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Introduction
Adenosine monophosphate-activated protein kinase
(AMPK), which serves as a metabolic master switch in
response to alterations in cellular energy charge, has been
reported to be implicated in the regulation of glucose and
lipid homeostasis and insulin
sensitivity[1_3]. It is activated by diverse stimuli that increase the AMP-to-ATP ratio (eg
exercise and hypoxia) as well as by hormones (eg adiponectin
and leptin)[4] and other antidiabetic agents (eg metformin
and Thiazolidinediones)[5]. In peripheral tissues, AMPK
activation by 5-amino-4-imidazolecarboxamide ribonucleotide
(AICAR) or other activators of AMPK may be a selective
tool to achieve normoglycemia by stimulating glucose
uptake[6_8] in muscles and inhibiting hepatic glucose
production[9,10]. In pancreatic β-cells, AMPK may modulate insulin
secretion response to different concentration of glucose in
INS-1 cells and isolated islets[11_13]. However,
the role of AMPK in β-cells under chronic lipotoxic conditions is poorly
understood.
Fenofibrate is an activator of the peroxisome
proliferator-activated receptor-α (PPARa), which has been reported to
upregulate genes of fatty acid b-oxidation pathways in
various tissues[14,15]. Clinically, fenofibrate has been used for
the treatment of dyslipidemia, mainly due to its ability to
lower triglyceride levels, raise high-density lipoprotein levels,
and decrease the levels of small, dense, low-density
lipoprotein particles[16]. In otsuka long evans tokushima fatty
(OLETF) rats, fenofibrate treatment may reduce adiposity,
improve peripheral insulin action, and exert beneficial effects
on pancreatic β-cells[17]. In primary human pancreatic islets,
fenofibrate may prevent the fatty acid-induced impairment
of glucose-stimulated insulin secretion (GSIS), apoptosis, and
triglyceride accumulation[18]. In addition,
PPARa activation was involved in insulin secretion in pancreatic
β-cells[16,19_22], but whether and why the action is protective is still disputed.
In the present study, we aimed to investigate the effect
of palmitate on AMPK expression and GSIS in isolated rat
pancreatic islets and INS-1 β-cells, as well as the effect of
fenofibrate on AMPK and GSIS in cells treated with
palmitate.
Materials and methods
Rat pancreatic islet isolation and
treatment Pancreatic islets were isolated from male Wistar
rats weighing 230_275 g with collagenase solution followed by
stationary in vitro digestion as
previously reported[23]. The
use of animals and the experimental protocols to which they
were subjected were approved by the Institutional Animal
Care and Use Committee of Shandong University (Jinan,
China). Freshly isolated islets were then transferred to
24-well plates (10 islets per well) for the secretory
experiment or a culture dish of 6 cm diameter (200 islets per
dish) for Western blotting. They were cultured for 24 h in
RPMI-1640 medium (Invitrogen, Grand island, NY, USA)
containing 11.1 mmol/L glucose supplemented with 10%
(v/v) fetal bovine serum (FBS; Invitrogen, USA), 100
IU/mL penicillin, and 100 µg/mL streptomycin in a
humidified atmosphere (5% CO2 and 95% air) at 37 °C. Before
commencing the experiment (24 h after seeding), the
medium was replaced with freshly prepared RPMI-1640
containing 11.1 mmol/L glucose and supplemented with either
bovine serum albumin (BSA; Sigma, St Louis, MO, USA)
alone or BSA coupled to palmitate (0.2 or 0.4
mmol/L)[24],in the presence or absence of 5 µmol/L fenofibrate (a gift
from Laboratories Fournier SA, Rue de Pres Potets,
Fontaine-les-Dijon, France) for 48 h. The BSA-coupled
palmitate was made in a molar ratio of 5:1.
Cell culture and treatment The rat insulinoma cell
line INS-1 (at passages below 40) were grown in a
monolayer as described previously[25] in the RPMI-1640 medium
containing 11.1 mmol/L glucose supplemented with 10
mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), 10% FBS, 1 mmol/L sodium pyruvate, 2
mmol/L L-glutamine, 50 µmol/L β-mercaptoethanol, 100
IU/mL penicillin, and 100 µg/mL streptomycin in a
humidified atmosphere (5% CO2 and 95% air) at 37 °C. The cells
were seeded at 2×105 per well in 1 mL medium in a
24-well plate for secretory experiment, and at
1×106 per well in a 6-well plate for the RNA detection, and at
4×106 cells in a culture dish of 10 cm diameter for Western blotting.
When the INS-1 cells were 80%_90% confluent, the
medium was replaced with fresh medium and the treatment
was the same as that of the isolated islets.
RNA isolation and cDNA synthesis The cultured cells
or islets were harvested in TRIzol, and RNA was extracted
according to a protocol of TRIzol isolation (Invitrogen,
USA). The extracted RNA was resuspended in diethyl
pyrocar-bonate-treated water and quantified by a DU640
nuclearic acid analyzer (Beckman, Fullerton, CA, USA). In
total, 2 µg RNA was reverse-transcribed (RT) into
the cDNA in a final reaction solution of 20 µL containing 5 mmol/L
MgCl2, 2 µL 10×RT buffer, 1 mmol/L dNTP, 20 U RNase
inhibitor, 5 U Avian myeloblastosis virus (AMV) reverse
transcriptase, 2.5 µmol oligo(dT) primer, and
2 µg RNA and RNase-free dH2O using a RNA PCR kit (TaKaRa, Otsu,
Shiga, Japan). The mixtures were heated as per the
following conditions: 10 min at 30 °C, 30 min at 42 °C, 5 min at
99 °C, and 5 min at 5 °C. The extracted cDNA was used at
real-time PCR or stored at -80 °C.
Real-time PCR Quantitative 3-step real time PCR was
performed using Quantitect SYBR green kit (Qiagen,
Hilden, Germany) following the manufacturer's
instructions on an ABI 7700 prism real-time PCR instrument and
software (Applied Biosystems, Branchburg, NJ, USA). The
reaction volume was 25 µL and contained 12.5 µL
2×QuantiTect SYBR green PCR master mix, 0.5 µmol/L primers, and 100 ng
cDNA and RNase-free water. The primers used for the PCR
are detailed in Table 1. The reaction conditions were as
follows: 1 cycle for 2 min at 50 °C, 1 cycle for 15 min at 95 °C,
40 cycles for 15 s at 95 oC, 30 s at 60 oC, and 30 s at 72 oC. All quantifications were performed with rat GAPDH as an
internal standard. The PCR products were visualized with gel
electrophoresis to confirm a single product of the correct
size (100 bp).
Protein analysis by Western blotting and enhanced
chemiluminescence (ECL) detection The cultured islets and
INS-1 cells were washed twice with ice-cold
phosphate-buffered solution (PBS) and placed immediately in Radio
immunoprecipitation assay (RIPA) lysis buffer containing 1×PBS,
1% nonylphenylpolyethylenglycol P-40 (NP-40), 0.1%
SDS, 5 mmol/L EDTA, 0.5% sodium deoxycholate, 1
mmol/L sodium orthovanadate, and 1 mmol/L phenylmethyl sulfonylfluoride.
The lysates were gently mixed for 10 min on ice and then
centrifuged at 10 000×g for 10 min at 4 °C. The protein
concentration of the extracts was determined according to the
bicinchoninic acid (BCA) protein assay method using BSA
as the standard. Then 60 µg of protein extracts were
separated by 10% SDS-PAGE and electroblotted onto
nitrocellulose membranes. The membranes were blocked with 5%
non-fat milk in TBST (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1%
Tween 20) for 1 h and then incubated with the specific
primary antibody of total AMPKa (T-AMPKa; at 1:1000 dilution,
cell signaling, Danvers, MA, USA), phosphorylated
AMPKα (P-AMPKα; cell signaling at 1:500 dilution), and
phosphorylated acetyl coenzyme A carboxylase (P-ACC; cell signaling
at 1:1000 dilution) overnight at 4 °C. After incubation with
the relative second antibody, immune complexes were
detected using the enhanced chemiluminescence (ECL) method
(Amersham Biosciences, Little Chalfont, Buckinghamshire,
United Kingdom); immunoreactive bands were quantified
using Alphaimager 2200 (Alpha Innotech, San Leandro, CA,
USA). Values were corrected with the absorbency of the
internal control (β-actin).
Insulin secretion The cultured cells or islets were
washed and pre-incubated for 30 min in Krebs-Ringer
bicarbonate buffer (KRB) medium with the following
composition: 143 mmol/L Na+, 5.8 mmol/L
K+, 2.5 mmol/L Ca2+, 1.2
mmol/L Mg2+, 124.1 mmol/L
Cl_, 1.2 mmol/L SO4 3-, and 25 mmol/L
CO32- (pH 7.4), supplemented with 10 mmol/L HEPES,
0.2% BSA, and 3 mmol/L glucose. Upon completion of the
incubation, the buffer was removed completely and replaced
with fresh KRB containing 20 mmol/L glucose for an
additional 20 min incubation. After 20 min incubation, the media
were collected for measuring GSIS using an insulin
radioimmunoassay (RIA) kit (Beijing Atom HighTech, Beijing,
China). For the total protein extraction, the cells were
homogenized in RIPA lysis buffer (Shenneng Bo Cai, Shanghai,
China) containing 1×PBS, 1% NP-40, 0.1% SDS, 5
mmol/L EDTA, 0.5% sodium deoxycholate, and 1 mmol/L sodium
orthovanadate. The protein concentration was determined
by BCA assay (Bio-Rad, Hercules, CA, USA). The insulin
level of the medium was normalized to its cellular protein
content.
Data analysis All of the experiments were repeated at
least 3 times. Values are given as mean±SD. Data
were analyzed using one-way ANOVA. Significance was
established at P<0.05.
Results
Expression of AMPKa1 and AMPKα2 in isolated rat
islets and INS-1 cells The catalytic subunit of AMPK is the
α subunit, which consists of 2 isoforms: α1 and α2.
Real-time results showed that in the islets (Figure 1A) and INS-1
cells (Figure 1B), the mRNA expression of AMPKa1 was
significantly higher than that of AMPKα2. The
AMPKα1 expression was 4.37-fold in the islets and 3.89-fold in the
INS-1 cells over that of AMPKα2, suggesting that
AMPKα1 was the main isoform of AMPKa in the β-cells. Our result is
consistent with a previous study of pancreatic
β-cells[26].
Inhibition effect of chronic exposure of b-cells to
palmitate on AMPKa expression and activity To determine the
effect of chronic exposure of b-cells to palmitate on
AMPKa expression and activity, we cultured isolated rat pancreatic
islets in RPMI-1640 medium containing 11.1 mmol/L glucose
supplemented with and without 0.2 and 0.4 mmol/L palmitate
for 48 h. The protein expression levels of
T-AMPKa, P-AMPKa, and P-ACC were detected by Western blotting.
The results demonstrated (Figure 2) that chronic exposure
of isolated rat pancreatic islets to palmitate induced a
significant decrease in P-AMPKa expression by 65% at 0.2
mmol/L palmitate and by 73% at 0.4 mmol/L palmitate treatment
(P<0.05) in a dose-dependent manner. Accordingly, the
expression of P-ACC, a downstream signal of AMPK, was also
reduced (P<0.05), indicating that chronic palmitate exposure
may inhibit the expression and activity of AMPKa.
Amelioration effect of fenofibrate on AMPKα
expression and activity in β-cells chronically exposed to
palmitate To observe the effect of fenofibrate on
AMPKa expression and activity in β-cells chronically exposed to
palmitate, we cultured the INS-1 cells in RPMI-1640
medium supplemented with and without 0.2 mmol/L palmitate
in the presence or absence of 5 µmol/L fenofibrate for
48 h. Then the mRNA levels of the AMPKα1 and α2 isoforms
were measured by real-time PCR; the protein levels of
T-AMPKα, P-AMPKα, and P-ACC and were detected by
Western blotting. The results (Figure 3A) showed that in the
palmitate-treated INS-1 cells, AMPKα1 mRNA expression
decreased by 22% (P<0.05) while the
AMPKα2 mRNA level was enhanced compared with the control. Compared with
the palmitate-treated cells, the AMPKα1 mRNA level was
enhanced by 26% (P<0.05) while there was no change to the
cells treated with palmitate and fenofibrate together.
Furthermore, the protein expression (Figure 3B) of
T-AMPKα decreased by 47% in the palmitate-treated cells.
Accordingly, the protein expressions of P-AMPKα and
P-ACC were reduced respectively by 34% and 81%
(P<0.05) in the palmitate-treated cells, respectively. Compared with the
palmitate-treated cells, the cells treated with fenofibrate and
palmitate together showed a remarkable increase in the
protein expression of T-AMPKα by 40%, P-AMPKα by 68%,
and P-ACC by 68% (P<0.05).
Effect of chronic exposure of α-cells to palmitate and
fenofibrate on glucose-stimulated insulin secretion Insulin secretion was measured in the isolated islets and INS-1 cells
by RIA induced by 20 mmol/L glucose as GSIS. As shown in
Figure 4, GSIS was markedly reduced by 30% in the isolated
islets pretreated for 48 h with 0.2 mmol/L palmitate.
Accordingly, in the palmitate-treated INS-1 cells, GSIS
decreased by 40%, implying the impairment role of palmitate on
insulin secretion. Compared with the palmitate-treated islets
and INS-1 cells, GSIS was restored to normal in the islets and
INS-1 cells pretreated with palmitate and fenofibrate,
indicating an improvement effect of fenofibrate on insulin
secretion.
Discussion
In the present study, we demonstrated that chronic
exposure of rat pancreatic β-cells to elevated palmitate
reduced the expression of AMPKα and activity and impaired
GSIS. Fenofibrate may potentiate GSIS associated with
enhanced AMPKa expression.
AMPK is composed of 3 subunits: a, b, and g. Among
them, the a subunit is the catalytic subunit, which contains
mainly 2 isoforms: α1 and α2. In our study, we first
detected the mRNA levels of AMPKα1 and AMPKα2. The
results revealed that not only AMPKα1 is more abundant
than AMPKα2 in the isolated islets and INS-1 cells, but in
the effect on the expression of AMPKα, the α1 isoform is
predominant. Our result is consistent with the study of da
Silva Xavier et al[26], in which an exclusive cytosolic
localized for the α1 isoform and weak staining was present for
α2 both in the cytosol and nucleus.
Next, we observed the effect of palmitate on
AMPKα expression and activity as well as GSIS. Our study found
that chronic exposure of islets and INS-1 cells to
palmitate decreased the expression and activity of
AMPKα and inhibited GSIS, indicating a possible role of
AMPKα in b-cell lipotoxicity. In accordance with our findings, Liu et al[27] demonstrated that
AMPKα expression and activity was decreased in the skeletal muscles of rats on a high-fat diet. The
decrease of AMPK activity may activate ACC via
dephos-phorylation, leading to an increase in the concentration of
the product of ACC, malony-CoA, and the reduction in the
carnitine palmitoyltransferase 1 expression to impair cellular
fatty acid oxidation and accordingly attenuate insulin
secretion. This suggests that the inhibition of AMPK
expression and activity could play a role in insulin release.
However, a recent study[28] found that chronic exposure of
MIN6 cells to elevated palmitate for 24 h showed a sustained
phosphorylation of AMPK. The results of the study appear
to contradict those of ours, but can be explained by the
difference in culture circumstances, such as different
glucose concentrations and insufficient palmitate treatment time.
Our results, at least partly, suggest that the decrease of
AMPKα may be one of the mechanisms of β-cell lipotoxicity.
As already known, fenofibrate is a PPARα synthesis
agonist. In several insulin-resistant rodent models,
the administration of the PPARα agonist was reported to improve
β-cell function[17]. Moreover, under conditions of
lipotoxicity induced by chronic fatty acid exposure, different
PPARα agonists significantly improved insulin secretion and the
stimulation index in primary human
islets[29,30]. In clinical experiments, fenofibrate can improve insulin secretion in
hypertriglyceridemic individuals[31]. In addition, the
activation of PPARα by the overexpression of
PPARα/retinoid X receptor a potentiated GSIS in the
rat islets and INS-1E rat β-cell
line[19]. Our study supports the above view that
PPARα activation can improve insulin secretion under conditions of
lipotoxicity and demonstrates that fenofibrate restored GSIS
impaired by palmitate in INS-1 cells, implying a beneficial
role in β-cell function under pathological conditions of
lipotoxicity.
However, we also observed that the improvement of
insulin secretion is accompanied by AMPKα expression
enhancement, implying a possible relationship between
fenofibrate improving insulin secretion and AMPKα activation. A similar relationship was found in human
umbilical vein endothelial cells by Murakami et
al[32], but they demonstrated that fenofibrate activating AMPK was
unrelated to the effect of PPARα. Although in our study we did
not verify whether the effect of fenofibrate on
AMPKα was related to PPARα, it at least partly provides evidence that
the promotion of fenofibrate on b-cell insulin secretion is
associated with the expression of AMPKα. In addition, our
study suggests a beneficial role of AMPKα activation in
the insulin secretion of INS-1 cells under lipotoxic conditions.
Some studies[27,33] have reported that
AMPKα activation stimulated by metformin, AICAR,
thiazolidinediones, leptin and so on may obviously ameliorate high-fat-induced
insulin resistance and improve b-cell function. Diraison et al[34] proved that AICAR, an agonist of AMPK, can reverse in
part the effects of sterol regulatory element binding
protein-1c (SREBP1c) overexpression on triacylglycerol
accumulation in transduction with SREBP1c of primary rat islets,
suggesting that AICAR may act under conditions of
β-cell lipid loading to favor the preservation of
β-cell function. Although some studies suggest the activation of AMPK by AICAR
(or by any other means) to exert deleterious or inhibitory
effects on β-cells[26,35], the potential interpretation of these
studies is the difference in the glucose concentrations and
relative action time of AICAR.
In conclusion, our present study demonstrates that the
α1 isoform expression, AMPKα expression and activity,
and GSIS decreased in rat β-cells under palmitate-induced
lipotoxic conditions. This disturbance could be
ameliorated by fenofibrate, which is associated with the enhanced
expression of AMPKα. Thus, our results suggest the
effect of AMPKα on insulin secretion in β-cells treated with
palmitate and the novel role of fenofibrate in improving
insulin secretion is associated with AMPKα activation.
Acknowledgements
The authors thank Professor Xiao HAN for providing us
with the INS-1 cells, and also the teachers at the Science
Center of Shandong Provincial Hospital (Ji-nan, China) for
excellent technical assistance.
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