AMP-Activated Protein Kinase–Deficient Mice Are Resistant to the Metabolic Effects of Resveratrol

Article (PDF Available)inDiabetes 59(3):554-63 · November 2009with44 Reads
DOI: 10.2337/db09-0482 · Source: PubMed
Abstract
Resveratrol, a natural polyphenolic compound that is found in grapes and red wine, increases metabolic rate, insulin sensitivity, mitochondrial biogenesis, and physical endurance and reduces fat accumulation in mice. Although it is thought that resveratrol targets Sirt1, this is controversial because resveratrol also activates 5' AMP-activated protein kinase (AMPK), which also regulates insulin sensitivity and mitochondrial biogenesis. Here, we use mice deficient in AMPKalpha1 or -alpha2 to determine whether the metabolic effects of resveratrol are mediated by AMPK. Mice deficient in the catalytic subunit of AMPK (alpha1 or alpha2) and wild-type mice were fed a high-fat diet or high-fat diet supplemented with resveratrol for 13 weeks. Body weight was recorded biweekly and metabolic parameters were measured. We also used mouse embryonic fibroblasts deficient in AMPK to study the role of AMPK in resveratrol-mediated effects in vitro. Resveratrol increased the metabolic rate and reduced fat mass in wild-type mice but not in AMPKalpha1(-/-) mice. In the absence of either AMPKalpha1 or -alpha2, resveratrol failed to increase insulin sensitivity, glucose tolerance, mitochondrial biogenesis, and physical endurance. Consistent with this, the expression of genes important for mitochondrial biogenesis was not induced by resveratrol in AMPK-deficient mice. In addition, resveratrol increased the NAD-to-NADH ratio in an AMPK-dependent manner, which may explain how resveratrol may activate Sirt1 indirectly. We conclude that AMPK, which was thought to be an off-target hit of resveratrol, is the central target for the metabolic effects of resveratrol.
AMP-Activated Protein Kinase–Deficient Mice Are
Resistant to the Metabolic Effects of Resveratrol
Jee-Hyun Um,
1
Sung-Jun Park,
1
Hyeog Kang,
1
Shutong Yang,
1
Marc Foretz,
2,3
Michael W. McBurney,
4
Myung K. Kim,
1
Benoit Viollet,
2,3
and Jay H. Chung
1
OBJECTIVE—Resveratrol, a natural polyphenolic compound
that is found in grapes and red wine, increases metabolic rate,
insulin sensitivity, mitochondrial biogenesis, and physical endur-
ance and reduces fat accumulation in mice. Although it is thought
that resveratrol targets Sirt1, this is controversial because res-
veratrol also activates 5 AMP-activated protein kinase (AMPK),
which also regulates insulin sensitivity and mitochondrial bio-
genesis. Here, we use mice deficient in AMPK1or-2to
determine whether the metabolic effects of resveratrol are me-
diated by AMPK.
RESEARCH DESIGN AND METHODS—Mice deficient in the
catalytic subunit of AMPK (1or2) and wild-type mice were
fed a high-fat diet or high-fat diet supplemented with resveratrol
for 13 weeks. Body weight was recorded biweekly and metabolic
parameters were measured. We also used mouse embryonic
fibroblasts deficient in AMPK to study the role of AMPK in
resveratrol-mediated effects in vitro.
RESULTS—Resveratrol increased the metabolic rate and re-
duced fat mass in wild-type mice but not in AMPK1
/
mice. In
the absence of either AMPK1or-2, resveratrol failed to
increase insulin sensitivity, glucose tolerance, mitochondrial
biogenesis, and physical endurance. Consistent with this, the
expression of genes important for mitochondrial biogenesis was
not induced by resveratrol in AMPK-deficient mice. In addition,
resveratrol increased the NAD-to-NADH ratio in an AMPK-
dependent manner, which may explain how resveratrol may
activate Sirt1 indirectly.
CONCLUSIONS—We conclude that AMPK, which was thought
to be an off-target hit of resveratrol, is the central target for the
metabolic effects of resveratrol. Diabetes 59:554–563, 2010
R
esveratrol is a natural polyphenolic compound
found in grapes and red wine and has been
shown to extend lifespan in many organisms,
including yeast (1), flies (2), and worms (2– 4).
Resveratrol extended lifespan in mice on a high-fat diet (5)
but not a regular diet (6). In mice with diet-induced
obesity, resveratrol reduced fat accumulation and im-
proved glucose tolerance and insulin sensitivity (5,7). In
addition, resveratrol increases mitochondrial biogenesis
and physical endurance. A resveratrol derivative with
higher bioavailability is being tested in clinical trials for
treating type 2 diabetes.
Given its potential as a lead molecule for the develop-
ment of drugs that treat metabolic disorders, it is critical to
understand how resveratrol modulates metabolism. It is
widely accepted that Sirt1, the founding member of the
Sirtuin family (8) of NAD-dependent deacetylase, is the
target of resveratrol (1,5,7). However, whether the puta-
tive Sirt1 activators such as resveratrol actually target
Sirt1 in vivo is controversial because resveratrol increases
Sirt1 activity in vitro only if the substrate is modified with
a fluorescent tag (9,10). Resveratrol appears to increase
the deacetylation rate by enhancing the affinity of Sirt1 for
fluorescent-tagged peptides.
Resveratrol also has a number of indirect effects (11),
including stimulation of 5 AMP-activated protein kinase
(AMPK) (5,12,13). AMPK is a heterotrimeric protein con-
sisting of an -catalytic subunit and two regulatory sub-
units, and (14). AMPK is a fuel-sensing kinase, which
is activated by ATP-depleting conditions such as physical
exercise, ischemia, and glucose deprivation. The catalytic
subunit of AMPK has two isoforms, 1 and 2, which have
different tissue expression patterns. Muscle expresses
predominantly the 2-isoform (15), whereas fat and brain
express predominantly the 1 isoform (16,17), and liver
expresses both 1 and 2 isoforms (18). AMPK1 and
AMPK2 knockout mice are viable, but AMPK1/2 dou-
ble knockout causes embryonic lethality. Like resveratrol,
activation of AMPK has been shown to reduce fat accu-
mulation and increase glucose tolerance, insulin sensitiv-
ity, mitochondrial biogenesis, and physical endurance
(19 –23). Therefore, it is possible that the metabolic effects
of resveratrol are mediated by AMPK. Supporting this
possibility, resveratrol-mediated extension of lifespan in
worms requires AMPK (24).
Resveratrol may activate AMPK in several different
ways. Resveratrol, as well as other polyphenols, can
reduce ATP levels by inhibiting ATP synthase (25). Res-
veratrol can also activate AMPK without altering the
AMP-to-ATP ratio. Dasgupta et al. (12) showed that, at
lower doses, resveratrol can activate AMPK through a
Sirt1-independent manner. Interestingly, Hou et al. (26)
and Lan et al. (27) reported that the activity of liver kinase
B (LKB)-1, one of the AMPK kinases that is important for
AMPK activity, is activated by resveratrol in a Sirt1-
dependent manner.
RESEARCH DESIGN AND METHODS
Mice and diet. Wild-type C57BL/6J mice were originally purchased from The
Jackson Laboratory. AMPK2
/
(22) and AMPK1
/
(28) mice were back
-
From the
1
Laboratory of Biochemical Genetics, National Heart, Lung, and
Blood Institute, National Institutes of Health, Bethesda, Maryland; the
2
Institut Cochin, Universite´ Paris Descartes, Centre National de la Recher
-
che Scientifique (UMR 8104), Paris, France; the
3
National de la Sante´etde
la Recherche Me´dicale, Paris, France; and the
4
Center for Cancer Thera
-
peutics, Ottawa Health Research Institute, Ottawa, Ontario, Canada.
Corresponding author: Jay H. Chung, chungj@nhlbi.nih.gov.
Received 1 April 2009 and accepted 30 October 2009. Published ahead of
print at http://diabetes.diabetesjournals.org on 23 November 2009. DOI:
10.2337/db09-0482.
J.-H.U. and S.-J.P. contributed equally to this article.
© 2010 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
See accompanying commentary, p. 551.
ORIGINAL ARTICLE
554 DIABETES, VOL. 59, MARCH 2010 diabetes.diabetesjournals.org
crossed to C57BL/6J for at least six generations before this study. Four- to
6-week-old male mice were housed with a 12-h light-dark cycle (light on 6:00
A.M. to 6:00 P.M.) and fed a high-fat diet (40% calories from fat; Bio-serv) or a
high-fat diet supplemented with resveratrol (400 mg kg
1
day
1
; Orchid
Chemicals and Pharmaceuticals) for 12 weeks as previously described (7). All
experiments were approved by the National Heart, Lung, and Blood Institute
Animal Care and Use Committee.
Metabolic measurements. Body weight and caloric intake were monitored
biweekly. Plasma glucose was measured by using a glucometer (Ascensia).
For the glucose tolerance test, mice were fasted for 16 h, and 1 mg/g glucose
was injected intraperitoneally. Blood glucose was measured at 0, 15, 30, 45, 60,
and 90 min after injection. For the insulin tolerance test, mice were fasted for
4 h before they were injected with 0.4 units/g i.p. of human insulin (Sigma).
Prior to the endurance test, the mice were accustomed and trained by running
on an Exer-3/6 mouse treadmill (Columbus Instruments) at 10 m/min for 5 min
for 3 days before endurance testing. For the endurance test, the treadmill was
set at a 15
o
incline, and the speed was increased in a stepwise fashion (10
m/min for 10 min followed by 14 m/min for 5 min and then the final speed of
18 m/min). The test was terminated when mice reached exhaustion, which
was defined as immobility for 30 s. Locomotor activity of mice was
measured by photobeam breaks using an Opto-Varimex-4 (Columbus Instru-
ments). Indirect calorimetry was performed using Oxymax chambers (Colum-
bus Instruments). All mice were acclimatized for 24 h before measurements.
Resting metabolic rate was determined by calculating the average energy
expenditure at each 30-min time point during a 24-h period.
Isoform-specific AMPK kinase assay. Muscle lysates (300 g protein) were
immnoprecipitated with specific antibody against 1or2 catalytic subunits
(Abcam) and protein G beads (Invitrogen). Kinase reaction was carried out in
40 mmol/l HEPES, pH 7.0; 1 g GST-SAMS; 0.2 mmol/l AMP; 80 mmol NaCl; 0.8
mmol/l dithiothretiol; 5 mmol/l MgCl
2
; and 0.2 mmol/l ATP (2Ci [-
32
P]ATP)
for 20 min at 30°C. Reaction products were dissolved in SDS sample buffer for
SDS-PAGE. Gels were dried and the kinase activity was quantified by Phospho
Imager (BAS-2500; Fuji Film).
Mitochondrial DNA quantification. Relative amount of nuclear DNA and
mitochondrial DNA (mtDNA) were determined by quantitative real-time RCR.
The ratio of mtDNA to nucleic DNA reflects the mitochondrial content in a
cell. Muscle tissue were homogenized and digested with protein K overnight
in a lysis buffer for DNA extraction by DNeasy kit (Qiagen). Quantities PCR
was performed using each primer (mtDNA-specific PCR, forward 5-CCG
CAAGGGAAAGATGAAAGA-3, reverse 5-TCGTTTGGTTTCGGGGTTTC-3;
and nuclear DNA–specific PCR, forward 5-GCCAGCCTCTCCTGATGT-3,
reverse 5-GGGAACACAAAAGACCTCTTCTGG-3; and SYBR Green PCR kit
in a prism 7900HT sequence detector (Applied Biosystem) with a program of
20 min at 95°C, followed by 50 60 cycles of 15 s at 95°C, 20 s at 58°C, and 20 s
at 72°C. mtDNA content was normalized with nuclear DNA content.
Fat index calculation. Fat mass was first measured by nuclear magnetic
resonance spectroscopy using Minispec (Bruker Biospin, Houston, TX). Fat
index was calculated by dividing the fat mass by total body weight.
Reactive oxygen species measurements. Reactive oxygen species (ROS)
levels were determined in muscle extracts using the ROS-sensitive fluorescent
dye dichlorodihydrofluorescein (DCF), as previously described (29,30).
Briefly, oxidation-insensitive dye (carboxy-DCFDA) was used as a control to
ensure that changes in the fluorescence seen with the oxidation-sensitive dye
(H
2
DCFDA) were due to changes in ROS production. Oxidation-insensitive
and oxidation-sensitive dyes were first dissolved at a concentration of 12.5
mmol/l and diluted with homogenization buffer to 125 mol/l immediately
before use. Diluted dyes were added to tissue extract (100 g) in a 96-well
plate to achieve a final concentration of 25 mol/l. The change in fluorescence
intensity was monitored at two time points (0 and 30 min) by using a
microplate fluorescence reader (Bio-Tek Instruments), at excitation 485
nm/emission 530 nm.
Serum analysis. Serum free fatty acid (MBL International) and triglyceride
(Cayman Chemical) levels were measured using the spectrophotometric
enzymatic assay kits. Serum insulin and adiponectin levels were determined
using the insulin and adiponectin enzyme-linked immunosorbent assay kits
(Alpco).
Diacylglycerol and ceramide quantification. After extracting total lipids
from skeletal muscle, kinase reaction was performed in kinase buffer contain-
ing 100 mmol/l imidazole HCl (pH 6.6), 100 mmol/l NaCl, 25 mmol/l MgCl
2
,2
mmol/l ethyleneglycolbis (B-aminoethyl ether)-NN-tetraacetic acid (pH 6.6), 2
l 100 mmol/l dithiothreitol in 1 mmol/l Detapac (pH 7.0), 5 g diglyceride
(DG) kinase (Sigma), and 8 l water. The reaction was initiated by the
addition of 10 l each of unlabeled 10 mmol/l ATP and [-
32
P]ATP (4.5 Ci per
sample) in 20 mmol/l imidazole (pH 6.6), 1 mmol/l Detapac, and incubation at
25°C for 45 min. The standards of diacylglycerol and ceramide (Sigma) were
run along with the samples by thin-layer chromatography on silica gel 60
plates by use of a solvent system consisting of chloroform/acetone/methanol/
acetic acid/water.
Cell culture and reagents. Sirt1
/
mouse embryonic fibroblasts (mefs) (31)
were maintained in DMEM supplemented with 10% fetal bovine serum.
Sirt1
/
mefs were stably transfected with either an empty vector or an
expression vector for V5-tagged mouse Sirt1 with lipofectamine to generate
Sirt1 and Sirt1 mefs, respectively. AMPK1/2 double knockout and
wild-type mefs were derived as previously described (32). Cells were treated
with 50 mol/l resveratrol (Sigma) or DMSO for the indicated time. C2C12
myoblast cells (ATCC) were maintained in DMEM and 10% FBS. To generate
C2C12 myotubes, a confluent culture of C2C12 cells were grown in DMEM and
2% horse serum for 3 days.
Immunoblotting. Cells were lysed in radioimmunoprecipitation assay buffer
and subjected to immunoblotting. For tissue extraction, samples were pulver-
ized in liquid nitrogen and homogenized in a lysis buffer. The following
antibodies were used: AMPK (Cell Signaling Technology), p-AMPK (T172)
(Cell Signaling Technology), phosphor–acetyl-CoA carboxylase (ACC), which
recognizes phosphorylated Ser 79 in ACC1 or phosphorylated Ser 22 in ACC2
(Cell Signaling Technology), ACC (Cell Signaling Technology), Sirt1 (Upstate
Biotechology), V5 (Invitrogen), cytochrome C (Cell Signaling Technology),
actin (Santa Cruz), and glyceraldehyde 3-phosphate dehydrogenase (BD
Bioscience). Peroxisome proliferator–activated receptor (PPAR)- coactiva-
tor (PGC)-1 acetylation was visualized by immunoprecipitating PGC-1
(antibody from Santa Cruz) from the nuclear extract (500 g) of skeletal
muscle and immunoblotting with antibody specific for acetylated lysine (Cell
Signaling Technology) or PGC-1. Levels of PGC-1 acetylation were then
quantified by scanning densitometry.
Real-time PCR. Total RNA was isolated by using the TRIzol reagent
extraction kit (Invitrogen), according to the manufacturer’s instructions. RNA
was subsequently reverse-transcribed to cDNA by using the high-capacity
cDNA archive kit (ABI). The mRNA levels were measured by real-time PCR
using the ABI Prism TM 7900HT Sequence Detection System (Applied
Biosystem).
Detection of PGC-1 acetylation. PGC-1 was immunoprecipitated from
skeletal muscle extract (500 g of nuclear protein) using with anti–PGC-1a
antibody (Santa Cruz). Immunoprecipitated PGC-1 was electrophoresed in
SDS-PAGE and immunoblotted with antibody specific for acetylated lysine
(cell signaling) to detect acetylation or antibody specific for PGC-1 to detect
total PGC-1 level.
NAD
-to-NADH ratio measurements. The NAD
-to-NADH ratio was mea
-
sured from whole cell extracts of C2C12 myotubes or skeletal muscle
(gastrocnemius) using the Biovision NAD/NADH Quantitation kit according to
the manufacturer’s instructions.
Statistical analysis. Comparisons between the treatment groups were
analyzed by Student’s t test, and comparisons involving repeated measure-
ments were analyzed by repeated-measures ANOVA followed by Bonferroni
posttest. Results are expressed as the means SE. Significance was accepted
at P 0.05.
RESULTS
Resveratrol-induced weight loss requires AMPK. We
examined AMPK activity in the skeletal muscle (gastroc-
nemius) and white adipose tissue (WAT) of wild-type mice
fed a high-fat diet (40% fat by calories). Supplementation
of resveratrol (400 mg kg
1
day
1
) in the diet-induced
phosphorylation of Thr 172 (p-AMPK), a marker of AMPK
activity in both skeletal muscle (Fig. 1A, left) and WAT
(Fig. 1A, right). Consistent with this, AMPK-mediated
phosphorylation of ACC is also increased with resveratrol.
The Sirt1 level, however, did not change with resveratrol.
Since resveratrol-treated mice have less fat, the effect of
resveratrol on AMPK in these tissues may not be cell
autonomous. To address this, we treated C2C12 myotubes
with resveratrol and examined AMPK activity. Resveratrol
treatment increased AMPK activity and ACC phosphoryla-
tion also in C2C12 myotubes (Fig. 1B), indicating that
resveratrol activates AMPK in a cell-autonomous manner.
To differentiate the effect of resveratrol on AMPK1 and
-2 activity, we immunoprecipitated them from WAT
and skeletal muscle of mice treated with resveratrol and
measured their kinase activity (Fig. 1C). In WAT, the
activity of AMPK1, but not AMPK2, was induced by
J.-H. UM AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, MARCH 2010 555
resveratrol, but in skeletal muscle, both the activity of
both AMPK1 and -2 was induced by resveratrol, al-
though the activity of AMPK2 was induced significantly
more than that of AMPK1. To determine whether AMPK
activation is required for the metabolic effects of resvera-
trol, we fed AMPK1
/
and AMPK2
/
mice that had
been backcrossed to C57BL/6J for at least six generations
a high-fat diet or a high-fat diet supplemented with res-
veratrol for 3 months. Food intake was similar for wild-
type, AMPK1
/
, and AMPK2
/
mice and was not
affected significantly by resveratrol (data not shown).
Resveratrol reduced the body weight of wild-type and
AMPK2
/
mice but not that of AMPK1
/
mice (Fig.
1D). Consistent with this, resveratrol decreased the fat
index in wild-type mice and AMPK2
/
mice (Fig. 1E).
These results suggest that AMPK1 is required for the
antiobesity effect of resveratrol.
To understand why AMPK1
/
mice did not lose
weight on resveratrol, we measured their O
2
consumption
(V
O
2
) and physical activity (Fig. 2
). Resveratrol increased
the metabolic rate of wild-type mice, but it did not increase
the metabolic rate of AMPK1
/
mice (Fig. 2A). Resvera
-
trol did not affect the physical activity levels (Fig. 2B)in
either wild-type or AMPK1
/
mice, indicating that res
-
veratrol increases the intrinsic metabolic rate through
AMPK. To determine the source of the increased meta-
bolic rate in wild-type mice treated with resveratrol, we
measured the expression levels of uncoupling proteins
(UCPs) in WAT and brown adipose tissue (BAT). As
shown in Fig. 2C and D, resveratrol increased the expres-
sion of UCP1, -2, and -3 in both WAT and BAT in wild-type
mice but not in AMPK1
/
or AMPK2
/
mice. More
-
over, UCP2 and UCP3 are not even detectable in
AMPK1
/
WAT. Increased uncoupling in adipose tissue
may increase the body temperature. However, the body
temperatures of wild-type, AMPK1
/
, and AMPK2
/
mice were not significantly different and did not change
with resveratrol.
Resveratrol-induced mitochondrial biogenesis and
muscle function requires AMPK. As reported previ-
ously (7), resveratrol increased physical endurance
(309.2 37 vs. 212.3 19 m) (Fig. 3A) of wild-type mice.
However, resveratrol did not increase the physical endur-
ance of AMPK1
/
or AMPK2
/
mice (Fig. 3A). Pro
-
longed activation of AMPK increases Glut4 expression
(33). To determine whether increased expression of Glut4
expression was involved in the resveratrol-mediated im-
provement of physical endurance, we measured Glut4
mRNA levels in skeletal muscle. As shown in Fig. 3B, Glut4
mRNA levels in resveratrol-treated wild-type mice were
similar to that in resveratrol-treated AMPK1
/
mice,
although it was higher than that in resveratrol-treated
AMPK2
/
mice, suggesting that Glut4 did not play a
significant role in resveratrol-mediated increase in physi-
cal indurance. It is believed that the resveratrol effects are
mediated by increased expression and function of PGC-1,
the master regulator of mitochondrial biogenesis and
oxidative phosphorylation (34). We measured the expres-
sion of PGC-1 and other resveratrol-induced genes criti-
cal for mitochondrial function in the skeletal muscle of
resveratrol-treated AMPK-deficient mice. As shown in Fig.
3C, resveratrol induced the expression of these genes in
wild-type mice but not in AMPK1
/
or AMPK2
/
mice. Consistent with this, resveratrol increased the mito-
0
0.1
0.2
0.3
0.4
**
#
A
RSVHF RSVHF
Skeletal muscle
p-AMPK
Sirt1
AMPK
p-ACC
ACC
p-AMPK
AMPK
p-ACC
ACC
B
RSV (hr):
013616
C2C12 myotubes
WAT
C
D
Actin
WAT Muscle
WT α1KO α2KO
HF
RSV
E
Fat index (g/g)
15
20
25
30
35
40
15
20
25
30
35
40
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
3
*
AMPK
α
1 activity
AMPK α1 activity
AMPK α2 activity
AMPK α2 activity
***
**
*
*
*
**
***
***
***
Body weight (g)
Body weight (g)
15
20
25
30
35
40
123456789101112
α2KO-HF
α2KO-RSV
α1KO-HF
α1KO-RSV
WT-HF
WT-RSV
**
*
*
*
Weeks of treatment
123456789101112
Weeks of treatment
123456 7891011
12
Weeks of treatment
Body weight (g)
FIG. 1. Resveratrol does not reduce fat mass in AMPK1
/
mice. A: AMPK Thr-172 phosphorylation (p-AMPK) status in skeletal muscle
(gastrocnemius, left) and epididymal fat (right) of wild-type mice fed with high-fat diet (HF) alone or with high-fat diet supplemented with
resveratrol (RSV, 400 mg kg
1
day
1
)(left) for 3 months. AMPK-mediated phosphorylation (Ser-79) of ACC and Sirt1 levels in the muscle are
also shown. B: AMPK phosphorylation and ACC phoshorylation in C2C12 myotubes treated with RSV (50 mol/l) in vitro are shown. C: Kinase
activity of AMPK1or-2 immunoprecipitated from WAT or skeletal muscle from A (n 5 per genotype). The activity level of the kinase isolated
from high-fat tissue was arbitrarily set to one for each isoform. Results are means SE. *P < 0.05; **P < 0.01; ***P < 0.001 between high-fat
diet and resveratrol. D: Body weight of wild-type (WT), AMPK1
/
(1KO), and AMPK2
/
(2KO), mice fed with high-fat diet alone or with
high-fat diet supplemented with resveratrol (HF-RSV) for 3 months (n 9–10 for each genotype). The body weight curves between high-fat diet
and resveratrol were statistically significant for wild-type (P 0.003) and AMPK2
/
(P 0.02) mice. Results are means SE. Bonferroni’s
post hoc analysis: *P < 0.05; **P < 0.01; ***P < 0.001 between high-fat diet and resveratrol for each genotype at the indicated time points. E:
Fat mass index (fat mass/body weight) of wild-type, AMPK1
/
, and AMPK2
/
mice (n 9 –10). Results are means SE. **P < 0.01 between
high-fat diet and resveratrol for wild-type mice. #P 0.07 between high-fat diet and resveratrol for AMPK2
/
mice.
METABOLIC EFFECTS OF RESVERATROL REQUIRE AMPK
556 DIABETES, VOL. 59, MARCH 2010 diabetes.diabetesjournals.org
chondrial content, as measured by cytochrome C (Fig. 3D)
and mitochondrial DNA (Fig. 3E) levels, in wild-type
muscle but not in AMPK1
/
or AMPK2
/
muscle. To
determine whether the requirement of AMPK for the
resveratrol effect is cell autonomous, we treated mefs
developed from AMPK1/2 double knockout (32) and
wild-type embryos with resveratrol for 6 –24 h. The cellular
content of cytochrome C increased after resveratrol treat-
ment in wild-type mefs, but it did not change signifi-
cantly in AMPK1/2 double knockout mefs (Fig. 3F).
Sirt1 levels were not affected by resveratrol treatment in
either wild-type or AMPK1/2 double knockout mefs.
In agreement with these findings, resveratrol induced
the expression of PGC-1, medium-chain acyl-CoA de-
hydrogenase (MCAD), and estrogen-related receptor
(ERR) in wild-type mefs but not in AMPK1/2 double
knockout mefs (Fig. 3G). Taken together, these findings
indicate that the resveratrol-activated signaling pathway
that increases mitochondrial biogenesis and physical
endurance is AMPK dependent.
Resveratrol-induced improvements in glucose ho-
meostasis require AMPK. Sirt1 overexpression (35–37),
AMPK activation (21,22,38), and resveratrol treatment
(5,7) all improve glucose tolerance and insulin sensitivity.
To determine whether the resveratrol-induced improve-
ments in glucose homeostasis required AMPK, we per-
formed glucose and insulin tolerance tests with wild-type,
AMPK1
/
, and AMPK2
/
mice fed a high-fat diet or a
high-fat diet supplemented with resveratrol. Glucose tol-
erance (Fig. 4A) and insulin sensitivity (Fig. 4B) were
similar between wild-type, AMPK1
/
, and AMPK2
/
mice on high-fat diet alone. However, glucose tolerance
was dramatically improved by resveratrol in wild-type
mice but only modestly in AMPK2
/
mice and not at all
in AMPK1
/
mice. Insulin sensitivity was also dramati
-
cally improved by resveratrol in wild-type mice but not in
AMPK1
/
or AMPK2
/
mice.
Insulin-resistant states are associated with increased
production of ROS, and reduction of ROS with antioxi-
dants improves insulin sensitivity (39,40). Since resvera-
trol is thought to have antioxidant properties, it is possible
that some insulin sensitizing of resveratrol effect may be
related to its antioxidant effect. We quantified the levels of
ROS in skeletal muscle to determine whether resveratrol
reduced ROS (Fig. 4C). Resveratrol reduced ROS levels in
wild-type mice by almost 30% but not in AMPK1
/
or
AMPK2
/
mice, suggesting that, at least in vivo, the
antioxidant effect of resveratrol is produced indirectly
through an AMPK-dependent pathway.
Insulin-resistant tissues are often associated with ele-
vated levels of intramyocellular lipids such as diacylglyc-
eride (DAG) and ceramide (41). Mitochondrial dysfunction
decreases fat oxidation, leading to accumulation of these
lipids, which decrease insulin sensitivity (41,42). Since
resveratrol increases mitochondrial function (7), we mea-
sured the DAG and ceramide levels in resveratrol-treated
skeletal muscle. As shown in Fig. 4D and E, resveratrol
decreased DAG and ceramide levels in wild-type mice but
not in AMPK1
/
and AMPK2
/
mice. Although res
-
veratrol decreased intramyocellular DAG and ceramide, it
did not decrease serum free fatty acid or triglyceride levels
(Fig. 4F and G), suggesting that resveratrol did not de-
crease the intramyocellular lipids by decreasing the deliv-
ery of lipids to the muscle but by increasing their
oxidation. Adiponectin, a cytokine that is produced by fat
and is decreased in obese and diabetic subjects (43),
increases insulin sensitivity by activating AMPK (38). To
determine whether resveratrol increased insulin sensitiv-
ity by increasing adiponectin levels, we measured serum
adiponectin levels in resveratrol-treated mice. Adiponectin
0
0.5
1
1.5
2
2.5
0
2
4
6
8
10
12
UCP1
UCP2
0
5
10
15
20
25
30
**
*
WT
A
B
C
D
BAT
E
WAT
HF
RSV
0
0.2
0.4
0.6
0.8
1
0
0.5
1
1.5
2
0
0.2
0.4
0.6
0.8
1
*
*
UCP1
UCP2
UCP3
*
UCP3
2000
2500
3000
3500
0
4000
8000
12000
16000
20000
33
34
35
36
37
38
VO
2
(ml/kg/hr)
WT
α1KO α1KO α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
HF
RSV
**
Activity level (B.B.)
WT
HF
RSV
Core temperature (
o
c)
HF
RSV
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
FIG. 2. Resveratrol does not increase the metabolic rate in AMPK1
/
mice. A: Twenty-four-hour oxygen consumption (VO
2
) of wild-type (WT)
and AMPK1
/
mice. Results are means SE. **P < 0.01 between high-fat diet (HF, ) and resveratrol (RSV, f) for wild-type mice (n 3 for
each genotype). B: Twenty-four-hour activity level as measured by beam breaks (B.B.). , High-fat diet; f, resveratrol. C: Transcription levels
of UCP1, UCP2, and UCP3 in WAT as measured by real-time PCR (n 4 –5 per genotype). Results are means SE. *P < 0.05; **P < 0.01 between
high-fat diet and resveratrol for wild-type mice. , High-fat diet; f, resveratrol. D: Transcription levels of UCP1, UCP2, and UCP3 in BAT as
measured by real-time PCR (n 4 –5 per genotype). Results are means SE. *P < 0.05 between high-fat diet and resveratrol for wild-type mice.
E: Core temperature was measured by a rectal thermometer (n 4–5 per genotype). , High-fat diet; f, resveratrol.
J.-H. UM AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, MARCH 2010 557
levels were similar for wild-type, AMPK1
/
, and
AMPK2
/
mice and were not changed by resveratrol
(Fig. 4H). An increase in insulin sensitivity usually de-
creases insulin levels. However, resveratrol dramatically
decreased the fasting insulin levels in wild-type,
AMPK1
/
, and AMPK2
/
mice even though resvera
-
trol increased insulin sensitivity only in wild-type mice
(Fig. 4I). Although this can be explained, at least in part,
by the observation that resveratrol inhibits insulin secre-
tion by inhibiting the electrical activity in the -cells (44),
more studies are required to fully understand the disparity
between the insulin levels and insulin sensitivity in res-
veratrol-treated AMPK-null mice.
Resveratrol modulates the NAD-to-NADH ratio in an
AMPK-dependent manner. A previous report (7) has
shown that resveratrol treatment leads to deacetylation of
PGC-1, suggesting that the catalytic activity of Sirt1 is
increased by resveratrol. Since AMPK activity increases
the NAD-to-NADH ratio and Sirt1 activity (45), we mea-
sured the NAD-to-NADH ratio in C2C12 myotubes treated
with resveratrol for 6 and 16 h. As shown in Fig. 5A,
resveratrol increased the NAD-to-NADH ratio by 30%.
0
1
2
3
4
0
0.5
1
1.5
2
0
0.5
1
1.5
2
MCAD
**
*
D
0
0.2
0.4
0.6
0.8
1
0
0.5
1
1.5
2
0
0.5
1
1.5
2
2.5
0
2
4
6
UCP3 ERR α NRF Tfam
0
1
2
3
4
5
Cyt C (A.U.)
**
0
0.5
1.0
1.5
Relative mtDNA
copy number
PGC-1α PGC-1β
*
A
B
*
Running distance (m)
HF
RSV
HF
RSV
HF
RSV
E
G
AMPK
Sirt1
WT AMPK KO
RSV (hr): 0 6 12 24 0 6 12 24
F
p-AMPK
Cyt C
Actin
0
0.5
1
1.5
2
0
0.5
1
1.5
2
0 6 12 24
PGC-1α
MCAD
0
1
2
3
4
ERR α
WT
AMPK KO
RSV (hr)
0 6 12 24
RSV (hr)
0 6 12 24
RSV (hr)
GAPDH
CytC
RSVHF RSVHF RSVHF
*
*
*
***
***
C
0
0.2
0.4
0.6
0.8
1
Glut-4
HF
RSV
0
100
200
300
400
*
HF
RSV
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
FIG. 3. Resveratrol-induced mitochondrial biogenesis and endurance require AMPK. A: Physical endurance of mice fed high-fat diet (HF, ) alone
or high-fat diet supplemented with resveratrol (RSV, f) as measured by treadmill running until exhaustion (n 4–5). Results are means SE.
*P < 0.05 between wild-type mice fed a high-fat diet (HF) alone and high-fat diet supplemented with RSV. B: Transcription levels of Glut4 in
skeletal muscle as measured by real-time PCR in arbitrary units (n 4 –5 per genotype). , High-fat diet; f, resveratrol. C: Transcription levels
of skeletal muscle genes induced by resveratrol (n 4–5 for each genotype). *P < 0.05 and **P < 0.01 between wild-type mice fed high-fat diet
alone and high-fat diet supplemented with resveratrol. , High-fat diet; f, resveratrol. D: Cytochrome C (Cyt C) levels induced by resveratrol
in skeletal muscle. Cytochrome C levels were detected by immunoblotting with Cytochrome C antibody and quantified by densitometry analysis
(n 4 –5 per genotype). **P < 0.01 between wild-type mice fed high-fat diet alone and high-fat diet supplemented with resveratrol. E: Relative
mtDNA copy number in skeletal muscle (n 4 –5 per genotype). The copy number in wild-type mice fed with high-fat diet alone was arbitrarily
set to 1. *P < 0.05 between wild-type mice fed high-fat diet alone and high-fat diet supplemented with resveratrol. , High-fat diet; f, resveratrol.
F: Cytochrome C and Sirt1 levels in wild-type and AMPK1/2 double knockout (AMPK KO) mefs after resveratrol treatment for the indicated
durations were detected by immunoblotting. G: The mRNA levels (arbitrary units) of PGC-1, MCAD, and ERR in wild-type () and AMPK1/2
double knockout (AMPK KO, f) mefs after resveratrol treatment were measured by real-time PCR (n 3). The statistical difference between
wild-type and AMPK KO mefs: P 0.0001 for PGC-1, P 0.07 for MCAD, and P 0.02 for ERR. Bonferroni’s posttests: *P < 0.05; ***P < 0.001
between the genotypes at the indicated time points. WT, wild type.
METABOLIC EFFECTS OF RESVERATROL REQUIRE AMPK
558 DIABETES, VOL. 59, MARCH 2010 diabetes.diabetesjournals.org
NAD and the NAD-to-NADH ratio in skeletal muscle were
increased by resveratrol in wild-type mice but not in
AMPK1
/
or AMPK2
/
mice (Fig. 5B). The magnitude
of the increase was modest but statistically significant. To
determine whether the resveratrol-mediated increase in
NAD and NAD-to-NADH ratio increased Sirt1 activity, we
quantified the acetylation level of PGC-1 in skeletal
muscle. As shown in Fig. 5C, resveratrol decreased the
acetylation level of PGC-1 only in wild-type mice.
PGC-1 is a coactivator for its own transcription (46),
and, as a result, deacetylation of PGC-1 by Sirt1 should
increase its ability to positively regulate PGC-1 transcrip-
Time (min)
0306090
Serum glucose level (%)
0
20
40
60
80
100
120
Time (min)
0306090
Serum glucose level (mg/dl)
0
100
200
300
400
500
600
700
Relative ROS level (%)
*
0
50
100
A
C
D
E
WT
Time (min)
0306090
Serum glucose level (%)
0
20
40
60
80
100
120
Time (min)
0306090
Serum glucose level (%)
0
20
40
60
80
100
120
Time (min)
0306090
Serum glucose level (mg/dl)
0
100
200
300
400
500
600
700
Time (min)
0306090
Serum glucose level (mg/dl)
0
100
200
300
400
500
600
700
B
HF
RSV
F
G
I
H
*
*
*
**
**
***
***
***
***
α1KO α2KO
α1KO α2KOWT
WT
0
500
1000
1500
2000
0
200
400
600
800
1000
DAG (pmol/mg)
**
HF
RSV
***
*** ***
***
HF
RSV
ceramide (pmol/mg)
HF
RSV
HF
RSV
HF
RSV
HF
RSV
HF
RSV
HF
RSV
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
2.5
2.0
1.5
1.0
0.5
0.0
Free fatty acid (mM)
HF
RSV
WT
α1KO α2KO
25
20
15
10
5
0
Adiponectin (µg/ml)
HF
RSV
WT
α1KO α2KO
2.0
1.5
1.0
0.5
0.0
Triglyceride (mM)
HF
RSV
WT
α1KO α2KO
10
8
6
4
2
0
Insulin (ng/ml)
HF
RSV
WT
α1KO α2KO
FIG. 4. Resveratrol does not improve glucose homeostasis in AMPK-deficient mice. A: Serum glucose levels after glucose injection (intraperi-
toneal) for wild-type (WT), AMPK1
/
, and AMPK2
/
mice fed high-fat diet (HF, F) alone or high-fat diet supplemented with resveratrol
(RSV, E)(n 4 –5 for each genotype). Results are means SE. Glucose tolerance curves between high-fat diet and resveratrol were significant
for wild-type mice (P 0.001) but not for AMPK1
/
(P 0.8) and AMPK2
/
(P 0.053) mice. Bonferroni’s posttests: *P < 0.05; **P < 0.01;
***P < 0.001 between high-fat diet and resveratrol for each genotype at the indicated time points. B: Serum glucose levels (% of basal glucose
levels) after insulin injection (intraperitoneal) (n 5 for each genotype). Results are means SE. Insulin sensitivity curves between high-fat
diet (F) and resveratrol (E) were significant for wild-type (WT) mice (P 0.01). Bonferroni’s post hoc analysis: *P < 0.05; **P < 0.01; ***P <
0.001 between high-fat diet and resveratrol for wild-type mice at the indicated time points. C: Relative levels of ROS in skeletal muscle extracts
from wild-type (WT) and AMPK1
/
mice fed high-fat diet () alone or high-fat diet supplemented with resveratrol (f) as measured by DCF
fluorescence (52) (n 4 –5 for each genotype). The ROS level in wild-type mice on high-fat diet alone was set to 1. Results are means SE. *P <
0.05 between wild-type mice fed high-fat diet alone or high-fat diet supplemented with resveratrol. D: DAG levels in skeletal muscle (n 4 –5 per
genotype). **P < 0.01 between wild-type (WT) mice fed high-fat diet () alone or high-fat diet supplemented with resveratrol (f). E: Ceramide
levels in skeletal muscle (n 4 –5 per genotype). ***P < 0.001 between wild-type (WT) mice fed high-fat diet () alone or high-fat diet
supplemented with resveratrol (f). Serum levels of free fatty acid (F), triglyceride (G), adiponectin (H), and fasting insulin (I) are shown (n
4 –5). , High-fat diet; f, resveratrol. ***P < 0.001 between wild-type mice fed high-fat diet alone or high-fat diet supplemented with resveratrol.
J.-H. UM AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, MARCH 2010 559
tion. A previous report (7) showed that resveratrol did not
induce PGC-1 transcription in Sirt1
/
mefs and con
-
cluded that Sirt1 is essential for resveratrol-induced tran-
scription of PGC-1. However, the PGC-1 mRNA levels
were measured only at one posttreatment time point (24 h)
in that report. We compared resveratrol-stimulated tran-
scription of PGC-1 and mitochondrial biogenesis in
Sirt1
/
mefs stably transfected with either an empty
vector (Sirt1) or Sirt1 expression vector (Sirt1). Con-
sistent with previous reports (47), Sirt1 mefs had higher
AMPK activity than Sirt1 mefs (Fig. 6A). Moreover,
resveratrol activated AMPK in Sirt1 mefs, indicating that
Sirt1 is not essential for AMPK activity or its induction
by resveratrol. Transcription of PGC-1 was stimulated by
resveratrol in both Sirt1 mefs and Sirt1 mefs, but by
12–24 h, it returned to basal levels in Sirt1 mefs, whereas
it remained elevated in Sirt1 mefs beyond 24 h (Fig. 6B).
Therefore, the difference in the expression patterns of
these genes between Sirt1 and –Sirt1 mefs was statisti-
cally significant only at the 24-h time point. Similar pat-
terns were also seen with MCAD and ERR. Therefore,
Sirt1 is not required for resveratrol to stimulate PGC-1
transcription but helps to prolong the duration of the
stimulated state. This stands to reason because the effect
of Sirt1-mediated deacetylation of PGC-1 on PGC-1
transcription will only occur after sufficient PGC-1 pro-
tein has accumulated.
DISCUSSION
Dietary control and exercise prevent metabolic disorders,
but they are not usually successful interventions. Drugs
A
B
NAD/NADH Ratio
NAD (nmol/µg protein)
C
NADH (nmol/µg protein)
0
2
4
6
0
0.5
1
1.5
0
1
2
3
4
5
0
0.8
1.6
2.4
3.2
0
1
2
3
4
0
0.4
0.8
1.2
0
4
8
12
-+
***
RSV
-+
RSV
RSV
-+
***
NAD (nmol/µg protein)
*
HF
RSV
NADH (nmol/µg protein)
NAD/NADH Ratio
*
Relative PGC1 acetylation
*
HF
RSV
C2C12
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
WT
α1KO α2KO
FIG. 5. The role of resveratrol on the NAD-to-NADH ratio. A: Intracellular levels of NAD, NADH, and NAD-to-NADH ratio in C2C12 myotubes
treated with vehicle () or resveratrol () (50 mol/l) for6h(n 5). Results are means SE. *P < 0.05; **P < 0.01; ***P < 0.001 between
the treatments. B: Intracellular levels of NAD, NADH, and NAD-to-NADH ratio in skeletal muscle of mice fed with high-fat diet (HF, ) alone or
supplemented with resveratrol (RSV, f)(n 4 –5). Results are means SE. *P < 0.05 between wild-type (WT) mice fed high-fat diet alone or
supplemented with resveratrol. C: Acetylation levels of PGC-1 in skeletal muscle (n 4 –5 per genotype). Results are means SE. *P < 0.05
between wild-type (WT) mice fed high-fat diet alone () and high-fat diet supplemented with resveratrol (f).
PGC-1α
0
0.2
0.4
0.6
0.8
1
0
0.5
1
1.5
0
0.5
1
1.5
2
2.5
ERRα
MCAD
+Sirt1 -Sirt1
AMPK
0 6 12 24 0 6 12 24
Actin
p-AMPK
RSV (hr):
Sirt1
B
A
+Sirt1
-Sirt1
***
*
0 6 12 24
RSV (hr)
0 6 12 24
RSV (hr)
0 6 12 24
RSV (hr)
FIG. 6. Sirt1 modulates resveratrol effect. A: AMPK activity (Thr-172 phosphorylation, p-AMPK) in Sirt1
/
mefs (Sirt1) and Sirt1
/
mefs with
restored Sirt1 (Sirt1) after resveratrol (RSV) treatment. B: PGC-1, MCAD, and ERR mRNA levels (AU) in Sirt1 (f) and Sirt1 () mefs
treated with resveratrol (RSV) (n 3). Results are means SE. The statistical difference between wild-type and Sirt1 knockout mefs: P 0.001
for PGC-1, P 0.07 for MCAD, and P 1.0 for ERR. Bonferroni’s posttests: *P < 0.05; ***P < 0.001 between the genotypes at the 24-h time
point.
METABOLIC EFFECTS OF RESVERATROL REQUIRE AMPK
560 DIABETES, VOL. 59, MARCH 2010 diabetes.diabetesjournals.org
that mimic calorie restriction and exercise are being
developed to combat metabolic disorders. One target for
treating metabolic disorders is Sirt1 (8), an NAD
-depen
-
dent protein deacetylase (48). Resveratrol, which was
discovered in a small-molecule screen to be a Sirt1 acti-
vator (1), has drawn a great deal of interest for its
therapeutic potential in treating metabolic disorders such
as type 2 diabetes. By increasing mitochondrial biogenesis
and metabolic rate, resveratrol reduced fat and increased
glucose tolerance and insulin sensitivity and physical
endurance.
If Sirt1 is the target of resveratrol, transgenic mice
overexpressing Sirt1 should have phenotypes very similar
to those induced by resveratrol. Thus far, three models of
whole-body Sirt1 gain of function have been reported. In
Sirt1 knockin mice (36), the Sirt1 transgene, which was
expressed from the -actin locus, is overexpressed in fat
but not in muscle or liver, key organs affected in metabolic
disorders. These mice have increased metabolic rate,
lower fat mass, and are more insulin sensitive than control
mice, resembling mice treated with resveratrol. More
recently, transgenic mice in which the Sirt1 transgene is
expressed from its own promoter have been reported by
two independent groups (35,37). These transgenic mice,
which have a more physiological expression pattern of the
Sirt1 transgene than the knockin mice (36), are more
insulin sensitive partly due to increased adiponectin levels
and decreased hepatic steatosis. Surprisingly, the trans-
genic mice developed by Banks et al. (35), which are
congenic in C57BL6/J background, have reduced meta-
bolic rate and body temperature and have the same
amount of fat despite eating less. It should be noted that
unlike the other Sirt1 gain-of-function studies, this study
measured the metabolic rate using regular diet not high-fat
diet. Nevertheless, this study shows that the function of
Sirt1 on energy balance may be opposite of what was
previously thought (7) and would be predicted if the
central target of resveratrol was Sirt1. One possible expla-
nation for the reduced fat mass in the knockin mice (36) is
that expression of the Sirt1 transgene from the -actin
locus impaired adipocyte differentiation during develop-
ment (49). Therefore, the Sirt1 knockin model (36) cannot
distinguish between a direct effect of Sirt1 and an indirect
one caused by reduced fat.
Resveratrol has been reported to affect the activities of
many enzymes (11) including AMPK (12). To evaluate the
possibility that the effects of resveratrol are mediated by
AMPK, we studied the effects of resveratrol in mice
deficient in AMPK1or-2. Our findings indicate that all
of the salient effects induced by resveratrol are abolished
in AMPK1- and/or AMPK2-deficient mice, suggesting
that the metabolic changes induced by resveratrol are
largely mediated through AMPK rather than Sirt1. Because
the wild-type mice used in our study were not littermates
of AMPK-null mice, it is possible that some differences
between wild-type mice and AMPK-null mice may be due
to differences in the genetic background. However, we feel
that this difference is minimal because the AMPK-null mice
used in this study have been backcrossed to C57BL6/J
mice, which we used as wild-type control, for at least six
generations. Therefore, we expect our AMPK-null mice to
be at least 98% congenic to the wild-type controls. More-
over, the dominant role of AMPK in the metabolic effects
of resveratrol is supported by our studies using Sirt1-
deficient mefs. Resveratrol-induced transcription of
PGC-1 and PGC-1 dependent genes was shorter in
duration but was not abolished in Sirt1-deficient mefs (Fig.
6), whereas it was abolished in AMPK-deficient mefs (Fig.
3). The ability of AMPK to increase NAD and the NAD-to-
NADH ratio (45) may also explain how resveratrol treat-
ment can lead to Sirt1 activation without directly
activating it. Moreover, AMPK can activate PGC-1, one of
Sirt1 substrates, by directly phosphorylating it (50,51),
indicating that activation of AMPK can affect the Sirt1-
dependent pathways in multiple ways. Thus, our findings
suggest that the direct target of resveratrol in vivo may not
be Sirt1 and supports the possibility that Sirt1 plays a
modulatory role, rather than a central role, in resveratrol
response. Whether it is the direct target of resveratrol or
not, it appears that Sirt1 can also function upstream of
AMPK in HepG2 hepatocytes and HEK293T cell line
(26,27). Since Sirt1 is not required for resveratrol-mediated
activation of AMPK activation in mefs (Fig. 6A), it is
possible that Sirt1 is upstream of AMPK only in certain cell
types.
Resveratrol-induced physical endurance has been
largely attributed to increased mitochondrial function (7).
Certainly, converting muscle fiber to mitochondria-rich,
slow-twitch fibers increases physical endurance (52).
However, it is also likely that the glycogen content in
skeletal muscle, which is known to be increased by AMPK
activity (34), also plays a role in resveratrol-induced
physical endurance.
Although the expression levels of UCPs in WAT and
BAT increased with resveratrol in wild-type mice, the body
temperature of wild-type or AMPK
/
mice did not in
-
crease with resveratrol. It is possible that the stress
associated with the rectal temperature measurement may
have masked any subtle differences in body temperature.
It should also be noted that unlike AMPK1
/
mice,
AMPK2
/
mice gained less weight on resveratrol even
though the expression levels of UCPs were not induced by
resveratrol in the adipose tissue. We do not have a clear
explanation for this, but one possibility is that the anti-
adipogenic function of Sirt1 (49) is being driven by the
resveratrol-AMPK1-Sirt1 pathway in AMPK2
/
mice.
The complex nature of the resveratrol effect is also
demonstrated by our observation that even though res-
veratrol induced weight loss in AMPK2
/
mice, it did
not improve their insulin sensitivity (Fig. 4B). One reason
resveratrol failed to improve insulin sensitivity in
AMPK2
/
mice may be that the failure to increase
mitochondrial biogenesis and fat oxidation in skeletal
muscle led to a build up of lipids that are known to inhibit
insulin action. Indeed, resveratrol failed to increase mito-
chondrial content and decrease DAG and ceramide in both
AMPK1
/
and AMPK2
/
mice. In skeletal muscle, the
AMPK1 isoform makes up only 20% of the total AMPK
activity (17), and, yet, resveratrol-induced mitochondrial
biogenesis (Fig. 3D and E) or reduction in ROS (Fig. 4C),
DAG (Fig. 4D), and ceramide (Fig. 4E) did not occur in the
skeletal muscle of AMPK1
/
mice. One possible expla
-
nation is that in addition to the AMPK2 activity, a
crosstalk between the skeletal muscle and either the
nervous system or fat, where the AMPK1 isoform is
predominant (17), is required for resveratrol-induced mi-
tochondrial biogenesis or reduction of ROS in skeletal
muscle. For example, the AMPK2 activity in skeletal
muscle and the low-energy signal from resveratrol-induced
weight loss, which requires AMPK1, may both be re-
quired for the full resveratrol effect. It is also likely that in
skeletal muscle, AMPK1 and AMPK2 have nonoverlap-
J.-H. UM AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, MARCH 2010 561
ping functions, both of which are required for responding
to resveratrol.
ACKNOWLEDGMENTS
This work was supported by the Intramural Research
Program, National Heart, Lung, and Blood Institute, Na-
tional Institutes of Health, and by the European Union FP6
Program (EXGENESIS Integrated Project LSHM-CT-
2004-005272).
No potential conflicts of interest relevant to this article
were reported.
We thank Dalton Saunders for his assistance with ani-
mal management.
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    • "AMPK has very similar role to SIRT1, when cellular energy charge decreases marked by increase in AMP levels and the decrease of ATP, AMPK is activated and shuts down energy-consuming anabolic processes and turns on mitochondrial oxidation and catabolism to replenish cellular energy [25, 48]. SIRT1 and AMPK not only display functional synergy, but their activity is cross-regulated [25,[49][50][51][52][53]. Several investigators have shown that AMPK and SIRT1 act on the same transcription of the indicated genes were determined in RT-qPCR reactions (n = 8 except for TBX-1, where n = 3, median and quartiles are plotted). "
    [Show abstract] [Hide abstract] ABSTRACT: Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when comparing control and AICAR-treated white adipocytes. Our data point out that in human pericardial hADMSCs the role of AMPK activation in controlling beige differentiation is restricted to morphological features, but not to actual metabolic changes.
    Full-text · Article · Jun 2016
    • "Preclinical studies have demonstrated a variety of protective effects in animal models of cardiovascular disease, including hypertension (Dolinsky et al., 2013; Dolinsky et al., 2009; Mizutani et al., 2000), hypercholesterolemia (Juhasz et al., 2011; Penumathsa et al., 2007), atherosclerosis (Do et al., 2008; Wang et al., 2005), ischemic heart disease (Andreadou et al., 2015; Novelle et al., 2015), diabetes (Su et al., 2006; Um et al., 2010) and metabolic syndrome (Novelle et al., 2015; Pechanova et al., 2015). These cardiovascular effects of resveratrol in laboratory animals have been reviewed in our previous article (Li et al., 2012) and in a recent publication (Zordoky et al., 2015). "
    [Show abstract] [Hide abstract] ABSTRACT: Antioxidant effects of resveratrol (3,5,4'-trihydroxy-trans-stilbene) contribute substantially to the health benefits of the compound. Resveratrol has been shown to be a scavenger of a number of free radicals. However, the direct scavenging activities of resveratrol are relatively poor. The antioxidant properties of resveratrol in vivo are more likely to be attributable to its effect as a gene regulator. Resveratrol inhibits NADPH oxidase-mediated production of reactive oxygen species by downregulating the expression and activity of the oxidase. The polyphenolic compound reduces mitochondrial superoxide generation by stimulating mitochondria biogenesis. By upregulating the tetrahydrobiopterin-synthesizing enzyme GTP cyclohydrolase I, resveratrol prevents superoxide production from uncoupled endothelial nitric oxide synthase. In addition, resveratrol increases the expression of a variety of antioxidant enzymes. Part of the gene-regulating effects of resveratrol are mediated by the histone/protein deacetylase sirtuin 1 or by the nuclear factor-E2-related factor-2. In this review article, we have also summarized the cardiovascular effects of resveratrol observed in clinical trials. This article is protected by copyright. All rights reserved.
    Article · Apr 2016
    • "Therefore, it is necessary to comprehensively establish the mechanism of respiration inhibition by RSV. [6,11] mTOR Activation of AMPK by resveratrol inhibits mTOR through TSC1/2 activation Inhibition of anabolism allows counteracting insulin resistance, cholesterol accumulation and dyslipidemia [11,52,54] Importantly, the information reviewed here indicates the toxicological potential of RSV supplementation. Therefore, more clinical trials targeted at specific diseases are needed to search for safe concentrations of RSV supplementation. "
    [Show abstract] [Hide abstract] ABSTRACT: Resveratrol (3,4',5-trihydroxy-trans-stilbene, RSV) has emerged as an important molecule in the biomedical area. This is due to its antioxidant and health benefits exerted in mammals. Nonetheless, early studies have also demonstrated its toxic properties toward plant-pathogenic fungi of this phytochemical. Both effects appear to be opposed and caused by different molecular mechanisms. However, the inhibition of cellular respiration is a hypothesis that might explain both toxic and beneficial properties of resveratrol, since this phytochemical: (1) decreases the production of energy of plant-pathogenic organisms, which prevents their proliferation; (2) increases adenosine monophosphate/adenosine diphosphate (AMP/ADP) ratio that can lead to AMP protein kinase (AMPK) activation, which is related to its health effects, and (3) increases the reactive oxygen species generation by the inhibition of electron transport. This pro-oxidant effect induces expression of antioxidant enzymes as a mechanism to counteract oxidative stress. In this review, evidence is discussed that supports the hypothesis that cellular respiration is the main target of resveratrol.
    Full-text · Article · Mar 2016
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