In this study, we investigated whether PPARc modulates visfatin expression in murine bone marrow-derived macrophage and human primary human resting macrophage, classical macrophage, alte
Trang 1receptor gamma in human macrophages
The´re`se He`rve´e Mayi1,2,3,4, Christian Duhem1,2,3,4, Corinne Copin1,2,3,4, Mohamed Amine
Bouhlel1,2,3,4, Elena Rigamonti1,2,3,4, Franc¸ois Pattou1,5,6, Bart Staels1,2,3,4and Giulia
Chinetti-Gbaguidi1,2,3,4
1 Univ Lille Nord de France, France
2 Inserm, Lille, France
3 UDSL, Lille, France
4 Institut Pasteur de Lille, France
5 Service de Chirurgie Ge´ne´rale et Endocrinienne, Centre Hospitalier Re´gional et Universitaire de Lille, France
6 Inserm ERIT-M 0106, Faculte´ de Me´decine, Lille, France
Keywords
adipocytokines; inflammation; macrophages;
nuclear receptors; visfatin
Correspondence
Bart Staels, Inserm UR 1011, Institut
Pasteur de Lille, 1, rue du Professeur
Calmette, BP 245, Lille 59019, France
Fax: +33 3 20 87 73 60
Tel: +33 3 20 87 73 88
E-mail: bart.staels@pasteur-lille.fr
(Received 15 January 2010, revised 27 April
2010, accepted 3 June 2010)
doi:10.1111/j.1742-4658.2010.07729.x
Obesity is a low-grade chronic inflammatory disease associated with an increased number of macrophages (adipose tissue macrophages) in adipose tissue Within the adipose tissue, adipose tissue macrophages are the major source of visfatin⁄ pre-B-cell colony-enhancing factor ⁄ nicotinamide phos-phoribosyl transferase The nuclear receptor peroxisome proliferator-acti-vated receptor gamma (PPARc) exerts anti-inflammatory effects in macrophages by inhibiting cytokine production and enhancing alternative differentiation In this study, we investigated whether PPARc modulates visfatin expression in murine (bone marrow-derived macrophage) and human (primary human resting macrophage, classical macrophage, alterna-tive macrophage or adipose tissue macrophage) macrophage models and pre-adipocyte-derived adipocytes We show that synthetic PPARc ligands increase visfatin gene expression in a PPARc-dependent manner in primary human resting macrophages and in adipose tissue macrophages, but not in adipocytes The threefold increase of visfatin mRNA was paralleled by an increase of protein expression (30%) and secretion (30%) Electrophoretic mobility shift assay experiments and transient transfection assays indicated that PPARc induces visfatin promoter activity in human macrophages by binding to a DR1–PPARc response element Finally, we show that PPARc ligands increase NAD+ production in primary human macrophages and that this regulation is dampened in the presence of visfatin small interfering RNA or by the visfatin-specific inhibitor FK866 Taken together, our results suggest that PPARc regulates the expression of visfatin in macro-phages, leading to increased levels of NAD+
Abbreviations
AcLDL, acetylated low-density lipoprotein; AP-1, activator protein 1; ATM, adipose tissue macrophages; EMSA, electrophoretic mobility shift assay; IL, interleukin; NAMPT, nicotinamide phosphoribosyl transferase; M1, classical pro-inflammatory macrophage phentotype; M2, alternative anti-inflammatory macrophage phenotype; NF-jB, nuclear factor-kappaB; NR, nuclear receptor; PBEF, pre-B-cell colony-enhancing factor; PPARc, peroxisome proliferator-activated receptor gamma; PPRE, peroxisome proliferator-activated receptor response elements; Q-PCR, quantitative PCR; ROS, reactive oxygen species; RM, resting macrophages; RSG, rosiglitazone; RXR, retinoic X receptor; siRNA, small interfering RNA; SIRN, sirtuin (silencing mating type information regulation 2 homolog); SMC, smooth muscle cells; TNF-a, tumor necrosis factor alpha.
Trang 2Originally discovered in liver, skeletal muscle and
bone marrow, and also known as pre-B-cell
colony-enhancing factor (PBEF), a cytokine acting in B-cell
differentiation [1], visfatin, is nicotinamide
phosphori-bosyl transferase (NAMPT) [2,3], a rate-limiting
enzyme in the synthesis of NAD+ from
nicotin-amide Visfatin⁄ PBEF ⁄ NAMPT is synthesized and
secreted in adipose tissue by adipocytes, and mostly
by macrophages, and circulates in the plasma of
humans and mice [4] Plasma concentrations of
visfa-tin are positively associated with cytokines such as
interleukin (IL)-6 and increase in morbidly obese
subjects Elevated circulating levels of visfatin have
been observed in many inflammatory diseases such
as rheumatoid arthritis, obesity, insulin resistance
and type 2 diabetes [5–7] Visfatin is secreted by
neutrophils in response to inflammatory stimuli and
is regulated in monocytes by pro-inflammatory
fac-tors such as IL-1b, tumor necrosis factor alpha
(TNF-a), IL-6 via nuclear factor-kappaB (NF-jB)
and AP-1-dependent mechanisms [8–10] Visfatin
acti-vates pro-inflammatory signalling pathways in human
endothelial and vascular smooth muscle cells (SMC)
through reactive oxygen species (ROS)-dependent
NF-jB activation or NAMPT activity, respectively,
and therefore could provide a link between obesity
and atherothrombotic diseases [11,12] Visfatin
func-tions as an extracellular and intracellular NAD
bio-synthetic enzyme that converts, in mammals,
nicotinamide (a form of vitamin B3) to NMN,
a NAD precursor Thus, the NAD pool is maintained,
at least in part, by visfatin, which is important, for
instance, in b-cell insulin secretion [2] Although still
controversial, visfatin is thought to have insulin
mimetic effects and, similarly to insulin, visfatin
enhances glucose uptake by myocytes and adipocytes
and inhibits hepatocyte glucose release in vitro [13,14]
Altogether, the pleiotropic role of visfatin suggests that
the regulation of NAD+synthesis is critical for several
aspects of cell physiology [15]
Macrophages, crucial cells in the development of
inflammatory and metabolic disorders such as
athero-sclerosis and obesity, are a heterogeneous cell
popula-tion that adapts and responds to a large variety
of microenvironmental signals [16] The activation
states and functions of macrophages are regulated
by several cytokines and microbial products T helper
1 cytokines, such as interferon-gamma, IL-1b or
lipopolysaccharide (LPS), induce activation of a
classi-cal pro-inflammatory macrophage phenotype (M1),
whereas T helper 2 cytokines, such as IL-4 and IL-13,
induce an alternative anti-inflammatory macrophage phenotype (M2) [17] In macrophages, many genes are regulated by transcription factors, such as the nuclear receptors (NRs), which translate physiological signals into gene regulation Peroxisome proliferator-activated receptor gamma (PPARc) is a NR that regulates genes controlling lipid, glucose metabolism and inflammation After activation by its ligands, PPARc forms a hetero-dimer with the retinoic X receptor (RXR) [18] The binding of this heterodimer to specific DNA sequences, called PPAR response elements (PPRE), results in the regulation of its target genes [18] In this way PPARc modulates crucial pathways of adipocyte differentiation and lipid metabolism, thus impacting on glucose metab-olism and insulin sensitivity Furthermore, activated PPARc inhibits inflammatory response genes by nega-tively interfering with the NF-jB, signal transducers and activators of transcription (STAT) and AP-1 signal-ing pathways in a DNA-bindsignal-ing independent manner [19] This trans-repression activity is probably the basis for the anti-inflammatory properties of PPARc
PPARc is activated by natural or synthetic ligands such as GW1929 and the antidiabetic thiazolidinedi-ones rosiglitazone (RSG) and pioglitazone [20] PPARc expression is very low in human monocytes, but is induced upon differentiation into macrophages and is present in foam cells of atherosclerotic lesions [21–23] More recently, PPARc has been shown to enhance the differentiation of monocytes into alterna-tive anti-inflammatory M2 macrophages [24,25] and to promote the infiltration of M2 macrophages into adi-pose tissue [26] Consistent with these results, selective inactivation of macrophage PPARc in BALB⁄ c mice results in an impairment in the maturation of alterna-tively activated M2 macrophages and in the exacerba-tion of diet-induced obesity, insulin resistance, glucose intolerance and expression of inflammatory mediators [24,27] All these studies provide evidence that macro-phage PPARc is a central regulator of inflammation and insulin resistance
Here, we identify visfatin as a novel PPARc-regu-lated gene in human macrophages Interestingly, PPARc activation enhanced visfatin gene expression in both M1 and M2 human macrophages, but not in murine macrophages or in human adipocytes Finally,
we show that intracellular NAD+concentrations cor-relate with visfatin protein expression upon PPARc ligand activation Reduction of visfatin expression and activity by small interfering RNA (siRNA) or a spe-cific inhibitor abolished the PPARc-mediated increase
of NAD+
Trang 3PPARc agonists induce visfatin gene expression
in human macrophages in a PPARc-dependent
manner
To investigate whether PPARc regulates visfatin gene
expression, quantitative PCR (Q-PCR) analysis was
performed in primary human resting macrophages
(RM) upon PPARc activation Time course
experi-ments showed that visfatin induction was already
observed after 9 h of stimulation with GW1929
(600 nm) or RSG (100 nm) and became maximal at
24 h (Fig 1A), with no significant further increase
after 48 h (data not shown) Treatment of RM with
increasing concentrations of the PPARc ligands
GW1929 (300, 600 and 3000 nm) or RSG (50, 100 and
1000 nm) for 24 h significantly increased visfatin
mRNA levels in a concentration-dependent manner
(Fig 1B) Expression of CD36, a known PPARc target
gene [23], was also induced to a similar extent in a dose-dependent manner (data not shown) Interest-ingly, visfatin regulation by PPARc was also observed
in macrophage foam cells, obtained by acetylated low-density lipoprotein (AcLDL) loading (Fig 1C) Moreover, GW1929 (600 nm) also regulated visfatin expression in infiltrated adipose tissue macrophages (ATM) derived from visceral fat depots (Fig 1D) To determine whether PPARc agonists up-regulate visfatin expression in a PPARc-dependent manner, the effect
of GW1929 (600 nm) was analysed in the presence or
in the absence of the PPARc inhibitor, T0070907 (1 lm) [28] T0070907 abolished GW1929-induced visf-atin mRNA expression (Fig 1E) Furthermore, infec-tion of RM with PPARc-expressing adenovirus resulted in a significant further increase of visfatin expression in the presence of the agonist (Fig 1F) Expression of two PPARc target genes, CD36 and FABP4 (aP2), used as positive controls, was also increased (Fig 1G,H) Taken together, these data
AcLDL
1
2
Visfatin/cyclophilin mRNA Visfatin/cyclophilin mRNA CD36/cyclophilin mRNA FABP4/cyclophilin mRNA
Visfatin/cyclophilin mRNA Visfatin/cyclophilin mRNA Visfatin/cyclophilin mRNA
3
4
3 h 6 h 9 h 12 h 24 h
GW1929
RSG
Control
A
** **
***
***
1 2 3 4
6 5
**
***
***
C
1 2 3 4
B
0
*
*
*
*
*
5
D
0.5 1 1.5 2
2.5
*
ATM
1
2
3
Control T00709
E
*
1 2 3
AdGFP AdPPAR
γ
AdGFP AdPPAR
γ
AdGFP AdPPAR
γ
F
4
RSG Control
***
***
*
Control GW1929
§
RSG Control
RSG
Control
Control GW1929
1 2
3 RSG Control
G
1 2 3 4 5 6 7
***
***
*
***
***
*
RSG Control H
Fig 1 PPARc agonists regulate visfatin gene expression in human macrophages in a PPARc-dependent manner Primary human
macrophag-es were incubated or not (control) with (A) GW1929 (600 n M ) or RSG (100 n M ), for the indicated periods of time, or (B) with GW1929 (300,
600 and 3000 n M ) or RSG (50, 100 and 1000 n M ) for 24 h, or (C) were transformed into foam cells by AcLDL (50 lgÆmL)1) loading before treat-ment with PPARc ligands (D) Human visceral ATM were treated with GW1929 (600 n M ) for 24 h (E) Primary human monocytes were differ-entiated in macrophages in the presence or absence of GW1929 (600 n M ), T0070907 (1 l M ), or both, which were added at the start of the differentiation Primary human macrophages were infected with recombinant adenovirus AdGFP or AdPPARc and treated with RSG (100 n M ) for 24 h Visfatin (F), CD36 (G) and FABP4 (H) mRNA were analyzed by quantitative PCR and normalized to cyclophilin mRNA The results are representative of those obtained from three independent macrophage preparations and are expressed relative to the levels in untreated cells set as 1 Each bar is the mean value ± SD of triplicate determinations Statistically significant differences between treatments and controls are indicated (t-test; *P < 0.05; **P < 0.01; ***P < 0.001; T00709 + G929 versus GW1929 § P < 0.05).
Trang 4demonstrate that PPARc ligands induce visfatin gene
expression in human macrophages through a
PPARc-dependent mechanism
PPARc agonists do not regulate visfatin gene
expression in murine macrophages or human
adipocytes
To determine whether regulation of visfatin also occurs
in mouse macrophages, experiments were performed in
murine bone marrow-derived macrophages that were
treated with GW1929 (1200 nm) or RSG (1000 nm) for
24 h PPARc activation did not increase visfatin gene
expression, although expression of CD36 was induced
(Fig 2A,B) Similar results were observed with the
murine macrophage cell line, RAW264.7, when
incu-bated with increasing concentrations of GW1929 and
RSG (data not shown) Furthermore, activation of
PPARc by exposure to GW1929 (600 nm) for 24 h did
not lead to an increased expression of visfatin in
human mature adipocytes derived from the
differentia-tion of primary pre-adipocytes in vitro, while the expression of CD36 was strongly induced (Fig 2C,D) Similar results were obtained in the murine pre-adipo-cyte cell line, 3T3L1, after treatment with RSG or pioglitazone (data not shown), in line with a previous report [29]
PPARc regulates visfatin gene expression at the transcriptional level
To determine whether visfatin is a direct PPARc target gene, the human visfatin promoter was examined by bio-informatic analysis Three putative DR1-like PPRE motifs were identified in the 2150-bp sequence upstream of the ATG start site of the visfatin gene [30] Among these sites, only the putative PPRE identi-fied at position -1501⁄ -1513 (AGGGCA A AGATCA) was found to be functional in electrophoretic mobility shift assay (EMSA) experiments (Fig 3A) Incubation
of the labeled -1501⁄ -1513 visfatin–PPRE oligonucleo-tide with in vitro-translated PPARc and RXRa resulted in the formation of a retarded complex (Fig 3A, lane 6) The binding specificity of PPARc to this DR1–visfatin–PPRE site was demonstrated by competitive inhibition with excess cold unlabeled wild-type (Fig 3A, lanes 7-11), but not mutated (Fig 3A, lanes 12-17), visfatin–PPRE oligonucleotide, as well as
by the supershift with a specific anti-human PPARc IgG1(Fig 3A, lane 18) Binding of RXRa and PPARc
to labelled DR1-consensus PPRE was assayed as a positive control (Fig 3A, lane 2)
To determine whether PPARc activates transcription from the (-1501⁄ -1513) PPRE site, six copies of this element were cloned in front of the heterologous her-pes simplex virus thymidine kinase promoter to obtain the (DR1–visfatin–PPRE)6x-Tk-Luc luciferase reporter vector Co-transfection of the pSG5–PPARc expres-sion vector with the (DR1–visfatin PPRE)6 reporter vector in primary human RM led to a significant induction of transcriptional activity compared with the pSG5 empty vector, an effect enhanced in the presence
of GW1929 (600 nm) (Fig 3B) The consensus DR1– PPRE site cloned in six copies (DR1–consensus PPRE)6, used as a positive control, was strongly induced by PPARc (Fig 3B) Taken together, these results indicate that visfatin is a direct PPARc target gene in human macrophages
PPARc activation induces visfatin gene expression in M1 and M2 macrophages
As macrophages are heterogeneous cells [16,17], we decided to investigate whether induction of visfatin
2
RSG
Control
GW1929
*
1.2
1.4
RSG GW1929
*
1
0.4 0.6 0.8 1
0.2
3
mCD36/cyclophilin mRNA mVisfatin/cyclophilin mRNA
1.4
2
2.5
1 1.2
***
Control
GW1929
Control GW1929
1
1.5
CD36/cyclophilin mRNA Visfatin/cyclophilin mRNA
0.4 0.6 0.8
Fig 2 PPARc agonists do not regulate visfatin gene expression in
murine macrophages or human adipocytes (A, B) Murine bone
marrow-derived macrophages (BMDM) were incubated or not
(con-trol) in the presence of PPARc ligands GW1929 (1.2 l M ) or RSG
(1 l M ) (C, D) Human mature adipocytes derived from the
differenti-ation of pre-adipocytes in vitro were incubated or not (control) in
the presence of PPARc ligands GW1929 (600 n M ) CD36 (A, B) and
visfatin (C, D) mRNA were analyzed using quantitative PCR and
normalized to cyclophilin mRNA The results are representative of
at least three independent cell preparations and are expressed
rela-tive to the levels in untreated cells set as 1 Each bar is the mean
value ± SD of triplicate determinations Statistically significant
differences between treatments and controls are indicated (t-test;
*P < 0.05; ***P < 0.001).
Trang 5also occurs after PPARc activation in classical (M1) or
alternative (M2) macrophages Human monocytes were
differentiated in vitro into RM macrophages and
acti-vated into inflammatory M1 macrophages with
recom-binant human TNF-a (5 ngÆmL)1), IL-1b (5 ngÆmL)1)
or LPS (100 ngÆmL)1) As expected [8], expression of
visfatin was strongly induced by pro-inflammatory
stimuli (Fig 4A) Interestingly, the effects of TNF-a
and LPS treatment were amplified in the presence of the PPARc agonist GW1929 (Fig 4A) Under the same experimental conditions, PPARc inhibited the induction of TNF-a or IL-1b induced by inflammatory stimuli, indicative of its anti-inflammatory activity (data not shown)
In parallel experiments, human monocytes were dif-ferentiated in vitro into M2 macrophages with recom-binant IL-4 (15 ngÆmL)1) in the absence or in the presence of the PPARc agonist GW1929 added at the start of the differentiation process [25] As shown in Fig 4B, the expression of visfatin was significantly decreased by IL-4 stimulation However, as with RM, the PPARc agonist GW1929 enhanced visfatin gene expression in M2 macrophages A similar regulation was observed in monocytes differentiated into M2 macrophages in the presence of IL-13 (data not shown)
PPARc activation regulates visfatin protein expression and secretion in human macrophages
To determine whether visfatin gene induction by PPARc agonists leads to an increased protein level,
A
Ab anti-PPAR γ Lysate RXR Lysate
α + PPAR
γ
PPAR γ RXR
α
RXR α + PPARγ Cold DR1 Visfatin-PPRE Cold DR1 Visfatin-PPRE
2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1
DR1-consensus
PPRE
DR1-Visfatin-PPRE wt
3
B
pSG5
**
2
4
6
8
10
12
14
(DR1-Visfatin PPRE)6
pSG5-PPAR γ
Control
GW1929
§§
(DR1-consensus PPRE)6
**
*
10 20 30 40 50
60
70 Control
GW1929
pSG5 pSG5-PPAR γ
§§§
Fig 3 PPARc binds to and activates a PPRE in the human visfatin
gene promoter (A) EMSAs were performed using the end-labeled
DR1–consensus–PPRE (lanes 1 and 2) or the DR1–visfatin–PPREwt
oligonucleotide in the presence of unprogrammed reticulocyte lysate
or in vitro-translated human PPARc and human RXRa (lanes 3–5).
Competition experiments were performed in the presence of excess
cold unlabeled wild-type (wt) (lanes 6–11) or mutated (mut) DR1–
visfatin–PPRE oligonucleotides (lanes 12-17) Supershift assays
were performed using an anti-human PPARc Ig (lane 18) (B) Primary
human macrophages were transfected with the indicated reporter
constructs (DR1–visfatin–PPRE) 6 or (DR1–consensus–PPRE) 6 , in the
presence of pSG5 empty vector or pSG5–PPARc Cells were treated
or not (Control) with GW1929 (600 n M ) and luciferase activity
was measured Statistically significant differences are indicated
(pSG5 versus pSG5-PPARc;§§P < 0.01,§§§P < 0.001; control versus
GW1929 *P < 0.05, **P < 0.01) b-gal, beta-galactosidase; RLU,
relative luciferase units.
1 2 Visfatin/cyclophilin mRNA Visfatin/cyclophilin mRNA
3 4 5 6 7
IL-1β
TN
F-α
LPS
2
GW1929 Control
*
*
***
***
**
**
ns
0.5 1 1.5 2 2.5
§§
Control
**
**
*
*
*
GW1929
Fig 4 PPARc agonists induce visfatin gene expression in M1 and M2 macrophages (A) Primary human monocytes were differenti-ated to RM and tredifferenti-ated for 24 h with GW1929 (600 n M ) Where indicated, RM were activated to M1 macrophages by incubation with recombinant human TNF-a (5 ngÆmL)1) or recombinant human IL-1b (5 ngÆmL)1) for 4 h or with LPS (100 ngÆmL)1) for 1 h after GW1929 treatment (B) Primary human monocytes were differenti-ated in RM or M2 macrophages in the presence of IL-4 (15 ngÆmL)1), and the PPARc agonist GW1929 (600 n M ) was added
or not during the differentiation process Visfatin mRNA was ana-lyzed using Q-PCR and normalized to cyclophilin mRNA The results are representative of those obtained from five independent macro-phage preparations and are expressed relative to the levels in untreated cells set as 1 Each bar is the mean value ± SD of triplicate determinations Statistically significant differences between treatments and controls are indicated (control versus PPARc agonists *P < 0.05, ***P < 0.001; control versus cytokines
§ P < 0.05, §§ P < 0.01).
Trang 6western blot analysis was performed on human RM
treated with GW1929 (600 nm) or dimethylsulfoxide
for 24 h Activation of PPARc caused a significant
increase (approximately 30%) of visfatin protein
expression (Fig 5A) To examine whether this
induc-tion was followed by an increased secreinduc-tion, we exam-ined the ability of PPARc to stimulate visfatin release
As shown in Fig 5B, GW1929 markedly increased (approximately 30%) the visfatin concentration in macrophage supernatants after 24 h of treatment
PPARc activation increases the intracellular NAD+ concentration in human macrophages
As visfatin is known as a nicotinamide phosphoribosyl transferase [2], we investigated whether the induction
of visfatin by PPARc affects the concentration of NAD+ Human RM were treated or not with GW1929 (600 nm) for 24 h and intracellular NAD+ levels were determined using an enzymatic assay Our results showed that PPARc activation significantly enhances the cellular NAD concentration (Fig 6), an effect in line with the observed induction of visfatin expression (Figs 1 and 5)
To determine whether the NAD+ enhancement by PPARc was dependent on visfatin induction, experi-ments were performed in RM macrophages in the absence or in the presence of a specific visfatin siRNA Q-PCR analysis showed a significant decrease in visfa-tin gene expression after siRNA (scrambled = 1 ± 0.019 versus siRNA visfatin = 0.27 ± 0.01), whereas PPARc activation increased visfatin gene expres-sion (scrambled + GW1929 = 2.04 ± 0.4 and siRNA visfatin + GW1929 = 0.51 ± 0.022) siRNA-mediated visfatin knockdown resulted in a reduction of the basal, as well as of the GW1929-induced, NAD+ con-centration (Fig 6A) Moreover, experiments performed
Visfatin β-actin
GW1929 Control
Control GW1929
0.4
0.8
1.2
1.6
GW1929 Control
0.2
0.6
1.0
0.5 1 1.5 2 2.5
Fig 5 PPARc regulates visfatin protein expression and secretion
in primary human macrophages Primary human macrophages were
treated or not (control) with GW1929 (600 n M ) for 24 h (A)
Intracel-lular visfatin and b-actin protein expression was analyzed by
wes-tern blotting and relative signal intensities were quantified using
Quantity One Software The results are representative of four
inde-pendent macrophage preparations and are expressed relative to the
levels in untreated cells set as 1 (B) Secretion of visfatin protein
was quantified in the macrophage supernatant using ELISA The
results are representative of three independent macrophage
prepa-rations Each bar is the mean value ± SD of triplicate
determina-tions Statistically significant differences between treatments and
controls are indicated (t-test; *P < 0.01; ***P < 0.001).
100 120 140
100 120 140
*
*
*
GW1929 Control
60 80
60 80
§
**
+ concentration (%)
20 40
Scrambled siRNA visfatin
20 40
+ concentration (%)
+ concentration (%)
Vehicle
150
RSG
Control GW1929
§
** **
§
GW1929 Control
130 140
§§
110 120
130
*
90
AdPPARγ 100
Fig 6 PPARc activation affects intracellular NAD concentrations in primary human macrophages Primary human macrophages were trans-fected or not with non-silencing control or silencing siRNA against human visfatin (A), or treated or not with the visfatin inhibitor FK866 (100 n M ) (B), or infected or not with PPARc-expressing (AdPPARc) or GFP (AdGFP) adenovirus (C) and subsequently treated with GW1929 (600 n M ), RSG (100 n M ) or dimethylsulfoxide for 24 h Cells were lysed in NAD extraction buffer and the NAD + concentrations were mea-sured using an enzymatic cycling reaction assay, normalized to protein levels and expressed as a percentage, the control non-stimulated cells being expressed as 100% The results are representative of those obtained from three independent macrophage preparations The val-ues are means ± SD of triplicates Statistically significant differences are indicated (t-test; control versus PPARc agonists, *P < 0.05,
**P < 0.01; scrambled versus siRNA visfatin or vehicle versus FK866, § P < 0.05, §§ P < 0.05; AdGFP + PPARc agonists versus AdPPARc + PPARc agonists, § P < 0.05) GFP, green fluorescent protein.
Trang 7in the presence of a specific noncompetitive inhibitor
of visfatin (FK866) in the presence or absence of
GW1929 demonstrated that the induction of NAD+
by GW1929 was inhibited in the presence of FK866
(Fig 6B) Finally, PPARc over-expression increased
NAD+ levels, an effect enhanced by its synthetic
ligands GW1929 and RSG (Fig 6C)
Discussion
Visfatin has been suggested to act as an
inflamma-tory mediator, being expressed in blood monocytes
and foam cell macrophages within unstable
athero-sclerotic lesions where it potentially plays a role in
plaque destabilization [8,31] Visfatin induces
leuko-cyte adhesion to endothelial cells by inducing the
expression of the cell-adhesion molecules intercellular
adhesion molecule 1 (ICAM-1) and vascular cell
adhesion molecule 1 (VCAM-1), thus potentially
contributing to endothelial dysfunction [11]
More-over, visfatin increases matrix metalloproteinase-9
activity and the expression of TNF-a and IL-8 in
THP-1 monocytes [8] These effects of visfatin were
abolished when insulin receptor signalling was blocked
[8], in line with the report that visfatin could bind and
activate insulin receptors [14] However, the
insulin-mimetic actions of visfatin are still debated [13] All
these data suggest that visfatin might be a player
linking several inflammatory pathologies, including
obesity-associated insulin resistance, diabetes mellitus
and vascular wall dysfunctions [9,32]
In this study we showed that PPARc activation
up-regulates the expression of visfatin in human
mono-cyte-derived macrophages and ATM This induction is
concentration dependent and does not occur during
the short incubation time generally required for
macro-phage activation, but requires an incubation period of
more than 9 h The maximum effect was obtained at
24 h with no significant further increase at 48 h (data
not shown) In addition, treatment with AcLDL
induced visfatin mRNA levels, and PPARc activation
further increased visfatin expression in these
AcLDL-loaded macrophages
By over-expressing PPARc with adenovirus
con-structs, or by inhibiting PPARc with a specific
antago-nist, we demonstrated that PPARc agonists induce
visfatin gene expression in a PPARc-dependent
man-ner [33] By bio-informatics analysis, we detected the
presence of three DR1-like motifs that might serve as
PPREs in the 2150-bp sequence upstream of the ATG
codon of the human visfatin gene [30] Using EMSA
and transient transfection experiments in primary
human macrophages, a functional PPRE was identified
at position -1501⁄ -1513 within the promoter This PPRE is distinct from the described AP-1 or NF-jB– response element (RE) like elements (located at posi-tion -1757⁄ -1767) within the human visfatin promoter [30] This can explain our observation that inflamma-tory cytokines and PPARc agonists have an additive effect on visfatin mRNA expression, an effect appar-ently in contrast to the known anti-inflammatory actions of PPARc in macrophages as a result of its ability to interfere with the NF-jB and AP-1 signaling pathways [19] This is similar to what has already been reported for other nuclear receptors, such as liver X receptor, for which short-term pretreatment with liver
X receptor agonists significantly reduced the LPS-induced inflammatory response, whereas 24-h pretreat-ment of macrophages with agonists resulted in an enhanced inflammatory response [34]
PPARc agonists induce visfatin protein expression and secretion in human primary macrophages Visfatin
is a secreted cytokine-like protein [35], although it has been speculated that the release of visfatin may be caused either by cell lysis or by cell death [36,37] However, it has been demonstrated in adipocytes and Chinese Hamster ovary (CHO) cells that visfatin is actively secreted through a nonclassical (non-Golgi⁄ endoplasmic reticulum system) secretory path-way [2] In our experiments we did not observe any cellular toxicity after treatment with PPARc agonists, suggesting that the secretion of visfatin in human mac-rophages may be an active process
As visfatin is the rate-limiting enzyme for the con-version of nicotinamide to NAD+ in mammals, the increased concentration of intracellular NAD+ induced by PPARc agonists is probably the conse-quence of visfatin induction NAD+ modulates vari-ous signalling pathways For instance, it regulates the transcription and function of NAD+-dependent
SIR-Ts, and increased expression of visfatin upregulates sirtuin 1(SIRT1) activity [2] The observed variation of intracellular NAD+concentrations after visfatin mod-ulation (by siRNA or PPARc activation) are in the same order of magnitude as previously reported in murine NIH-3T3 fibroblasts transduced with visfatin-specific small hairpin RNA (shRNA) The reduction of intracellular visfatin protein in these cells led to a reduction of NAD+levels from 20% to 40%, whereas cells over-expressing visfatin displayed a 15–25% increase in total intracellular NAD+ levels [38] By using the pharmacological visfatin inhibitor, FK866, a significant decrease in the intracellular NAD+ concen-tration was observed, even in the presence of PPARc ligand, confirming the role of the enzymatic activity of visfatin and the possibility that PPARc can modulate
Trang 8intracellular NAD+ levels via an increase of visfatin
expression Indeed, a small increase in the
concentra-tion of NAD+in response to GW1929 in siRNA
visf-atin treated-macrophages was observed, suggesting
that an additional PPARc-related pathway might
mod-ulate NAD+levels
Moreover, we have shown that PPARc agonists
increase the expression of visfatin in macrophages
irre-spective of their M1 or M2 polarization
Visfatin-depen-dent recycling of nicotinamide to NAD+may represent
a physiologically important homeostatic mechanism to
avoid depletion of the intracellular NAD+pool during
its active use as a substrate by sirtuins, cADP-ribose
synthases or PARPs [15] It has recently been shown
that pharmacological SIRT1 activators exert broad
anti-inflammatory effects in macrophages [39]
Con-versely, SIRT1 knockdown leads to an increase in the
basal expression of TNF-a, monocyte chemoattractant
protein 1 (MCP-1) and keratinocyte-derived chemokine
(KC) The activity of SIRT1 requires an increase of
visfatin expression to compensate for the consumption
of NAD+ Van Gool et al have identified SIRT6,
another member of the sirtuin family, as the
NAD-dependent enzyme able to increase TNF-a production
in macrophages by acting post-transcriptionally [40]
Taken together, these observations suggest that NAD+
can exert pro- and⁄ or anti-inflammatory properties
depending on the activated sirtuins
It is also possible that macrophage-produced visfatin
has a local paracrine effect on surrounding cells, such
as SMC, within atherosclerotic plaques, because in
vascular SMC, over-expression of visfatin promotes
cell maturation by regulating NAD+-dependent SIRT
deacetylase activity [41] Visfatin has been reported as
a longevity protein that extends the life span of human
SMC, suggesting that visfatin allows vascular cells to
resist stress and senescence, a hallmark of
atheroscle-rotic lesions [42] The ability of visfatin to prolong the
longevity of vascular SMC might contribute to the
sta-bilization efficiency of a developing atherosclerotic
lesion by SMC Treatment of humans with PPARc
ligands does not alter adipose visfatin gene expression
and circulating visfatin levels, as reported in several
publications [43–45] However, other authors reported
that in lean as well as in lean-HIV-infected patients,
RSG treatment increased the amounts of circulating
visfatin [46,47] It thus appears that the effect of
treat-ment with PPARc ligand on circulating visfatin levels
is highly dependent on the patient phenotype
How-ever, in such studies the net contribution of visfatin
from adipocytes or macrophages cannot be evaluated
and cell-specific PPARc regulation of visfatin may
have a local effect
Adipose tissue is composed not only of adipocytes, but also of several other types of cells, including macrophages, lymphocytes and endothelial cells It has been shown that PPARc agonists induce the expression of visfatin in the visceral fat of OLETF rats [48] The authors analyzed whole adipose tissue, and thus it cannot be determined whether PPARc regulation of visfatin occurred in macrophages or in adipocytes Here we show that PPARc activation leads to an increased expression of visfatin in ATM However, this regulation does not occur in human primary mature adipocytes derived from pre-adipocyte differentiation in vitro It has been shown recently that PPARc binding in macrophages occurs at genomic locations different from those in adipocytes, showing that PPARc-binding sites are cell type-specific [49] These results are in agreement with a previous report showing that in humans, PPARc has distinct func-tions in different cell types because treatment with pioglitazone induces apoptotic cell death specifically
in macrophages, whereas differentiated adipocytes did not show any significant increase in apoptosis [50] Furthermore, treatment with pioglitazone for 3 weeks did not alter visfatin gene expression in adipose cells,
in either non-diabetic or diabetic individuals [43] Altogether, these results may allow some light to be shed on the regulation of visfatin expression by PPARc in human adipose tissue, an effect limited to ATM
In conclusion, our results identify visfatin as a novel PPARc target gene in human macrophages and dem-onstrate that PPARc activation induces visfatin gene and protein secretion in different types of human macrophages This induction of visfatin by PPARc in macrophages contributes to enhanced concentrations
of intracellular NAD+
Materials and methods
Cell culture Mononuclear cells were isolated from blood (buffy coats; thrombopheresis residues) of human healthy
normolipidem-ic donors by Fnormolipidem-icoll gradient centrifugation [21] Briefly, after Ficoll gradient centrifugation, peripheral blood mono-nuclear cells were suspended in RPMI-1640 (Gibco,
(0.05%) (both from Gibco, Invitrogen) Cells were cultured, depending on the experiment, at a density of 1 or
2· 106
cells per well in six-well plastic culture dishes (Pri-maria; Becton Dickinson Labware) Selection of a pure monocyte population occurred spontaneously after 2 h of cell adhesion to the culture dish After two washing
Trang 9steps with NaCl⁄ Pi, cells were cultured in RPMI-1640
con-taining gentamycin (40 lgÆmL)1), glutamine (0.05%) and
10% pooled human serum (Biowest, Nuaille´, France)
Dif-ferentiation of monocytes into macrophages is completed
after 7 days, characterized by immunocytochemistry or flow
cytometry analysis using macrophage marker anti-CD68
antibody [21] These primary human macrophages, also
called RM, were used for experiments after 7 days of
differ-entiation RM were incubated for 3, 6, 9, 12 or 24 h in the
presence of the PPARc ligands GW1929 (300, 600,
3000 nm) or RSG (50, 100, 1000 nm), or with
dimethylsulf-oxide as a control Where indicated, RM were transformed
and treated with the PPARc ligands GW1929 (600 nm) or
RSG (100 nm), or with dimethylsulfoxide as a control
Where indicated, the PPARc antagonist T0070907 (1 lm)
(Tocris Bioscience, Bristol, UK) or the NAMPT inhibitor
FK866 (100 nm) (Cayman Chemical, Tallinn, Estonia) were
added In other experiments, RM were treated with
GW1929 (600 nm) or dimethylsulfoxide for 24 h and then
activated into M1 macrophages by incubation with
(5 ngÆmL)1) (Promokines, Heidelberg, Germany) for 4 h or
with LPS (100 ngÆmL)1) (Sigma, Saint-Quintin Fallavier,
France) for 1 h M2 macrophages were obtained by
differ-entiating monocytes in the presence of recombinant human
IL-4 (15 ngÆmL)1) (Promokines)
Visceral adipose tissue biopsies were obtained from
consenting obese patients undergoing bariatric surgery
This study was approved by the Ethics Committee of the
University Hospital of Lille, France After removing all
fibrous materials and visible blood vessels, adipose tissue
was cut into small pieces and digested in Krebs buffer, pH
7.4, containing collagenase (1.5 mgÆmL)1; Roche
Diagnos-tic, Meylan, France) The cell suspension was filtered
through a 200-lm pore-size filter and centrifuged at 300 g
for 15 min to separate floating adipocytes The stromal
vascular fraction was pelleted, treated with erythrocyte
pH 7.4) for 10 min and filtered through meshes with a pore
size of 70 lm The stromal vascular fraction was then
sub-jected to magnetic-activated cell sorting of CD14+ cells
(Miltenyi Biotec, Paris, France) using CD14-labelled
mag-netic beads and MS columns (Miltenyi, Paris, France),
according to the manufacturer’s instructions, to yield
cytometry analysis ATM were cultured for 24 h in
endo-thelial cell basal medium, supplemented with 0.1% BSA,
before treatment with GW1929 (600 nm) or
dimethylsulfox-ide for 24 h
The CD14 negative fraction was cultured in
pre-adipo-cyte basal medium (Promocell, Heidelberg, Germany) for
24 h, then washed with NaCl⁄ Pi to remove floating cells
Adherent pre-adipocytes were then cultured in
pre-adipo-cyte growth medium (Promocell), according to the
manu-facturer’s instructions, until confluence After confluence, pre-adipocytes were cultured in pre-adipocyte differentia-tion medium (Promocell) for 72 h To complete the differ-entiation process into mature adipocytes, cells were fed every 2–3 days for 12 days with adipocyte nutrition med-ium (Promocell) At the end of the differentiation, mature adipocytes were treated with the PPARc ligand GW1929 (600 nm)
Murine bone marrow-derived macrophages were pre-pared from C57BL⁄ 6J mice Bone marrow cell suspensions were isolated by flushing the femurs and tibias with NaCl⁄ Pi and cells were cultured as previously described [51] Bone marrow-derived macrophages were treated with the PPARc ligands GW1929 (1.2 lm) and RSG (1 lm) for
24 h
RNA extraction and analysis Total cellular RNA was extracted from human macrophages using Trizol (Invitrogen, France) for RM or the RNeasy micro kit (Qiagen, Courtaboeuf, France) for ATM For Q-PCR, total RNA was reverse transcribed and cDNAs were quantified by the Q-PCR on an MX 4000 apparatus (Strata-gene) using specific primers for human visfatin (forward, GCC AGC AGG GAA TTT TGT TA-3¢; and reverse, 5¢-TGA TGT GCT GCT TCC AGT TC-3¢), mouse visfatin (forward, 5¢-TCCGGCCCGAGATGAAT-3¢; and reverse, 5¢-GTGGGTATTGTTTATAGTGAGTAACCTTGT-3¢), human CD36 (forward, 5¢-TCAGCAAATGCAAAGAAG GGAGAC-3¢; and reverse, 5¢-GGTTGACCTGCAGCCGT TTTG-3¢), mouse CD36 (forward, 5¢-GGATCTGAAATC GACCTTAAAG-3¢; and reverse, 5¢-TAGCTGGCTTGAC CAATATGTT-3¢), human FABP4 (forward, 5¢-TACTGG GCCAGGAATTTGAC-3¢; and reverse, 5¢-GTGGAAGT GACGCCTTTCAT-3¢) and human ⁄ mouse cyclophilin (for-ward, 5¢-GCA TAC GGG TCC TGG CAT CTT GTC C-3¢; and reverse, 5¢-ATG GTG ATC TTC TTG CTG GTC TTG C-3¢) Visfatin mRNA levels were subsequently normalized to those of cyclophilin
Adenovirus preparation and cell infection The recombinant adenoviruses AdGFP and AdPPARc were obtained by homologous recombination in Escherichia coliafter insertion of the cDNAs into the pAdCMV2 vector (Q.BIOgene, Illkirch, France) Viral stocks were created as previously described [52] Viral titers were determined by plaque assay on HEK 293 cells and defined as plaque-form-ing unitsÆmL)1 For the infection experiments, primary human macrophages were seeded in six-well Primaria plates
at a density of 106cells per well and viral particles were added at a multiplicity of infection of 100 for 12 h Cells were subsequently incubated for 24 h with RSG (100 nm)
or dimethylsulfoxide
Trang 10In vitro translation and EMSA
PPARc and RXRa were in vitro transcribed from the
pSG5–hPPARc and pSG5–hRXRa plasmids, respectively,
using T7 polymerase, and subsequently translated using the
transcription and translation (TNT)-coupled transcription⁄
translation system (Promega, Madison, WI, USA) Proteins
were then incubated for 10 min at room temperature in a
binding buffer (10 mm Hepes, pH 7.8, 100 mm NaCl,
contain-ing 1 lg of poly(dI-dC) and 1 lg of herrcontain-ing sperm DNA in
a total volume of 20 lL Double-stranded oligonucleotides
containing the wild-type DR1–PPRE, present at
end-labeled using T4 polynucleotide kinase and [32P]dATP[cP],
were added as a probe to the binding reaction For
compe-tition experiments, increasing amounts (5, 10, 50, 100 and
200-fold excess) of unlabeled visfatin–PPREwt (5¢-CAAT
ACAGGGCAAAGATCATGGAAG-3¢) or
visfatin–PPRE-mut (5¢-CAATACAGGAAAAAGAAAATGGAAG-3¢)
oli-gonucleotides were added to the mixture 10 min before the
DR1–visfatin–PPREwt The binding reaction was incubated
for a further 15 min at room temperature For supershift
assays, 2 lL of monoclonal mouse anti-human PPARc IgG
(Sc-7273; Santacruz Biotechnology, Heidelberg, Germany)
was added to the binding reaction DNA–protein complexes
were resolved by 6% nondenaturing PAGE in 0.25· Tris ⁄
Borate⁄ EDTA
Plasmid cloning and transient transfection
experiments
was generated by inserting six copies of the double-strand
oligonucleotides (forward, 5¢-CAATACAGGGCAAAGAT
CATGGAAG-3¢; and reverse, 5¢-CTTCCATGATCTTTG
CCCTGTATTG-3) into the pTK–pGL3 plasmid Primary
human macrophages were transfected overnight in RPMI
containing 10% human serum with reporter plasmids and
expression vectors (pSG5–empty or pSG5–hPPARc) using
expression vectors were used as an internal control of
trans-fection efficiency Subsequently, cells were incubated for an
additional 24 h in RPMI containing 2% human serum in
the presence of GW1929 (600 nm) or dimethylsulfoxide At
the end, cells were lysed, and luciferase and b-galactosidase
activities were measured on cell extracts using a luciferase
buffer (Promega)
siRNA
siRNA specific for human PBEF1 (Visfatin NAMPT), and
nonsilencing control siRNA (siScrambled) were purchased
from Dharmacon Seven-day-old human macrophages were
transfected with siRNA using the transfection reagent DharmaFECT Reagent 4 Sixteen hours after transfection, cells were incubated in the presence of GW1929 (600 nm)
or vehicle (dimethylsulfoxide) and harvested 24 h later
Protein extraction and western blot analysis Cells were washed twice with ice-cold NaCl⁄ Piand harvested
in ice-cold protein lysis buffer (RIPA) Cell homogenates were collected by centrifugation at 13 000 rpm at 4C for 30 minutes and protein concentrations were determined using the bicinchoninic acid assay (Pierce Interchim, Rockford, IL, USA) Ten micrograms of protein lysate was separated by
mem-branes (Amersham, Saclay, France) Equal loading of pro-teins was verified by Ponceau red staining Membranes were then subjected to immunodetection using rabbit polyclonal antibodies against visfatin (ab24149; Abcam, Paris, France)
or against b-actin (I-19; Santacruz Biotechnology) After incubation with a secondary peroxidase-conjugated antibody (Cell Signaling Technology, Denver, MA, USA), immunore-active bands were revealed using a chemiluminescence ECL detection kit (Amersham) and the intensity of signals was subsequently analyzed by densitometry and quantified using Quantity One software
Measure of visfatin protein secretion by ELISA Human RM were treated with the PPARc ligand GW1929 (600 nm, or with dimethylsulfoxide, for 24 h Supernatants were collected and extracellular visfatin concentrations were measured using a commercially available ELISA kit with a human visfatin (COOH-terminal) enzyme immunometric assay (Phoenix Pharmaceuticals, Karlsruhe, Germany), according to the manufacturer’s instructions
Measurement of cellular NAD content Total nicotinamide adenine dinucleotide (NADt = NAD + NADH) levels were determined in cell lysates using the
manufac-turer’s instructions (Biovision research products, Mountain View, CA, USA) Briefly, human RM, treated or not with FK866 (100 nm), infected or not with adenovirus (AdGFP, AdPPARc) and transfected or not with siRNA (siScrambled, siVisfatin) were treated with the PPARc ligands GW1929 (600 nm) or RSG (100 nm), or with dimethylsulfoxide, for
24 h Cells were lysed in NAD+extraction buffer after wash-ing three times with ice-cold NaCl⁄ Pi The NAD⁄ NADH
were normalized to protein content The results are expressed
as a percentage, with the control unstimulated cells being expressed as 100% All assays were performed in triplicate in
at least three independent experiments