Adiponectin Decreases C-Reactive Protein Synthesis and
Secretion From Endothelial Cells
Evidence for an Adipose Tissue-Vascular Loop
Sridevi Devaraj, Natalie Torok, Mohan R. Dasu, David Samols, Ishwarlal Jialal
Downloaded from http://atvb.ahajournals.org/ by guest on June 17, 2017
Background and Objective—Inflammation is pivotal in atherosclerosis. C-reactive protein (CRP), in addition to being a
cardiovascular risk marker, may also be proatherogenic. We have previously shown that in addition to the liver, human
aortic endothelial cells (HAECs) synthesize and secrete CRP. Whereas CRP levels are increased in obesity, metabolic
syndrome, and diabetes, levels of adiponectin are reduced in these conditions. We tested the hypothesis that adiponectin
reduces CRP synthesis and secretion in HAECs under normoglycemic (5.5 mmol/L glucose) and hyperglycemic
conditions (15 mmol/L glucose).
Methods and Results—Adiponectin dose-dependently reduced CRP mRNA and protein from HAECs. Adiponectin
treatment of HAECs significantly decreased I␬B phosphorylation and NF␬B binding activity. There was no effect of
adiponectin on STAT or C/EBP transcriptional activity. Adiponectin also activated AMP kinase resulting in decreased
NF␬B activity and decreased CRP mRNA and protein. These effects of adiponectin were mimicked by AICAR, an
activator of AMPK, and reversed by inhibition of AMPK. Thus, adiponectin reduces CRP synthesis and secretion from
HAECs under hyperglycemia via upregulation of AMP kinase and downregulation of NF␬B. Similar findings were
observed in rat primary hepatocytes.
Conclusions—Thus, in obesity and diabetes, the hypoadiponectinemia could exacerbate the proinflammatory state by
inducing CRP production. (Arterioscler Thromb Vasc Biol. 2008;28:1368-1374)
Key Words: CRP 䡲 adiponectin 䡲 endothelium 䡲 adipose
I
nflammation is pivotal in all phases of atherogenesis.1,2
C-reactive protein (CRP), the prototypic marker of inflammation in man, has been shown in several studies to be a
cardiovascular risk marker with high levels of CRP predicting
cardiovascular events.1–3 Much recent data challenge the
dogma that CRP is exclusively produced by the liver.3
Indeed, cogent data suggest that it is produced in atherosclerotic lesions, the kidney, neurons, and alveolar macrophages.4,5 mRNA and protein for CRP is expressed in arterial
plaque tissue, and both CRP mRNA and protein levels are
10-fold higher in plaque when compared to the normal artery,
suggesting that CRP is produced in atherosclerotic lesions.6
Furthermore, we showed that human aortic endothelial cells
synthesize and secrete CRP.7 The most potent agonist for
CRP production from HAECs is the combination of interleukin (IL)-1 and IL-6.8 In addition, the secretion of CRP is
augmented 100-fold in presence of macrophage conditioned
media (MCM).7 Thus, stimulated synthesis and secretion of
CRP by cells in the atherosclerotic lesion by paracrine/
autocrine loops could result in local concentrations of CRP
far in excess of plasma concentrations and could contribute to
proinflammatory proatherogenic effects. This could contribute to the poorer prognosis in patients with elevated CRP
levels and acute coronary syndromes.9,10 Furthermore, the
adipose tissue, previously thought to be an inert triglyceride
depot, has been shown to produce numerous adipokines
including adiponectin.
See accompanying article on page 1219
Adiponectin is a potent adipocytokine.11–16 Decreased levels
of adiponectin are found in obesity, type 2 diabetes, and
coronary artery disease. Low levels of adiponectin are inversely associated with high levels of CRP. Furthermore,
adiponectin has been shown to upregulate eNOS and decrease
cytokine and chemokine synthesis from HAECs. Recent
studies have shown that recombinant globular adiponectin, a
proteolytic cleavage product of total adiponectin is pharmacologically active, upregulates eNOS in vascular endothelial
cells, and decreases atherosclerosis in apo E⫺/⫺ mice.13,16,17
Also, there is emerging data indicating that CRP impairs
insulin signaling and adiponectin is able to improve insulin
sensitivity.18,19 A strong and inverse correlation has also
Original received January 18, 2008; final version accepted April 18, 2008.
From the Laboratory for Atherosclerosis and Metabolic Research (S.D., M.R.D., I.J.) and the Department of Internal Medicine (N.T.), UC Davis
Medical Center, Sacramento, Calif; and the Department of Biochemistry (D.S.), Case Western Reserve University, Ohio.
Correspondence to Ishwarlal Jialal, MD, PhD, Director, Laboratory for Atherosclerosis and Metabolic Research, Research Bldg I Rm 3000, 4635
Second Avenue, Sacramento, CA 95817. E-mail [email protected]
© 2008 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1368
DOI: 10.1161/ATVBAHA.108.163303
Devaraj et al
been documented between CRP and adiponectin mRNA in
human adipose tissue.20 Thus, adiponectin could be regulating CRP. However, there is a paucity of data examining
the effect of adiponectin on CRP synthesis and secretion
from HAECs. In this study, we provide novel data that
CRP synthesis and secretion from HAECs is augmented
under hyperglycemia and that pretreatment with adiponectin significantly downregulates synthesis and secretion of
CRP from HAECs.
Methods
Cell Culture
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HAECs were used between passages 3 to 5 and cultured in
normoglycemic media (NG, 5.5 mmol/L glucose) in presence or
absence of globular adiponectin (0 to 10 ␮g/mL) in 12-well culture
plates. Also, total adiponectin was used in some studies. Both total
and globular adiponectin were obtained from Peprotech Inc. HAECs
were incubated in serum- and growth factor–free media for the
expression of CRP under normoglycemic conditions (5.5 mmol/L
glucose) or hyperglycemic conditions (15 mmol/L) for 24 or 48
hours, and RNA or protein/media were collected for CRP measurements, respectively. Mannitol was used as hyperosmolar control. All
media were endotoxin-free, and adiponectin was passed through
Detoxigel.
For experiments with adiponectin, endothelial cells were pretreated with adiponectin, (0, 2.5, 5, and 10 ␮g/mL) for 2 hours
before incubating in hyperglycemic conditions. Concentrations of
adiponectin that have been used are physiological as reported
previously.12–14,18
CRP Protein Expression
The cells were collected in M-PER (Pierce Biotechnologies).
Western blotting for CRP was performed as described previously7
using rabbit antihuman CRP (Calbiochem) antibody. Western
blotting for phospho p65, phospho p38MAPK, and phospho
AMPK was performed with monoclonal antibodies (Santa Cruz
Biotechnology and Abcam Inc, respectively) and ␤-actin (mouse
monoclonal antibody; Sigma Aldrich) or the respective unphosphorylated total antibody (antip65, antip38 MAPK from Santa
Cruz Biotechnology and anti-AMPK antibody from Abcam Inc)
was used as an internal control.7
Secreted CRP by ELISA
CRP levels in the supernatants of HAECs (pooled from 3 different
wells and concentrated) were measured using an enzyme-linked
immunosorbent assay (ELISA) specific for human CRP (Alpco
Laboratories) as described previously and reported as ng/mg cell
protein.7
CRP mRNA
CRP mRNA was assessed by semi-quantitative RT-PCR and realtime RT-PCR. RNA was isolated from the cells using TRIzol
(Invitrogen). For the RT reaction, 1 ␮g of total RNA was used based
on the standard curve to synthesize the cDNA. For semiquantitative
PCR, the primers used for human CRP were: forward: 5⬘TCG TAT
GCC ACC AAG AGA CAA GAC A 3⬘ and reverse: AAC ACT
TCG CCT TGC ACT trichloroacetic acid (TCA) TAC T 3⬘ and 18s
RNA primer pair was obtained from R&D. PCR reaction was
performed using Invitrogen master mixes as previously described7,21 using 50 ng of cDNA. CRP and 18s RNA yield a band
between 400 to 600 bp, were resolved on a 2% agarose gel. Band
intensities were determined using Image quant software (GE
Healthcare and Biosciences).7 For real-time RT-PCR, we used 25
ng of cDNA for PCR reaction. Human CRP (amplicon size 80bp,
assay id# Hs00357041_m1) and 18s (amplicon size 187bp; assay
id#:Hs99999901-s1) gene expression assays were purchased from
Applied Biosystems and followed the recommended protocol. No
Adiponectin Downregulates CRP
1369
RT, no template, and positive controls were run in parallel. The
optimal amount of RNA was determined by generating a standard
curve using serial dilutions of control RNA (concentration of RNA
versus Ct values) with fixed number of cycles, and subjecting the
PCR products to gel electrophoresis for further confirmation using
18s RNA as the reference. Data were calculated using the 2-⌬⌬CT
method and presented as CRP/18s mRNA ratio normalized to
mannitol/low glucose.
NF␬B, STAT, C/EBP Binding Activity Assay
Activation of NF␬B, STAT, C/EBP in nuclear extracts was determined using TransAM assay (Active Motif). Nuclear extracts were
suspended in TransAM lysis buffer and nuclear proteins (5 ␮g total
protein) were incubated with immobilized oligonucleotides containing the NF␬B consensus DNA-binding site (5⬘-GGGACTTTCC-3⬘)
for 1 hour at room temperature. After washing, 100 ␮L of p65
subunits monoclonal antibody (1:1000 dilutions) were added for 1
hour at room temperature. After 3 washes, 100 ␮L of horseradishperoxidase– conjugated secondary antibody (1:1000 dilutions) were
added to each well for 1 hour at room temperature. The absorbance
at 450 nm was determined using a standard for NF␬B. STAT and
C/EBP activities were examined similarly using the respective
Trans-AM kits.21,22
Luciferase Transactivation Assay
Deletion promoter constructs and mutants were obtained from the
Samols laboratory. HAECs (50% to 80% confluent) were transfected
with 2 ␮g of DNA (1 ␮g luciferase reporter-CRP promoter construct
and 1 ␮g transcription factor expression vector). Luciferase transactivation assays were performed using CRP promoter constructs with
mutated NF␬B sites to evaluate their role in mediating the effect of
HG or adiponectin. After 24 hours, luciferase assays were performed
(Promega). Luciferase activity was normalized to the protein concentration of the extract measured by using a BioRad DC protein
assay kit.
Isolation of Rat Primary Hepatocytes
Primary cultures of rat hepatocytes were prepared by the method
of Gores et al.23 Briefly, rats were anesthetized, and isolated
livers were perfused via the portal vein with Ca2⫹- and Mg2⫹-free
HEPES buffer. Subsequent perfusion included 1.0 mmol/L Ca2⫹
and 0.02% collagenase. The livers were then gently raked, the cell
suspension was centrifuged, and the resulting cell pellet was
resuspended in DMEM containing 0.1% BSA, 200U/mL penicillin, and 200 mg/mL streptomycin. The cells were plated at a
density of 5⫻105 cells on rat tail collagen-coated plates in
absence and presence of adiponectin and challenged with IL1⫹IL-6 as described previously.7
Statistical analyses was performed using GraphPad Prizm software. Analysis of variance followed by paired t tests were used to
determine significant differences between treatments, and significance was set at P⬍0.05.
Results
Adiponectin Decreases CRP Synthesis and
Secretion Induced by HG in HAECs
We demonstrate that CRP mRNA and protein are significantly increased under hyperglycemic (HG) conditions and
that CRP protein and mRNA are inhibited by pretreatment
with globular adiponectin (Figure 1a and 1b). Furthermore,
secreted CRP is significantly increased in HG conditions, and
treatment with both globular (dose-dependently) and total
adiponectin inhibited CRP secreted protein induced by hyperglycemia (Figure 1c).
1370
Arterioscler Thromb Vasc Biol
July 2008
a
b
NG
+
HG
Adipo µg/ml)
(µ
+
-
+
2.5
+
5
+
10
+
5(total)
NG
+
HG
Adipo (µ
µ g/ml)
+
-
+
2.5
+
5
+
10
+
5(total)
CRP
CRP
18s RNA
1
2.2*
1.4
± 0.3 ±0.35
1.2#
1.1#
± 0.2
± 0.4 ± 0.4
β-actin
1.3#
CRP/18SRatio
0.6
± 0.1
1.3*
1.0
0.5#
0.1#
0.4#
± 0.3
±0.6
± 0.2
± 0.03 ± 0.1
CRP/Actin Ratio
*
16
14
12
10
8
6
4
2
0
(1
+T
0u
g/
ot
m
al
L)
Ad
ip
o
(5
ug
/m
L)
G
H
G
+A
di
po
+
5
H
L)
(5
ug
/m
L)
+
2.5
+A
di
po
G
+
-
H
H
(2
.5
ug
/m
G
N
-
+A
di
po
+
+
-
G
#
H
G
Mannitol
NG
HG
Adipo
(µ
µ g/ml)
#
#
M
an
ni
to
l
(ng/mg cell protein)
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Secreted protein
c
+
10
Figure 1. Effect of Adiponectin on HG-induced CRP synthesis and secretion (n⫽5). HAECs were incubated with mannitol, NG/HG (5.5/15mMglucose) in absence and presence of
globular or total adiponectin. a, Representative RT-PCR with
CRP/18S densitometric ratios. b, Representative Western
blot with CRP/␤-actin ratios. c, Secreted CRP. *P⬍0.01 vs
NG; #P⬍0.05 vs HG.
+
5(total)
Effect of Adiponectin on Transcriptional Factors
Involved in CRP Synthesis
Because the promoter for CRP in hepatocytes contains
binding elements for NF␬B, STAT, and C/EBP, we examined
the effect of HG on these 3 transcriptional factor activities, in
absence and presence of adiponectin. Whereas HG upregulated STAT-1 (Figure 2a), C/EBP-␤ (Figure 2b), and NF␬B
(Figure 2c) activities in nuclear extracts of HAECs, adiponectin failed to affect STAT and C/EBP-␤ activities. However
there was a significant downregulation of NF␬B transcriptional activity after pretreatment with adiponectin (Figure 2c).
Furthermore, in nuclear extracts, adiponectin pretreatment of HAECs (0 to 10 ␮g/mL for 24 hours) significantly decreased phosphorylation of p65 induced by HG
(Figure 2d).
Loss of NF␬B Abrogates HG-Induced CRP
Synthesis in HAECs
To confirm the role of NF␬B, we transfected HAECs with
dominant negative NF␬B or control vector. When NF␬B was
inhibited, there was significant decrease in CRP synthesis
and secretion with HG (Figure 3 a and 3b). Furthermore,
luciferase transactivation assays performed using CRP promoter constructs with mutated NF␬B sites (PC3) resulted in
loss of CRP promoter activity in presence of HG indicating
the critical involvement of NF␬B in CRP transcription in
HAECs (please see http://atvb.ahajournals.org for supplemental materials).
Effect of Adiponectin on MAPK in HAECs
Because HG activated p38MAPK but not extracellular signal
regulated kinase (ERK)/JNK, we tested the effect of adi-
ponectin on HG-induced phosphorylation of p38MAPK and
adiponectin failed to have any significant effect (data not
shown).
Adiponectin Upregulates AMPK Activity
in HAECs
Several reports indicate that adiponectin also activates
AMP kinase, we tested the effect of adiponectin on AMPK
activity and subsequent CRP release under HG conditions.
Adiponectin pretreatment resulted in significant upregulation in AMPK activity, and this was mimicked by AICAR,
250 ␮mol/L (AICAR is a cell-permeable adenosine analogue that can be phosphorylated to ZMP, an AMP
analogue and known AMPK activator; Figure 4a). In
presence of Compound C, 100 nmol/L (AMPK inhibitor,
Calbiochem), there was a significant reversal of the effects
of adiponectin on AMP kinase phosphorylation, NF␬B
activity and CRP mRNA and protein expression (Figure 4a
through 4d). Also pretreatment with either adiponectin or
AICAR resulted in decreased NF␬B binding (Figure 4d)
and decreased CRP mRNA and protein (Figure 4b and
Figure 4c, respectively), and this was reversed with Compound C (Figure 4b through 4d).
Adiponectin Decreases CRP Synthesis in Primary
Rat Hepatocytes
Lastly, because a large part of transcriptional regulation of
CRP in hepatocytes has been studied in hepatoma cell lines
(HepG2 or Hep3B cells), we isolated primary rat hepatocytes
and examined the effect of adiponectin on IL-1- and IL-6 –
mediated CRP synthesis and secretion in these primary cells.
Devaraj et al
a
50
40
30
20
10
(ng/mg protein)
60
70
CEBP/B Activity
STAT (ng/mg protein)
STAT1
STAT3
*
*
70
*
*
+
HG,15mM
+
HG+Adipo
5
60
50
40
30
20
10
0
HG (15mM)
LG, _
5mM
Adipo (5ug/mL) _
+
HG,15mM
+
HG+Adipo
5
_
0
HG (15mM)
+
c
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Binding Activity
1371
b
80
Nuclear NFKb-
Adiponectin Downregulates CRP
Adipo (5ug/mL) _
_
+
d
*
800
700
600
500
_C
#
NG +
HG
Adipo (µg/ml)
+
-
+
10
+
5
+
2.5
400
300
200
100
0
HG (15mM)
pp65
p65
C
HG,15m M
_
Adipo (5ug/mL) _
HG+Adipo 5
+
+
0.21
_
+
±0.11
0.6*
0.17#
0.19#
± 0.16 ± 0.08 ± 0.08
0.23#
pp65/p65 Ratio
±0.09
Figure 2. Effect of Adiponectin on HG-induced nuclear transcription factor activity: HAECs were incubated with NG/HG⫾adiponectin. a,
STAT1 and STAT3; b, C/EBP-␤; c, NF␬B. *P⬍0.01 vs control. *P⬍0.001 vs NG and #P⬍0.01 vs HG. d, A representative blot of phospho p65 expression with densitometric ratios of pp65/p65 (n⫽3).
As shown in Figure 5a, the combination of IL-1 and IL-6
resulted in significant upregulation in secreted CRP. This was
abrogated in presence of adiponectin. Similar results were
observed for CRP mRNA (data not shown). Furthermore, the
combination of IL-1⫹IL-6 resulted in significant increase in
C/EBP-␤, STAT 3, and NF␬B activities, and adiponectin
preincubation decreased NF␬B activity (Figure 5b).
Discussion
We report 2 novel findings in this article. First, high glucose
results in increased synthesis and secretion of CRP from
human aortic endothelial cells, and this appears to be via
upregulation of NF␬B. Secondly, we demonstrate that adiponectin abrogates the HG-induced CRP synthesis and secre-
tion via upregulation of AMP kinase and downregulation of
NF␬B activity. Furthermore, we confirm these inhibitory
effects of adiponectin on CRP synthesis in primary rat
hepatocytes.
Conditions associated with diabetes and obesity exhibit
increased cardiovascular morbidity and mortality. Thus, it is
important to clarify the molecular pathways between fat
accumulation and vascular disease. Whereas adiponectin is
expressed largely in adipose tissue, circulating levels of
adiponectin are significantly decreased in obesity, diabetes,
metabolic syndrome, and CAD.11–17 Conversely, high sensitivity CRP (hsCRP) levels are higher in these conditions of
hypoadiponectinemia. High levels of CRP are associated with
increased cardiovascular events.2,3 Also, CRP is produced
Figure 3. Loss of NF␬B abrogates HG-induced CRP: HAECs were transiently transfected with control or dominant negative I␬B vector
for 24 hours in presence of NG or HG. a, Secreted CRP. b, Representative RT-PCR gel of CRP with ratios. *P⬍0.001 compared to
mannitol/NG; #P⬍0.01 vs HG, n⫽3.
1372
Arterioscler Thromb Vasc Biol
July 2008
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Figure 4. Effect of adiponectin on HG-induced AMPK and NF␬B activity in HAECs. a, Representative Western blot for pAMPK/AMPK
with densitometric ratios. b, Representative CRP RT-PCR gel and densitometric ratios. c, Secreted CRP. d, Nuclear NF␬B DNA binding
activity. *P⬍0.01 compared to C, NG and #P⬍0.01 compared to HG, n⫽4 experiments.
locally in vascular tissue, and this leads to concentrations
10-fold excess of that in plasma.1,4,20 CRP has been shown to
be produced in macrophages, in neurons, and in the kidney
and adipocytes. We have previously shown that CRP is
synthesized and secreted by HAECs and that this is augmented several fold in presence of macrophage conditioned
media, indicating that there is cross-talk between vascular
cells, ie, macrophages and endothelial cells resulting in
increased inflammation.7 Ouchi has previously shown a
strong inverse correlation between CRP and adiponectin
mRNA.20 However, this does not imply cause and effect. In
a recent report, it has been suggested that CRP inhibits
adiponectin secretion in 3T3 cells.24 Because there are many
contaminants associated with CRP and the authors do not
appear to purify their CRP, this finding needs to be interpreted with caution. If confirmed, it will suggest that both
adiponectin and CRP can modulate each other’s secretion and
thus account for the proinflammatory burden of obesity and
diabetes. We were unable to confirm the inhibitory effect of
CRP on adiponectin using carefully purified CRP25 (data not
shown).
Diabetes and metabolic syndrome are associated with
increased inflammation.26 –28 Both IL-1 and IL-6 are potent
inducers of CRP synthesis and secretion.8 In this study, we
show that under HG conditions, HAECs synthesize and
secrete increased levels of CRP compared to normoglycemia.
In addition, we demonstrate that adiponectin significantly
abrogates HG-induced synthesis and secretion of CRP.
Adiponectin modulates endothelial function, improves endothelial NO, and has an inhibitory effect on proliferation of
vascular smooth muscle cells induced by growth factors,
inhibits collagen-induced platelet aggregation, suppresses
foam cell formation via inhibiting scavenger receptor A, and
has direct effects on improving liver and muscle insulin
sensitivity.10 –15,29 Overexpression of human adiponectin attenuated plaque formation in ApoE⫺/⫺ mice. Thus, adiponectin could have a protective effect on atherosclerosis, and it
may be the link between obesity, type 2 diabetes, and
atherosclerosis. Yamauchi et al13,30 showed that globular
adiponectin, a proteolytic cleavage product of adiponectin,
has a greater potency in reversing insulin resistance than
native uncleaved adiponectin. In endothelial cells, globular
adiponectin inhibits expression of cytokines, chemokines,
intracellular cell adhesion molecule, vascular cell adhesion
molecule, upregulates eNOS, and results in decreased
monocyte-endothelial cell adhesion and NF␬B.13,16,17,30 Although there appears to be a reciprocal relationship between
levels of adiponectin and CRP,20 especially in patients with
the metabolic syndrome or diabetes, there exist no data on the
regulation of CRP synthesis and secretion in ECs with
adiponectin. Also, previously another adipokine, leptin has
been shown to upregulate CRP synthesis and secretion in
cultured HAECs.31 Here, we present novel data that adiponectin downregulates CRP synthesis and secretion in
HAECs.
In liver-derived cell lines, the main regulators of cytokine
induced CRP synthesis are C/EBP-␤, STAT 3, and NF␬B,
Devaraj et al
Secreted CRP
(ng/mg protein)
a
500
450
400
350
300
250
200
150
100
50
0
#
*
C
IL-1+IL-6
IL-1+IL-6+Adipo 5
IL-1+IL-6+Adipo 10
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Nuclear DNA Binding Activity
b
C
IL-1+IL-6
IL-1+IL-6+Adipo 5
IL-1+IL-6+Adipo 10
1200
1000
800
*
600
400
*
* *
*
*
#
#
*
200
0
C/EBP
STAT 1
STAT 3
NFkb
Figure 5. Effect of adiponectin on CRP secretion in primary rat
hepatocytes incubated with IL-1b and IL-6 in absence and presence of adiponectin. a, Secreted CRP levels. *P⬍0.001 vs
IL-1⫹IL-6 and #P⬍0.05 vs IL-1⫹IL-6. b, Nuclear C/EBP-beta,
STAT1, STAT3, and NF␬B activity. *P⬍0.001 vs Control and
#P⬍0.05 vs IL-1⫹IL-6; n⫽3.
transcriptional regulation in HAECs has not been delineated
previously.8,32,33 Here, we show that among the transcription
factors affecting the CRP promoter, in presence of HG, there
is increased STAT1, C/EBP-␤, and NF␬B activity. In
addition, using promoter deletion constructs for CRP, we
show that CRP transcription under HG conditions in
HAECs is NF␬B-centric. Furthermore, transfection of
HAECs with dominant negative NF␬B abrogated the
HG-induced CRP synthesis and secretion, confirming the
crucial role of NF␬B in HG-induced transcription of CRP.
Previously, adiponectin has been shown to downregulate
NF␬B activity in endothelial cells resulting in decreased
expression of the cell adhesion molecules and IL-8.34,35 In
this study, we provide evidence that adiponectin downregulates CRP synthesis and secretion in HAECs under HG
conditions via inhibition of NF␬B.
AMPK is a highly conserved heterotrimeric signaling
kinase responsive to hypoxia, exercise, and cellular stress.36,37
Adiponectin metabolic signaling in liver, skeletal muscle, and
adipose has been shown to be mediated via AMPK activation.38 – 40 Also, adiponectin has been shown to increase nitric
oxide synthesis via activation of AMPK.41 Under HG conditions, apoptosis in ECs as well as diminished ability of insulin
to activate Akt was prevented by activation of AMPK by
AICAR, suggesting that AMPK may play a key role in
protecting ECs from the adverse effects of HG.39
In support of these studies, we also show that adiponectin
upregulates AMPK phosphorylation in HAECs under HG
conditions, these effects are mimicked by AICAR, a known
activator of AMPK activity, and inhibited by addition of
compound C, a specific inhibitor of AMPK phosphorylation.
Adiponectin decreases macrophage phagocytic capacity
through cross-talk between AMPK and NF␬B signaling
Adiponectin Downregulates CRP
1373
pathways.42,43 We show in HAECs that adiponectin upregulates AMPK, resulting in decreased NF␬B activity, and
subsequent synthesis and secretion of CRP under HG
conditions.
The liver is the primary source of CRP synthesis, thus we
also examined the effect of adiponectin in primary rat
hepatocytes that were induced to produce CRP with the
combination of IL-1 and IL-6, because such treatment has
previously been shown to induce CRP synthesis in Hep3B
and HepG2 cells.8,33 We provide novel data that adiponectin
decreases IL-1– and IL-6 –induced CRP synthesis and secretion in primary rat hepatocytes and that this may be attributable to downregulation of C/EBP and NF␬B activities.
Further studies in this model will elucidate the molecular
pathways by which adiponectin exerts these effects.
In addition to the growing antiatherogenic and antidiabetic properties of adiponectin, we provide novel evidence
that adiponectin abrogates HG-induced CRP synthesis and
secretion via upregulation of AMPK and downregulation
of NF␬B. Our observations provide a fundamental mechanism for the link between adiposity and endothelial
dysfunction and suggest that adiponectin upregulation
could be beneficial in modulating the proinflammatory/
prothrombotic effects of CRP.3
Acknowledgments
We appreciate the gift from Dr Samols of the CRP promoter
deletion constructs.
Sources of Funding
This work was supported by NIH K24AT00596 and HL74360.
Disclosures
None.
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Adiponectin Decreases C-Reactive Protein Synthesis and Secretion From Endothelial
Cells: Evidence for an Adipose Tissue-Vascular Loop
Sridevi Devaraj, Natalie Torok, Mohan R. Dasu, David Samols and Ishwarlal Jialal
Arterioscler Thromb Vasc Biol. 2008;28:1368-1374; originally published online May 1, 2008;
doi: 10.1161/ATVBAHA.108.163303
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Supplement
Figure
Luciferase Reporter Activity
(Fold over basal RLU)
120
100
80
60
40
*
20
0
HG+PC1
HG+PC2
HG+PC3
Cells were transiently transfected with the promoter deletion constructs
(1μg) along with luciferase reporter and luciferase activity was performed as
described in Methods.
Promoter Construct 1(PC1)- has all 3 , NFκb,C/EBP and STAT 3 promoter
elements; PC2- C/EBP deletion construct; PC3- NFκb deletion construct
*p<0.001 compared to HG+PC1 and HG+PC2.