JBC Papers in Press. Published on October 27, 2010 as Manuscript M110.188144 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M110.188144
Up-regulation of Adiponectin by Resveratrol: The Essential Roles of the Akt/FOXO1 and AMPK Signaling Pathways and DsbA-L
Anping Wang1,2, Meilian Liu2,5, Xianling Liu1, Lily Q. Dong3, Randolph D. Glickman4, Thomas J. Slaga2, Zhiguang Zhou1 and Feng Liu1, 2,5
1
The Metabolic Syndrome Research Center, the Second Xiangya Hospital, Central South
University, Hunan, China Departments of 2Pharmacology, 3Cellular and Structural biology, and 4Ophthalmology, UTHSCSA,
5
Correspondence should be addressed to M.L. or F.L. (e-mail:
[email protected] or
[email protected])
1
Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
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San Antonio, TX 78229
The
natural
polyphenol
regulated by both the Akt/FOXO1 and the
resveratrol
AMPK signaling pathways. Lastly, we show
(RSV) displays a wide spectrum of health beneficial
activities,
yet
the
that DsbA-L plays a critical role in the
precise
promoting effect of RSV on adiponectin
mechanisms remain to be fully elucidated.
multimerization and cellular levels.
Here we show that RSV promotes the of
Adiponectin is an adipocyte-derived
The
hormone that plays an important role in the
stimulatory effect of RSV was not affected by
regulation of insulin sensitivity and energy
knocking out Sirt1, but was diminished by
homeostasis. Adiponectin exists in cells and
suppressing the expression levels of DsbA-L, a
the plasma in three major forms: trimers,
recently
hexamers, and the high-molecular-weight
multimerization
and
adiponectin
3T3-L1
in
identified
cellular
levels
adipocytes.
adiponectin-interactive
(HMW) forms, and the HMW form of
multimerization. Suppression of the Akt
adiponectin has been shown to be the most
signaling pathway resulted in an increase in the
bioactive with respect to insulin action (1-3).
expression levels of DsbA-L and adiponectin.
Reduction of the HMW form, rather than the
On the other hand, knocking out FOXO1 or
total levels of adiponectin, has been shown to
suppressing the activity or expression levels of
be associated with various metabolic disease
the AMP activated protein kinase (AMPK)
states (1,3-5).
that
promotes
down-regulated DsbA-L and adiponectin. The
How adiponectin multimerization is
stimulatory effect of RSV on adiponectin and
regulated is not yet completely understood. We
DsbA-L expression was completely diminished
have
in FOXO1-suppressed and AMPK-inactivated
interactive protein DsbA-L (Disulfide-bond-A
3T3-L1 adipocytes. Taken together, our results
oxidoreductase-like protein) that promotes
demonstrate that RSV promotes adiponectin
adiponectin
multimerization in 3T3-L1 adipocytes via a
adipocytes (6,7). The expression levels of
Sirt1-independent mechanism. In addition, we
DsbA-L are significantly reduced in obese
show that the stimulatory effect of RSV is
human subjects and mice (6). On the other 2
recently
identified
an
multimerization
adiponectin
in
3T3-L1
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adiponectin
protein
hand, the expression levels of DsbA-L are
adipocytes in which the expression levels of
stimulated by the PPARγ agonist rosiglitazone
DsbA-L are suppressed by RNAi. Our study
(6). However, it is unknown whether DsbA-L
thus uncovers a novel mechanism by which
expression could be stimulated by other
RSV exerts its health beneficial effect.
insulin-sensitizing factors. EXPERIMENTAL PROCEDURES Resveratrol (RSV) is a polyphenol with and
Materials and Antibodies-RSV, AICAR
(8-11).
and insulin were purchased from Sigma.
Some studies suggest that the beneficial health
Sirtinol, Akti VIII, and compound C were
effects of RSV are mainly mediated by the
purchased
deacetylase Sirt1 (9-13). However, a number of
A-sepharose
recent studies demonstrate that RSV could
Amersham-Pharmacia Biotech; Anti-beta actin
exert
antibody
anti-oxidant, anti-insulin
resistance
function
properties
via
Sirt1-independent
from
Calbiochem.
beads
was
from
Protein
were
BD
from
Transduction
mechanisms (9,14-16). There are some studies
Laboratories. Antisera to adiponectin were
showing that RSV has a stimulatory effect on
generated by our lab as described as previously
the expression levels (17,18) or the secretion
(6). Antibodies to Sirt1, DsbA-L and tubulin
(19) of adiponectin.
were
underlying
However, the precise
mechanisms
remain
Upstate
Biotechnology,
PhosphoSolutions and Sigma, respectively. All
largely
other antibodies were from Cell Signaling.
unknown. In the present report, we show that RSV stimulates
from
adiponectin
expression
Cell
Lines,
Cell
Culture,
Cell
Differentiation-3T3-L1 CAR cells stably
and
multimerization in 3T3-L1 adipocytes via a
expressing
Sirt1-independent mechanism. In addition, we
coxasckievirus and adenovirus (CAR) was a
demonstrate that the stimulatory effect of RSV
gift of Dr. Jianhua Shao (University of
is regulated by both the Akt/FOXO1 and the
California at San Diego). Sirt1 and PDK1
AMPK signaling pathways. The promoting
knockout MEF cells and their wild-type control
effect of RSV is greatly diminished in 3T3-L1
cells were described previously (20). To 3
a
truncated
receptor
for
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its
anti-inflammation,
C2C12
(6). The distribution of adiponectin complex
myoblasts, a plasmid encoding the AMPK α2
was determined by separating cell lysates using
short hairpin RNA construct cloned in pSM2
a Superdex 200 10/30 column (GE Healthcare
vector (catalog number RMM1766-96744125,
Bio-Sciences). 200 µL fractions were collected
Open Biosystems) was co-transfected with a
and separated by SDS/PAGE followed with
plasmid encoding the puromycin resistance
Western blot.
generate
AMPK
α2-suppressed
gene (pBSpacΔp) into C2C12 cells. Cells containing the AMPK α2 shRNA construct
Western
Blot
immunoprecipitation-The
and
expression
and
phosphorylation levels of various proteins in
previously (21). A stable cell line expressing
cell lysates or immunoprecipitates were
the pSM2 vector alone was used as the control.
detected by Western blot with specific
The
encoding
antibodies as described (6,21). Quantification
Foxo1-specific RNAi or scramble RNAi were
of the relative change in protein levels
a generous gift of Dr. Henry Dong (University
(arbitrary unit; expressed as percentage of
of Pittsburgh School of Medicine). The
control protein levels) was performed by
adenovirus encoding GFP or mouse DsbA-L
analyzing Western blots using the Scion Image
and DsbA-L-suppressed cells were generated
Alpha 4.0.3.2 program (Scion Corporation,
as described previously (6). The culture and
Frederick, MD) and was normalized for the
differentiation of 3T3-L1 cells, 3T3 CAR cells
amount
and the culture of C2C12 cells were described
experiment.
in
PGC-1α
FOXO1
our
previous
adenoviruses
studies
(6,22,23).
The
of
protein The
were
expression
in
each
immunoprecipitation
performed
by
using
of an
differentiation of wild-type and Sirt1 ko MEF
anti-PGC-1α specific antibody as described
cells into adipocytes were performed as
previously (21).
described previously (24). Quantitative Size Exclusion Chromatography-A Size exclusion
chromatographic
studies
real-time
PCR
(qRT-PCR)-Adiponectin mRNA was purified
were
from 3T3-L1 adipocytes using the TRIZOL
performed as described in our recent studies
reagent (Invitrogen). Total RNAs were reverse 4
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were selected with puromycin as described
(AIN-93G), which were either a normal,
First-strand synthesis supermix (Invitrogen).
maintenance diet, or a diet supplemented with
PCR reactions were carried out in duplicates in
0.1% resveratrol (w/w), for 14 weeks.
96 microtiter plates (Applied Biosystems) in an
Resveratrol (Batch #7044J; MP Biomedical,
ABI PRISM 7700 real-time PCR apparatus.
Solon, OH) was of natural origin, extracted
Each PCR reaction (20 μl total volume)
from Canadian Polygonum root, and was
contained: 10 μl of SYBR Green PCR Master
98.01% pure by HPLC analysis. Both chows
Mix (Applied Biosystems), 2.5 pmols of each
were prepared by Bio-Serv (Frenchtown, NJ).
primer and 250ng of cDNA. The PCR primers
By analysis, the maintenance diet had the
for adiponectin were: 5’-CCC AAG GGA ACT
following composition (the nominal content is
TGT GCA GGT TGG ATG-3’ and 5’-GTT
shown in parentheses): 17.20% (18.00%)
GGT ATC ATG GTA GAG AAG AAA
protein, 6.99% (7.00%) fat, 4.18% (5.00%)
GCC-3’. The PCR primers for DsbA-L were:
fiber, 2.41% (2.80%) ash, 6.56% (10.00%)
5’-GCG CCG CGC ATC CTG GAA CTC TTC
moisture, and 62.66% (59.00%) carbohydrate.
TAC-3’ and 5’-GTC TGG CAT TCG CAG
The vitamin and mineral content was as
TTG GGG GTA CAG-3’.The mRNA of
specified by the AIN-93G diet. The chows
glyceraldehyde 3-phosphate dehydrogenase
were supplied as pellets, which were placed in
(GAPDH) was quantified as an endogenous
the food hoppers in the animals' cages. Tissues
control, using the following primers:5’-ACC
were isolated from overnight fasted mice,
ACA GTC CAT GCC ATC AC-3’ and 5’-TCC
homogenized and used for Western blot to
ACC ACC CTG TTG CTG TA-3’. Reactions
determine the expression levels of DsbA-L and
without a template or without the reverse
adiponectin.
using
the
superscript
transcriptase were used as negative controls.
Data Analysis- Statistical analysis of the
The effects of RSV on adiponectin and
data was performed using analysis of variance
DsbA-L expression in mouse adipose tissue.
(ANOVA) (for cellular studies) or unpaired t
C57BL6 mice (2-month old) were fed ad
test (for animal studies). Data are presented as
libitum custom-prepared laboratory chows
mean ± S.E.M. Statistical significance was set 5
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III
transcribed
at P values of *
