L1 adipocytes.
Keywords Apelin  Insulin resistance  Glucose uptake 
Akt  GLUT4
Introduction
The prevalence of diabetes has dramatically increased in
the world. It is estimated that the number of diabetics will
rise to 380 million worldwide by 2025, and 80% of dia-
betics will be living in the developing world. Among dif-
ferent types of diabetes, type 2 diabetes is characterized by
insulin resistance, which is a state where insulin has a
reduced ability to mediate glucose homeostasis in its major
target tissues, such as skeletal muscle, fat, and liver. Insulin
resistance is not only the key pathophysiological abnor-
mality of type 2 diabetes but also the primary cause of
many related complications, such as hyperglycemia, dysl-
ipidemia, abdominal obesity, hypertension, and athero-
sclerosis. Accumulating evidence has indicated that these
disorders mainly stem from impaired insulin sensitivity and
the low-grade inflammatory state of insulin resistance
[1, 2]. Thus, improving both insulin sensitivity and the
inflammatory state is a critical goal for treating diabetes
and reducing related complications. These strategies to
alleviate insulin resistance have mainly focused on phar-
macological and nutritional supplements to improve insulin
sensitivity and mediate an inflammatory response; how-
ever, they have not shown satisfactory efficacy. Apelin, a
cytokine mainly secreted by adipocytes, is closely related
to glucose metabolism and was proposed to be a promising
therapeutic agent for treating insulin resistance.
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Apelin is the endogenous ligand for the orphan G pro-
tein-coupled receptor APJ and was first isolated from
bovine stomach extracts [3]. Apelin was identified as an
adipokine because it is mainly produced and secreted by
adipocytes [4]. The apelin gene encodes a 77 amino acid
preproprotein that is cleaved to shorter biologically active
peptides, including apelin-19, apelin-17, apelin-16, apelin-
13, and apelin-12. The 13-amino acid peptide (apelin-13)
possesses a pyroglutamate substitution at the N terminus, a
common post-translational modification that preserves
biological activity by rendering the peptide more resistant
to enzymatic cleavage. Studies have suggested that the
most potent isoform of apelin is the pyroglutamated form
of apelin-13 (Pyr-apelin-13), which is considered the
principal active biological ligand [5, 6]. In a number of
previous studies, this novel peptide has been shown to exert
various biological effects on several systems and organs,
including the central nervous system, adipose, heart, lung,
liver, kidney, and skin [7]. Several studies have also doc-
umented the altered level of serum apelin in type 2 diabetic
patients, but the results remain controversial [4, 8]. Thus,
determining how apelin affects insulin resistance and
whether it could be used as an anti-diabetic drug are of
great value.
In this study, we chose the commonly used pre-adipo-
cyte cell line 3T3-L1 to investigate whether apelin could
directly act on insulin-resistant adipocytes. We also
investigated the underlying molecular mechanism of
apelins actions on insulin resistance.
Materials and methods
Reagents
Dulbeccos Modified Eagles Medium (DMEM) and fetal
calf serum (FCS) were purchased from GIBCO (Gaithers-
burg, MD, USA). Pyr-apelin-13 was purchased from
Anaspec (Fremont, CA, USA). Isobutylmethylxanthine,
dexamethasone, insulin, wortmannin, TNF-a, and cytocha-
lasin B were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Antibodies against protein kinase B (Akt) and phos-
phorylated Akt (Ser473) were purchased from Cell Signaling
Technology (Danvers, MA, USA). Antibodies against glu-
cose transporter 4 (GLUT4) and clathrin were purchased
from Abcam (Cambridge, UK), and b-actin and FITC-con-
jugated secondary antibodies were purchased from Dingguo
(Beijing, China). 2-Deoxy-D-[3H] glucose (3H-2-DG) was
purchased from GE healthcare (Waukesha, WI, USA). A
bicinchoninic acid (BCA) protein assay kit was acquired
from Dingguo (Beijing, China), and the membrane protein
extraction kit was from Bestbio (Shanghai, China). Oil red O
was purchased from Dingguo (Beijing, China).
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Mol Cell Biochem (2011) 353:305313
Cell culture and assessment
Mouse 3T3-L1 pre-adipocyte cells were purchased from
American Type Culture Collection (ATCC, Manassas, VA,
USA) and were grown in high glucose (4.5 mM) DMEM
with 10% FCS at 37C in 5% CO2 atmosphere. These cells
were induced to differentiate into adipocytes as described
[9]. Briefly, 2 days after confluence, medium was changed
to 10% FCS DMEM supplemented with 0.5 mM isobu-
tylmethylxanthine, 1 lM dexamethasone, and 10 lg/ml
insulin. Subsequently, cells were treated with 10 lg/ml
insulin in 10% FCS DMEM for an additional 24 h and then
maintained with 10% FCS fresh media every other day for
8 days. With this protocol, more than 80% of the pre-adi-
pocytes differentiated into adipocytes. For Oil red O
staining, differentiated adipocytes grown on glass cover
slips were washed with PBS for three times. Then these
cells were put into ice-acetone for 30 min. After twice
wash of PBS, cells were stained in Oil red O for 2 h. Then
cells were washed with PBS and visualized using an
Olympus microscope.
Assessment of insulin resistance
To induce insulin resistance, the differentiated adipocytes
were starved in serum-free, 0.5% (w/v) BSA DMEM for
3 h before incubation in 10% FCS DMEM with 10 ng/ml
Tumor necrosis factor alpha (TNF-a) for 24 h. To evaluate
insulin resistance, 3H-2-DG radioactivity taken up by the
cells was determined by a scintillation counter. TNF-a-
treated cells showed significantly lower radioactivity than
untreated cells and were considered to be insulin resistant.
These insulin-resistant cells were used for the following
experiments.
3
H-2-DG uptake measurement
This assay was performed using a modified version of a
protocol previously described [10]. Briefly, insulin-resistant
adipocytes were starved in serum-free, 0.5% (w/v) bovine
serum albumin (BSA) DMEM for 3 h before treatment. To
confirm insulin resistance, 100 nM insulin was added to the
medium for 30 min and then 3H-2-DG radioactivity taken up
by the cells was determined. To investigate the effects of
apelin on glucose uptake or insulin-stimulated glucose
uptake, various concentrations of apelin (for different time
points) were added to the medium alone or followed by 100
nM insulin for 30 min. To determine the involvement of the
PI3K/Akt pathway, the PI3K-specific inhibitor wortmannin
(100 nM) was added to the medium 30 min before apelin
treatment. After washing three times with KrebsRinger
phosphate buffer (KRP, 1.32 mM NaCl, 4.71 mM KCl2,
47 mM CaCl2, 1.24 mM MgSO4, 2.48 mM Na3PO4, and
Mol Cell Biochem (2011) 353:305313
10 mM HEPES (pH 7.4)), a final concentration of 1 lCi/ml
3
H-2-DG was added to the cells. The medium was aspirated
10 min later, and the cells were washed three times with ice-
cold KRP to terminate the reaction. Next, the cells were lysed
with 0.1 N NaOH, and the radioactivity taken up by the cells
was determined using a scintillation counter (LS 6500,
Beckman Instruments, Fullerton, CA, USA). Nonspecific
glucose uptake was measured by subtracting values for 3H-2-
DG in the presence of 100 nM cytochalasin B. The DPM
value was corrected by protein content in each well, which
was measured with a BCA protein assay kit.
Protein extract and Western blot analysis
For total protein extract, the cells were washed twice with
ice-cold Phosphate Buffered Saline (PBS) and harvested in
lysis buffer (50 mM TrisHCl, 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
SDS, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leu-
peptin, and 0.1 mM sodium orthovanadate (pH 7.4)) on ice
with gentle shaking. After centrifugation at 12,000g for
10 min at 4C, the protein content of the supernatant was
determined with the BCA protein assay kit. For GLUT4
translocation analysis, membrane protein was extracted
following the manufacturers protocol. Briefly, cells were
lysed using 12 passes through a 22-gauge needle followed
by 6 passes through a 27-gauge needle in HES buffer
(20 mM HEPES, 10 mM EDTA, and 250 mM sucrose pH
7.4) containing complete protease inhibitor mixture and
phosphatase inhibitors (2 mM sodium orthovanadate,
1 mM pyrophosphate, 1 mM ammonium molybdate, and
10 mM sodium fluoride) at 4C. The lysate was then
centrifuged at 500g for 10 min to remove unbroken cells
and at 10,000g for 10 min, 15,000g for 20 min, and
175,000g for 75 min at 4C to obtain the membrane protein
fraction.
Cell lysates were then loaded onto 10% SDS-PAGE for
analysis of pAkt, total Akt, GLUT4, clathrin, and b-actin.
Clathrin was used as loading control for assessment of
GLUT4 plasma membrane (PM) fraction. Proteins were
transferred onto nitrocellulose (NC) membranes (Millipore,
Bedford, MA), which were blocked in 5% nonfat dry milk
in Tris-buffered saline with 1% Tween 20 (TBS-T, Ding-
guo, Beijing, China) for 1 h. Primary antibodies were
diluted in 5% nonfat dry milk and added to the membrane
overnight at 4C. The membranes were washed thoroughly
and incubated with horseradish peroxidase-conjugated
secondary antibodies. After washing, protein bands were
visualized using enhanced chemiluminescence (ECL,
Dingguo, Beijing, China) and an imaging system (BioRad,
Hercules, CA) according to the manufacturers instructions.
307
Immunocytochemistry staining for GLUT4
3T3-L1 pre-adipocytes grown on glass cover slips were
induced to insulin resistance as described above. After
three washes with PBS, the cells were either treated with
apelin or insulin at different concentrations. Immunocyto-
chemistry staining for GLUT4 was performed as described
with some modification [11]. Briefly, after treatment, the
cells were washed with ice-cold PBS. Next, they were fixed
for 15 min in cold acetone and washed using ice-cold PBS.
After blocking in 10% normal goat serum at 37C for 1 h,
the cells were washed and incubated at 37C with a rabbit
anti-GLUT4 antibody for another 1 h. After three PBS
washes of 10 min each, the FITC-conjugated goat anti-
rabbit secondary antibody was applied to the samples at
37C for 1 h. After washing thoroughly with PBS, the cells
were fixed and visualized using an Olympus confocal
microscope.
Quantitative real-time PCR for IL-6 and adiponectin
Total RNA was extracted using the TRIzol reagent (Gibco,
Gaithersburg, MD, USA). The cDNA was synthesized from
RNA using a PrimeScriptTM RT reagent Kit (TaKaRa, Shiga,
Japan). The cDNA specimens were amplified using SYBR
Premix Ex TaqTM II (TaKaRa, Shiga, Japan). Interleukin-6
(IL-6) primers had the following sequences: forward
50 -TTCTTGGGACTGATGCTG-30 and reverse 50 -AGGT
CTGTTGGGAGTGGT-30 . The sequences of the adiponec-
tin primers were as follows: forward 50 -CTGTTCCCAA
TGTACCCA-30 and reverse 50 -AAGAGGCTCACCTTC
ACA-30 . In addition, the forward and reverse primers of the
internal control b-actin were 50 -CAGCCTTCCTTCTTG
GGTAT-30 and 50 -GGTCTTTACGGATGTCAACG-30 ,
respectively. Polymerase chain reaction (PCR) amplification
was performed on the ABI 7500 system (Applied Biosys-
tems) using SYBR Green II (TaKaRa, Shiga, Japan). We
used levels of b-actin mRNA to normalize the data. Relative
quantitation of mRNA expression levels was determined
using the relative standard curve method according to the
manufacturers instructions (Applied Biosystems).
Statistical analysis
Data are presented as mean SEM. Dunnett t test or Two-
way ANOVA were used for statistics analysis as appro-
priate. The main factors of dosage and time are significant.
Comparisons between groups were made using Dunnett
t test or Two-way ANOVA. P value B 0.05 was considered
significant.
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Results
Assessment of adipocytes by Oil red O staining
3T3-L1 pre-adipocytes were induced to adipocytes and
stained by Oil red O as described in Materials and
Methods. Oil red O staining confirmed that more than
80% of the pre-adipocytes differentiated into adipocytes
(Fig. 1).
TNF-a caused insulin resistance in 3T3-L1 adipocytes
To induce insulin resistance, differentiated 3T3-L1 adipo-
cytes were starved in serum-free, 0.5% (w/v) BSA DMEM
for 3 h before being incubated in DMEM with 10 ng/ml
TNF-a for 24 h. 3H-2-DG was added to the medium, and
glucose uptake was assessed as described in the Materials
and Methods section. Levels of radioactivity showed that
after TNF-a treatment, basal and insulin-stimulated glucose
uptake were suppressed by 28 and 47%, respectively
(P \ 0.05; Fig. 2). The TNF-a-treated cells were consid-
ered to be insulin-resistant cells and were used for the
following experiments.
Fig. 1 3T3-L1 pre-adipocytes were successfully differentiated to
adipocytes. 3T3-L1 pre-adipocytes were induced as described in
Materials and Methods. a 3T3-L1 pre-adipocytes were differenti-
ated to adipocytes after 1012 days of induce. Lipid drops were found
inside the cells. b Oil red O staining confirmed that more than 80% of
the pre-adipocytes were differentiated into adipocytes
123
Mol Cell Biochem (2011) 353:305313
Apelin increased basal and insulin-stimulated 3H-2-DG
uptake in insulin-resistant 3T3-L1 adipocytes
The effect of apelin on glucose transport in insulin-resistant
3T3-L1 adipocytes was evaluated using the 3H-2-DG
uptake assay. After incubation with 10 ng/ml TNF-a for
24 h, cells were washed three times in PBS and incubated
with apelin. Treatment with different concentrations of
apelin for 60 min resulted in increased glucose uptake in
insulin-resistant 3T3-L1 adipocytes. Apelin exerted an
initial significant effect on glucose uptake at 100 nM
(P \ 0.05; Fig. 3a) and a maximal effect at 1,000 nM
(P \ 0.01; Fig. 3a). Higher concentrations of apelin (104
and 105 nM) showed a decreased effect on glucose uptake
compared to low concentrations of apelin. To perform a
time course analysis, 1,000 nM apelin was added to the
insulin-resistant adipocytes at different time points. Results
showed that glucose uptake was maximally augmented at
60 min after incubation with apelin (P \ 0.01; Fig. 3b);
apelin still retained its effect at 120 min (P \ 0.05;
Fig. 3b). After 120 min, the improvement in glucose
uptake declined and was no longer significant compared to
basal levels. Furthermore, we examined whether apelin
could increase insulin sensitivity in insulin-resistant cells.
Administration of 100 nM insulin for 30 min after stimu-
lation with 100 or 1,000 nM apelin significantly increased
insulin-stimulated 3H-2-DG uptake, and 1,000 nM apelin
showed greater effects (P \ 0.05 vs. apelin alone,
P \ 0.01 vs. apelin alone, and P \ 0.05 for 1,000 nM
apelin vs. 100 nM apelin; Fig. 3c). These results indicated
that apelin alone could enhance glucose uptake in insulin-
resistant cells. Apelin may also serve as an insulin-
sensitizing agent because it can markedly improve
Fig. 2 TNF-a induced insulin resistance in 3T3-L1 adipocytes.
Differentiated 3T3-L1 adipocytes were incubated with 10 ng/ml
TNF-a for 24 h. Insulin-stimulated 3H-2-DG uptake was calculated
by liquid scintillation. Radioactivity showed that TNF-a treatment
suppressed control and insulin-stimulated glucose uptake by 28 and
47%, respectively. Results were corrected for protein content by a
BCA protein assay kit. (*P \ 0.05 10 ng/ml TNF-a vs. control)
Mol Cell Biochem (2011) 353:305313 309

insulin-stimulated glucose uptake in insulin-resistant

3T3-L1 adipocytes.

Apelin improved glucose uptake in insulin-resistant

3T3-L1 adipocytes through the PI3K/Akt pathway

To examine whether the PI3K/Akt pathway was responsible

for the apelin-mediated improvements in glucose uptake,
the cells were pre-treated with 100 nM wortmannin, a
PI3K-specific inhibitor, for 30 min before apelin treatment.
Wortmannin pre-incubation significantly decreased apelin-
induced glucose uptake as well as insulin-stimulated
glucose uptake in insulin-resistant 3T3-L1 adipocytes
(P \ 0.05 vs. 100 nM wortmannin, P \ 0.01 vs. 100 nM
wortmannin; Fig. 4a). We also determined the activation of
phosphorylated Akt (Ser 473) in the presence or absence of
wortmannin. As expected, insulin stimulation resulted in
augmentation of pAkt (Ser 473). Interestingly, apelin also
significantly enhanced Akt phosphorylation (Ser 473). In
regards to enhancement of Akt phosphorylation, apelin was
almost as potent as insulin. Pre-incubation with wortmannin
blocked the enhancement of phosphorylated Akt (Ser 473)
(Fig. 4b).
Apelin promotes GLUT4 translocation
from the cytoplasm to the plasma membrane
We observed plasma membrane (PM) localization of
GLUT4 using an immunofluorescence confocal microscopy
technique to determine whether the observed improve-
ment in glucose uptake was due to increased GLUT4
Fig. 4 Apelin improved glucose uptake in insulin-resistant 3T3-L1
adipocytes through the PI3K/Akt pathway. Cells were incubated with
100 nM wortmannin (wort) for 30 min before apelin incubation.
a Radioactivity showed that treatment with 1,000 nM apelin for
60 min increased glucose uptake. Wortmannin, a PI3K-specific
inhibitor, blocked both the beneficial effects of apelin and insulin.
(*P \ 0.05 vs. 100 nM wort, **P \ 0.01 vs. 100 nM wort).
b Western blotting analysis confirmed that apelin up-regulated
phosphorylated Akt (Ser 473), which was consistent with the changes
in glucose uptake
translocation. Apelin and insulin increased the translocation
of GLUT4 from the cytoplasm to the plasma membrane in
insulin-resistant 3T3-L1 adipocytes (Fig. 5a). In regards to
GLUT4 translocation, apelin was almost as potent as that of
insulin. Furthermore, apelin pre-incubation significantly
increased insulin-stimulated GLUT4 translocation (Fig. 5a).

Fig. 3 Apelin enhanced

glucose uptake in insulin-
resistant 3T3-L1 adipocytes.
a Apelin exerted an initial
significant effect on glucose
uptake at 100 nM and a
maximal effect at 1,000 nM
(*P \ 0.05 vs. control;
**P \ 0.01 vs. control).
b glucose uptake was
maximally augmented at 60 min
after incubation (**P \ 0.01 vs.
control) and still effective at
120 min (*P \ 0.05 vs.
control); c The pre-incubation
of 1,000 nM apelin had a
greater effect on insulin-induced
glucose uptake than that of
100 nM apelin (*P \ 0.05
1,000 nM apelin vs. 100 nM
apelin). Results were corrected
for protein content by a BCA
protein assay kit

123
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Consistent with immunofluorescence images, western blot-
ting analysis of GLUT4 in the plasma membrane fraction
also confirmed that apelin and insulin could increase GLUT4
expression on the plasma membrane (Fig. 5b).
Apelin mediates the mRNA expression of inflammatory
factors in insulin-resistant 3T3-L1 adipocytes
We investigated the effects of apelin on the mRNA
expression of the pro-inflammatory factor IL-6 in insulin-
resistant 3T3-L1 adipocytes. Cells were treated with or
without 1,000 nM apelin for 60 min, and total RNA was
extracted using TRIzol reagent. Real-time PCR showed
that mRNA expression of IL-6 was significantly down-
regulated by apelin (P \ 0.01; Fig. 6a). In addition, apelin
significantly up-regulated mRNA expression of the anti-
inflammatory adipokine adiponectin (P \ 0.05; Fig. 6b).
These results suggest that apelin could improve the chronic
low-grade inflammatory state of insulin resistance.
Discussion
Apelin, which was first isolated from bovine stomach
extracts [3], is the endogenous ligand for the orphaned G
protein-coupled receptor APJ. Apelin is considered to be an
adipokine because it is mainly produced and secreted by
adipocytes [4]. A number of previous studies have shown
that this novel peptide exerts various biological effects [12]
on several systems and organs, including the central ner-
vous system [13], heart [1416], lung [17], liver [1820],
and kidney [21]. Apelin has also been shown to be closely
related to insulin resistance [4, 8] and glucose metabolism
[22], but the underlying mechanism of how apelin affects
insulin resistance is poorly understood.
Fig. 5 Apelin increased
GLUT4 translocation from the
cytoplasm to the plasma
membrane. a Confocal
microscopy images showed that
apelin enhanced the
translocation of cytoplasmic
GLUT4 to the plasma
membrane. In addition, apelin
pre-incubation further enhanced
insulin-simulated GLUT4
translocation. b Western
blotting analysis of the plasma
membrane (PM) fraction also
demonstrated that GLUT4
protein was up-regulated by
apelin
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Mol Cell Biochem (2011) 353:305313
Diabetes and metabolic syndromes have become a glo-
bal epidemic, specifically type 2 diabetes, which is char-
acterized by insulin resistance. In insulin-resistant
condition, normal insulin levels are unable to produce the
desired response in insulin target tissues and cells,
including fat, muscle, and liver cells. Because insulin
resistance is the major abnormality in type 2 diabetes [23],
the search for a treatment has focused on identifying
insulin-sensitizing agents to counteract insulin resistance.
Adipocytes are one of the major peripheral targets of
insulin and have been commonly used as a model for
studying insulin resistance in vitro. It is commonly
accepted that obesity, especially visceral adiposity, is
negatively correlated with insulin sensitivity. Adipose tis-
sue not only plays a vital role in energy homeostasis but
also acts as an endocrine organ by secreting adipokines and
cytokines, which regulate whole-body metabolism. Thus,
adipose tissue has been recognized as a promising thera-
peutic target for drug development. This study was
undertaken to explore a potential mechanism for apelin in
treating type 2 diabetes using insulin-resistant 3T3-L1
adipocytes, which mimic the pathophysiological abnor-
mality of type 2 diabetes. Elucidating a molecular role for
apelin in insulin resistance is extremely important because
this information can help us to determine whether apelin
can be used as a therapeutic agent for type 2 diabetes.
Several mechanisms, such as hyperglycemia [24, 25],
hyperinsulinism [26], high levels of free fatty acids (FFA)
[27], dexamethasone [28], and TNF-a [2830], have been
characterized for inducing insulin resistance at the cellular
level. In our experiment, we choose TNF-a to simulate the
low-grade inflammatory state in systemic insulin resis-
tance. For In vitro TNF-a-induced insulin resistance in
adipocytes, the concentration of TNF-a vary from 5 to
20 ng/ml, while the treatment time vary from 6 to 48 h
Mol Cell Biochem (2011) 353:305313
Fig. 6 Apelin modulates inflammatory responses in insulin-resistant
3T3-L1 adipocytes. a Real-time PCR showed that mRNA expression
of the pro-inflammatory factor IL-6 was markedly decreased by
[3135]. Among these treatment dose and time, 10 ng/ml
TNF-a for 24 h was most commonly used for inducing
insulin resistance in 3T3-L1 adipocytes [34, 35]. We also
observed whether apelin could affect the mRNA expression
of other adipokines or cytokines. Insulin-stimulated glu-
cose uptake, which was defined as insulin sensitivity, is
considered to be the gold standard for evaluating insulin
resistance at the cellular level. Cells with a significant
reduction in insulin-stimulated glucose uptake have com-
monly been accepted to be in an insulin-resistant state. In
this study, an insulin-resistant cell model was successfully
constructed by treatment with TNF-a, which significantly
reduced insulin-stimulated glucose uptake by 47%.
Through dosage and time course experiments, we
determined that a 60 min treatment with 1,000 nM apelin
optimized glucose uptake. High concentrations of apelin
(104 and 105 nM) showed a decreased effect on glucose
uptake compared to low concentrations of apelin. Addi-
tionally, 100 and 1,000 nM apelin for 60 min both mark-
edly increased insulin-stimulated glucose uptake and
1,000 nM apelin had a greater effect. After 120 min, the
improvement in glucose uptake declined and was no longer
significant compared to basal levels. To understand the
mechanism underlying the apelin-mediated improvement
in glucose uptake, we examined whether this effect was
occurring through the PI3K/Akt pathway.
In the insulin signaling cascade for glucose uptake,
activation of the insulin receptor b subunit results in sub-
sequent activation of the downstream signaling molecule
IRS-1. This leads to phosphorylation of Akt, which is
downstream of PI3K [36]. The phosphorylation of Akt can
induce the translocation of GLUT4 from the cytoplasm to
the plasma membrane, which promotes the transport of
glucose into the cell [37]. By pre-incubating cells with the
specific PI3K inhibitor wortmannin, we demonstrated that
the improvement of glucose uptake in insulin-resistant
3T3-L1 adipocytes was dependent on the PI3K/Akt
pathway.
In addition, GLUT4, which usually resides in the intra-
cellular vesicles in its basal state, plays a major role in
311
apelin (**P \ 0.01 vs. untreated). b Apelin significantly increased
mRNA expression of the anti-inflammatory factor adiponectin
(*P \ 0.05 vs. untreated)
glucose transport in muscle and adipocytes. Studies have
shown that insulin stimulates glucose uptake primarily by
inducing GLUT4 translocation to the plasma membrane
[37]. Immunocytochemical studies identified that apelin
could also induce the translocation of GLUT4 to the
plasma membrane, and apelin was almost as potent as
insulin. Apelin also markedly enhanced insulin-mediated
GLUT4 translocation. These results suggest that apelin
could be used as an insulin-sensitizing agent.
Recent reports suggest a close link between insulin
resistance and chronic inflammation [30, 38, 39]. As an
endocrine organ, adipose tissue secretes a variety of adi-
pokines and cytokines that exert regulatory functions in
inflammatory responses and energy metabolism though
autocrine or paracrine mechanisms. However, the balance
between pro-inflammatory and anti-inflammatory factors is
altered in insulin resistance due to the chronic low-grade
inflammatory state. In this study, we also examined whe-
ther apelin could play a role in mediating inflammatory
responses in insulin-resistant adipocytes. Similar to the
commonly used insulin-sensitizing agent pioglitazone
[40], apelin treatment significantly enhanced the mRNA
expression level of the commonly considered insulin-sen-
sitizer [41, 42] and anti-inflammatory adipokine [4345],
adiponectin. In addition, apelin markedly reduced the gene
expression of the pro-inflammatory cytokine IL-6. These
results indicated that apelin has anti-inflammatory proper-
ties in insulin-resistant 3T3-L1 adipocytes.
In conclusion, our data demonstrated that apelin stimu-
lates glucose uptake by inducing GLUT4 translocation in a
PI3K/Akt-dependent manner. In addition, apelin mediates
the inflammatory response in insulin-resistant 3T3-L1
adipocytes. Our findings suggest that apelin may be a
promising therapeutic agent for type 2 diabetes because it
improves glucose uptake and mediates the inflammatory
response. However, further studies to investigate both the
protein and mRNA level of the anti-insulin-resistant effect
of apelin, and in vivo studies of apelin in insulin-resistant
animals are necessary to elucidate its pharmaceutical
potential.
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Acknowledgments This study was supported by National Nature
Science Foundation of China (F.C., NO. 81090274, NO. 30970845;
C.W. NO. 30900611) and Xijing Research Boosting Program (F.C.,
NO. XJZT08Z04).
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