Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2

Article (PDF Available)inNature 447(7147):959-65 · July 2007with217 Reads
DOI: 10.1038/nature05844 · Source: PubMed
Abstract
Adipocyte fatty-acid-binding protein, aP2 (FABP4) is expressed in adipocytes and macrophages, and integrates inflammatory and metabolic responses. Studies in aP2-deficient mice have shown that this lipid chaperone has a significant role in several aspects of metabolic syndrome, including type 2 diabetes and atherosclerosis. Here we demonstrate that an orally active small-molecule inhibitor of aP2 is an effective therapeutic agent against severe atherosclerosis and type 2 diabetes in mouse models. In macrophage and adipocyte cell lines with or without aP2, we also show the target specificity of this chemical intervention and its mechanisms of action on metabolic and inflammatory pathways. Our findings demonstrate that targeting aP2 with small-molecule inhibitors is possible and can lead to a new class of powerful therapeutic agents to prevent and treat metabolic diseases such as type 2 diabetes and atherosclerosis.
ARTICLES
Treatment of diabetes and atherosclerosis
by inhibiting fatty-acid-binding protein aP2
Masato Furuhashi
1
,Gu
¨
rol Tuncman
1
, Cem Z. Go
¨
rgu
¨
n
1
, Liza Makowski
1,4
, Genichi Atsumi
1
{, Eric Vaillancourt
1
,
Keita Kono
1
, Vladimir R. Babaev
2
, Sergio Fazio
2
, MacRae F. Linton
2
, Richard Sulsky
3
, Jeffrey A. Robl
3
, Rex A. Parker
3
&Go
¨
khan S. Hotamisligil
1
Adipocyte fatty-acid-binding protein, aP2 (FABP4) is expressed in adipocytes and macrophages, and integrates
inflammatory and metabolic responses. Studies in aP2-deficient mice have shown that this lipid chaperone has a significant
role in several aspects of metabolic syndrome, including type 2 diabetes and atherosclerosis. Here we demonstrate that an
orally active small-molecule inhibitor of aP2 is an effective therapeutic agent against severe atherosclerosis and type 2
diabetes in mouse models. In macrophage and adipocyte cell lines with or without aP2, we also show the target specificity of
this chemical intervention and its mechanisms of action on metabolic and inflammatory pathways. Our findings demonstrate
that targeting aP2 with small-molecule inhibitors is possible and can lead to a new class of powerful therapeu tic agents to
prevent and treat metabolic diseases such as type 2 diabetes and atherosclerosis.
Lipids and lipid signals are critical in the integration of metabolic and
inflammatory response systems and consequently play significant
parts in the pathogenesis of a cluster of chronic metabolic diseases,
including type 2 diabetes, fatty liver disease and atherosclerosis
1
.
However, how lipids couple to target signalling pathways or meta-
bolic processes and how their intracellular trafficking is regulated
are poorly understood. Cytoplasmic fatty-acid-binding proteins
(FABPs) are a family of 14–15-kDa proteins that bind with high
affinity to hydrophobic ligands such as saturated and unsaturated
long-chain fatty acids and eicosanoids such as hydroxyeicosatetra-
enoic acid, leukotrienes and prostaglandins
2
. The adipocyte FABP,
aP2 (FABP4), is highly expressed in adipocytes and regulated by
peroxisome-proliferator-activated receptor-c (PPARc) agonists,
insulin and fatty acids
2–5
.
Studies in aP2-deficient mice have shown that aP2 has a significant
role in many aspects of metabolic syndrome. Deficiency of aP2 par-
tially protects mice against the development of insulin resistance
associated with genetic or diet-induced obesity
6,7
. Adipocytes of
aP2
2/2
mice have reduced efficiency of lipid transport in vitro and
in vivo, and yet exhibit only minor changes in serum lipids
8
.
Interestingly, recent studies demonstrated that aP2 is also expressed
in macrophages and regulated by phorbol 12-myristate 13-acetate,
lipopolysaccharide, oxidized low-density lipoproteins and PPARc
ligands
9–12
. The macrophage is a critical site of FABP action, and total
or macrophage-specific aP2-deficiency leads to a marked protection
against early and advanced atherosclerosis in apolipoprotein
E-deficient (Apoe
2/2
) mice
9,13
.
These findings indicate an important role for aP2 in the develop-
ment of major components of metabolic syndrome through its dis-
tinct actions in adipocytes and macrophages of integrating metabolic
and inflammatory responses. Hence, pharmacological agents that
modify FABP function may offer therapeutic opportunities for many
components of metabolic syndrome, such as insulin resistance, type 2
diabetes, and atherosclerosis. Here, we demonstrate the first evidence
of the efficacy of a novel chemical aP2 inhibitor in experimental
models.
Inhibition of aP2 in cellular models
BMS309403 (Fig. 1a) is a rationally designed, potent, and selective
inhibitor of aP2 that interacts with the fatty-acid-binding pocket
within the interior of the protein and competitively inhibits the
binding of endogenous fatty acids. In a fluorescent 1,8-anilino-
8-naphthalene sulphonate (ANS) binding displacement assay,
BMS309403 exhibited K
i
values ,2 nM for both mouse and human
aP2, compared with 250 nM for muscle FABP (FABP3) and 350 nM
for mal1 (FABP5)
14
. In this assay, the endogenous fatty acids palmi-
tic acid and oleic acid exhibited aP2 K
i
values of 336 and 185 nM,
respectively. Results of X-ray crystallography studies suggested the
specific interactions of BMS309403 with key residues in the fatty-
acid-binding pocket are the basis of its high in vitro binding affinity
and selectivity for aP2 over other FABPs
14
.
To test the specificity of aP2 inhibition by BMS309403, we
developed and used a cellular system with aP2
1/1
and aP2
2/2
macrophage cell lines
9,15
. In addition, we reconstituted aP2 express-
ion in the aP2
2/2
cells (aP2
2/2
R). As shown in Fig. 1b, aP2 protein
was expressed in the THP-1 (a human monocytic leukaemia cell
line), aP2
1/1
, and aP2
2/2
R macrophages but was not detected in
the aP2
2/2
macrophages. In all of the cell lines, mal1 was present
(Fig. 1b, c). Similarly, aP2 messenger RNA was readily detectable in
THP-1, aP2
1/1
and aP2
2/2
R but not in the aP2
2/2
macrophages
(Fig. 1c). In this system, we examined the impact of aP2 inhibition on
production of monocyte chemoattractant protein (MCP)-1 (also
known as CCL2), an important aP2-regulated atherogenic prod-
uct
9,15
, in macrophages.
Treatment with BMS309403 significantly decreased MCP-1 pro-
duction from THP-1 macrophages in a dose- and time-dependent
manner (Fig. 1d, Supplementary Fig. 1). To address whether this
effect is specific, we next investigated MCP-1 production using
1
Department of Genetics and Complex Diseases, Harvard Schoo l of Public Health, Boston, Massachusetts 02115, USA.
2
Department of Medicine, Vanderbilt University Medical
Center, Nashville, Tennessee 37232, USA.
3
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA.
4
Present address: Department of Medicine,
Division of Endocrinology, Metabolism, and Nutrition, Duke University Medical Center, Durham, North Carolina 27704, USA. {Present address: Clinical Molecular Biology, Teikyo
University, Kanagawa 199-0195, Japan.
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aP2
1/1
, aP2
2/2
or aP2
2/2
R mouse macrophage cell lines. In a sim-
ilar way to THP-1 cells, production of MCP-1 from macrophages was
decreased in aP2
1/1
cells in a dose-dependent fashion. In contrast,
BMS309403 had no effect on MCP-1 production in aP2
2/2
cells at
any dose tested. However, re-expression of aP2 (aP2
2/2
R) rendered
the aP2
2/2
cells responsive to BMS309403 treatment, resulting in a
dose-dependent reduction in MCP-1 production and demonstrating
the target specificity of this compound (Fig. 1e).
The impact of aP2 inhibition on atherosclerosis
To address whether inhibition of aP2 can alter the development of
vascular lesions, we performed early and late intervention studies in
the Apoe
2/2
mouse model of atherosclerosis on a western diet. In
the early intervention study, a western diet and the aP2 inhibitor
BMS309403 were started simultaneously in 5-week-old mice (Sup-
plementary Fig. 2a). In the late intervention paradigm, the aP2 inhib-
itor was administered after 8 weeks of a western diet (at 12 weeks
of age), when significant atherosclerosis has developed (Fig. 2a).
Analysis of the en face aorta demonstrated marked reductions in
atherosclerotic lesion area in the aP2-inhibitor-treated group com-
pared with the vehicle group in both the early (52.6%, Supplemen-
tary Fig. 2a, b) and late (51.0%, Fig. 2a, b) intervention studies.
Staining of cross-sections of the proximal aorta with Oil Red O
revealed fatty streak lesions (Fig. 2c and Supplementary Fig. 2c).
These were almost exclusively macrophage-derived foam cells, as
determined by immunohistochemical staining with MOMA-2
(Fig. 2d and Supplementary Fig. 2d). Macrophages were located
predominantly on the luminal surface of the lesions. The extent of
atherosclerotic lesion area in the proximal aorta was significantly
reduced in the aP2-inhibitor-treated group compared with vehicle-
treated controls in both the early (Supplementary Fig. 2e) and late
(Fig. 2e) intervention studies.
The aP2 inhibitor did not influence body weight, systemic glucose
or lipid metabolism in Apoe
2/2
mice (Supplementary Table 1).
Examination of the distribution of cholesterol among the serum
lipoprotein fractions by size-exclusion chromatography revealed
similar lipoprotein profiles between the groups with a large peak in
the very low density lipoprotein fractions and a reduced high density
lipoprotein peak that was due to APOE-deficiency in both the early
(Supplementary Fig. 2f) and late (Fig. 2f) intervention studies. No
significant difference in glucose levels during glucose tolerance tests
was observed between the vehicle and aP2 inhibitor groups (Sup-
plementary Fig. 3a, b). These results are consistent with previous
observations made in mice with genetic deficiency of aP2 in the
Apoe
2/2
background
9,13
.
Cholesterol and inflammatory responses in macrophages
Macrophage foam cell formation has a critical role in the patho-
genesis of atherosclerosis and is a process regulated by FABPs
9
.
Transformation of THP-1 macrophage to foam cells was significantly
reduced in the presence of aP2 inhibitor (25 mM) (Fig. 3a). The aP2-
inhibitor-treated THP-1 macrophages exhibited 44% reduction in
ab
c
d
THP-1
a
P2
+
/+
aP2
–/–
aP2
/–
R
THP-1 aP2
+/+
aP2
–/–
aP2
–/–
R
aP2
+/+
aP2
–/–
aP2
–/–
R
aP2
mal1
Actin
BMS309403
Et
Ph
Ph
N
N
O
COOH
e
0
2
4
6
8
10
12
14
16
MCP-1 (ng mg
–1
cellular protein)
THP-1
0101
aP2 inhibitor
(µM)
aP2 inhibitor
(µM)
25
*
**
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
aP2
mal1
aP
2
m
al1
aP
2
mal1
aP
2
mal1
aP2/18s or mal1/18s (AU)
0
20
40
60
80
100
120
MCP-1 production
(per cent of control)
010125010125010125
*
**
**
**
Figure 1
|
Target-specific effects of aP2 inhibition on MCP-1 production in
macrophages. a
, Structure of the compound, BMS309403. b, Protein levels
of aP2 and mal1 in human THP-1 macrophages and mouse macrophage cell
lines, aP2
1/1
, aP2
2/2
and aP2
2/2
R. c, aP2 and mal1 mRNA levels analysed
by quantitative real-time PCR.
d, MCP-1 production in human THP-1
macrophages treated with aP2 inhibitor at the indicated concentrations for
24 h.
e, MCP-1 production in mouse cell lines treated with the aP2 inhibitor
at the indicated concentrations for 24 h. Data are shown as the mean 6 s.e.m.
*P , 0.05, **P , 0.01 compared with the control (each untreated cell line).
AU, arbitrary units.
ac
d
Vehicle aP2 inhibitor
b
10 15 20 25 30 35 40
Fraction number
0
10
20
30
40
50
Cholesterol
(µg per fraction)
VLDL
IDL/LDL
HDL
Vehicle
aP2 inhibitor
f
aP2 inhibitorVehicle
aP2 inhibitorVehicle
aP2 inhibitorVehicle
Oil Red O
MOMA-2
e
Bleed
Euthanize
aP2 inhibitor (15 mg kg
–1
d
–1
)
or vehicle
Western diet
Apoe
–/–
4Week: 12 17 18
Bleed GTT
0
4
8
12
16
Lesion area
(per cent of total)
aP2 inhibitorVehicle
*
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Lesion area
(µm
2
× 10
5
)
*
Figure 2
|
Atherosclerosis in Apoe
2/2
mice treated with the aP2 inhibitor.
a
, Experimental design of the late intervention study and en face aortas
stained with Sudan IV.
b, Quantitative analyses of the atherosclerotic lesion
areas (per cent of total aorta surface area) in the vehicle (n 5 16) and aP2
inhibitor (n 5 15) groups.
c, d, Oil Red O (c) and MOMA-2 (d) stainings of
atherosclerotic lesions in the aortic root at the level of the aortic valves.
Magnification, 340.
e, Quantitative analyses of the proximal aorta
atherosclerotic lesion areas in the vehicle (n 5 11) and aP2 inhibitor (n 5 6)
groups.
f, Lipoprotein profile in Apoe
2/2
mice treated with vehicle (red) and
aP2 inhibitor (blue) in the late intervention study. Data are presented as an
average (n 5 3) per cent distribution of total cholesterol for each group. Data
are expressed as the mean 6 s.e.m. *P , 0.01. VLDL, very low density
lipoprotein; IDL, intermediate density lipoprotein; LDL, low density
lipoprotein; HDL, high density lipoprotein. GTT, glucose tolerance test.
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cholesterol ester accumulation compared with vehicle-treated
macrophages (Fig. 3b). In aP2
1/1
and aP2
2/2
R macrophages treated
with the aP2 inhibitor, total cellular cholesterol ester content was
significantly lower than in macrophages treated with vehicle (Fig.
3c). The aP2 inhibitor did not affect cholesterol ester content in
the aP2
2/2
macrophages, again demonstrating the specificity of
aP2 inhibitor action in this context.
To determine the potential mechanism for the reduction in cho-
lesterol ester accumulation on inhibition of aP2, we examined
APOA1-mediated cholesterol efflux in these cells. Cholesterol efflux
from human THP-1 macrophages was significantly increased on
treatment with the aP2 inhibitor (Fig. 3d). There was a significant
increase in both mRNA and protein levels of the ATP-binding cas-
sette A1 (ABCA1) protein, a critical mediator of cholesterol efflux in
macrophages, in the aP2-inhibitor-treated THP-1 cells compared
with vehicle-treated controls (Supplementary Fig. 4a, b). Consis-
tent with earlier observations
15
, cholesterol efflux in aP2
2/2
macro-
phages was substantially higher than that of the aP2
1/1
cells (Fig. 3e)
and was completely abrogated with reconstitution of aP2 expression.
Similar to genetic deficiency, treatment with the aP2 inhibitor sig-
nificantly increased cholesterol efflux in the aP2
1/1
and aP2
2/2
R
macrophages but not in the aP2
2/2
cells. We also examined the
impact of aP2 inhibition on principal target molecules that regulate
cellular cholesterol ester synthesis and hydrolysis in macrophages.
There was a modest reduction in acyl-coenzyme A: cholesterol-acyl-
transferase 1 (ACAT1), a key enzyme of cholesterol esterification, in
the aP2-expressing macrophages (Fig. 3f) but no effect of aP2 inhibi-
tion on the expression of hormone-sensitive lipase, which acts as the
neutral cholesterol esterase, in macrophages (Supplementary Fig. 5).
Macrophages participate in the pathogenesis of atherosclerosis not
only through the formation of foam cells but also by the production
of inflammatory mediators. Hence, we determined the impact of aP2
inhibition on several critical chemoattractant and inflammatory
cytokines, including MCP-1, interleukin (IL)1b, IL6 and tumour
necrosis factor (TNF) in macrophages. Expression of these cytokines
was significantly reduced in the aP2-expressing macrophages treated
with the aP2 inhibitor compared with those treated with vehicle
(Fig. 3g–j). No regulation was evident in aP2
2/2
cells on treatment
with the inhibitor, demonstrating the target specificity of the aP2
inhibitor.
Inhibition of aP2 in adipocytes
The main site of aP2 expression is the adipocyte and although this site
does not play a major part in atherosclerosis, it does significantly
a
cd
b
e
THP-1
DMSO aP2 inhibitor
THP-1
0
4
8
12
16
Cholesterol efflux
(per cent efflux
per mg protein)
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Il6/18s (AU)
*
*
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Il1β/18s (AU)
*
*
*
fgh
ij
0
40
80
120
160
200
Cholesterol efflux
(per cent efflux
of control)
**
**
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
THP-1
Acat1/18s (AU)
*
*
*
THP-1
0
2
4
6
8
10
+
Cholesterol ester
(µg mg
–1
protein)
Cholesterol ester
(µg mg
–1
protein)
**
aP2 inhibitor
*
aP2 inhibitor aP2 inhibitor–+
aP2
inhibitor
aP2
inhibitor
–+ –+ –+ –+
aP2
inhibitor
–+ –+ –+ –+
aP2
inhibitor
–+ –+ –+ –+
–+ –+ –+ –+
aP2
inhibitor
–+ –+ –+ –+
–+ –+
aP2 inhibitor –+ + –+–+
0
5
10
15
20
25
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Mcp-1/18s (AU)
** **
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Tnf/18s(AU)
*
*
*
aP2
+/+
aP2
–/–
aP2
–/–
R
aP2
+/+
aP
2
–/–
aP2
–/–
R
THP-1
aP2
+/
+
aP2
–/–
aP2
–/–
R
THP-1
aP2
+/
+
aP2
–/–
aP2
–/–
R
THP-1
aP2
+/
+
aP2
–/–
aP2
–/–
R
THP-1
aP2
+/
+
aP2
–/–
aP2
–/–
R
aP2
+/+
aP2
–/–
aP2
–/–
R
Figure 3
|
Effects of aP2 inhibitor on lipid accumulation, cholesterol efflux
and inflammatory responses in macrophages. a
, Oil Red O staining of
THP-1 macrophage foam cells loaded with acetylated low density
lipoprotein (50 mgml
21
) in the absence or presence of aP2 inhibitor
(25 mM). Magnification, 3400.
b, c, Cholesterol ester levels normalized
to cellular protein content in human THP-1 macrophages (
b) and
mouse macrophage cell lines, aP2
1/1
, aP2
2/2
and aP2
2/2
R(c).
d, e, APOA1-specific cholesterol efflux in THP-1 macrophages (d) and
mouse cell lines (
e) in the absence or presence of aP2 inhibitor (25 mM).
fj, Expression of Acat1 (f) and chemoattractant and inflammatory
cytokines, Mcp-1 (
g), Il1b (h), Il6 (i), and Tnf (j) in macrophages normalized
to 18s rRNA levels. Data are normalized to untreated cells and expressed as
the mean 6 s.e.m. *P , 0.05, **P , 0.01 compared with the control (each
untreated cell line). DMSO, dimethyl sulphoxide.
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contribute to systemic insulin resistance and type 2 diabetes
6,7,9
.To
begin to address the specific action of aP2 inhibition in adipocytes,
we generated wild-type (aP2
1/1
mal1
1/1
) and FABP-deficient
(aP2
2/2
mal1
2/2
) pre-adipocyte cell lines as well as FABP-deficient
cells reconstituted with exogenous aP2 or with control empty vector.
These cell lines fully differentiate into adipocytes and in all properties
tested, behave in a similar way to commonly used 3T3-L1 or 3T3-
F442A adipocytes (Fig. 4a). In genetic aP2-deficiency, the principal
alteration observed in adipocytes thus far is a reduction in fatty
acid transport
8
. Hence, we asked whether the chemical inhibition
of aP2 could mimic this action in adipocytes and do so in an aP2-
dependent fashion. Treatment with the aP2 inhibitor resulted in a
dose-dependent decrease in fatty acid uptake in wild-type adipocytes
(Fig. 4b). In contrast, there was no action of this inhibitor at any dose
in FABP-deficient adipocytes. However, when aP2-deficient cells
were reconstituted with aP2, they were rendered responsive to aP2
inhibition in a dose-dependent manner. Hence, the action of the
synthetic aP2 inhibitor of regulating lipid transport in adipocytes
was specific to its target, aP2.
Inhibition of aP2 in obese and diabetic mice
Having established the target specificity of BMS309403 in adipocytes,
we administered the compound into a genetic model of obesity and
insulin resistance, the leptin-deficient ob/ob (also known as Lep
ob/ob
)
mouse, and investigated insulin sensitivity and glucose metabolism.
During the course of the 6-week treatment, there was no significant
difference in body weight between animals receiving vehicle or aP2
inhibitor (Supplementary Fig. 6a). Similarly, per cent body fat, rates
of oxygen consumption and carbon dioxide production, food intake,
and physical activity were not different between the vehicle and aP2
inhibitor treatment groups (Supplementary Fig. 6b–f). In contrast,
blood glucose levels in both the fed and fasted state were decreased
after treatment with the aP2 inhibitor (Fig. 4 c). Similar to genetic
aP2-deficiency on the ob/ob background
7
, free fatty acid levels
showed a trend towards an increase after treatment with the aP2 inhi-
bitor (Supplementary Table 2, P 5 0.07). The aP2 inhibitor decreased
insulin and triglyceride levels and increased adiponectin concentra-
tion (Fig. 4d, e, and Supplementary Table 2), suggesting a potential
increase in systemic insulin sensitivity. In fact, glucose tolerance tests
revealed a significant improvement in glucose metabolism in the aP2-
inhibitor-treated group (Fig. 4f). Similarly, insulin tolerance tests
showed significantly increased insulin sensitivity in the ob/ob mice
treated with the aP2 inhibitor (Fig. 4g). At the end of the treatment
period, we analysed islet morphology in the pancreas. There was no
difference in the pancreatic morphology such as the size, shape, and
organization of the non-b-cell mantle between the vehicle- and aP2-
inhibitor-treated ob/ob mice (Supplementary Fig. 7).
We also investigated the effect of aP2 inhibition in a diet-induced
obesity model using both wild-type and FABP-deficient mice. The
aP2-inhibitor-treated wild-type mice showed a significant decrease
in glucose levels during glucose tolerance tests compared with
vehicle-treated animals, but there was no change in glucose levels
between the vehicle- and aP2-inhibitor-treated FABP-deficient mice
on a high-fat diet (Supplementary Fig. 8). These results demonstrate
that the insulin-sensitizing effects of the aP2 inhibitor in vivo are
target-specific and effective in two independent models of obesity
and insulin resistance.
Furthermore, we performed hyperinsulinaemic–euglycaemic
clamp studies in ob/ob mice after 4 weeks of treatment. There was
no significant difference in basal hepatic glucose production between
the vehicle and aP2-inhibitor groups, but clamp hepatic glucose
production was significantly suppressed in the aP2-inhibitor-treated
ob/ob mice compared with vehicle-treated controls (Fig. 4h). Both
whole-body glucose disposal and glucose infusion rates were also
significantly increased after treatment with the aP2 inhibitor (Fig.
4i). These data demonstrate that the aP2 inhibitor improves whole-
body insulin sensitivity through the suppression of hepatic glucose
production and enhancement of insulin-stimulated glucose disposal
in peripheral tissues. To explore this further, we determined the rate
of glucose uptake in gastrocnemius muscle and epididymal fat during
the clamp procedure. In the aP2-inhibitor-treated mice, glucose
uptake in muscle and adipose tissues was significantly increased
compared with that in the vehicle-treated controls (Fig. 4j).
Effects of aP2 inhibition on adipose tissue in ob/ob mice
Adipocyte size in ob/ob mice treated with the vehicle or aP2 inhibitor
was comparable, but macrophage infiltration in adipose tissue was
more severe in the vehicle-treated group (Fig. 5a). Expression of
two macrophage markers, F4/80 (Emr1) and Cd68, was significantly
0
50
100
150
200
250
300
350
400
Glucose (mg dl
–1
)
Fed state Fasting state
*
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
aP
2
inhibit
o
r
Vehicle
*
0
5
10
15
20
25
30
Insulin (ng ml
–1
)
*
a
cd
b
f
g
e
hi
WT KO
KO + aP2 KO + GFP
*
0
0 306090120
100
200
300
400
500
Time after glucose injection (min)
0 306090120
Time after insulin injection (min)
Glucose
(per cent initial glucose)
Glucose
(per cent initial glucose)
Vehicle
aP2 inhibitor
Vehicle
aP2 inhibitor
60
80
100
120
140
**
j
0
1
2
3
4
5
6
7
Adiponectin (µg ml
–1
)
**
100806040200
40
60
80
100
120
WT
KO
KO + aP2
KO + GFP
Fatty acid uptake
(per cent of control)
aP2 inhibitor (µM)
*
*
*
*
00
2
4
6
8
10
12
Basal Clamp
HGP (mg kg
–1
min
–1
)
Rate (mg kg
–1
min
–1
)
*
0
2
4
6
8
10
12
14
Gastrocnemius
Epididymal
fat
Glucose uptake
(µg g
–1
min
–1
)
**
*
4
8
12
16
20
Rd
**
**
GIR
Figure 4
|
Metabolic studies in aP2-inhibitor-treated adipocytes and ob/ob
mice. a
, Oil Red O staining of wild-type (WT), FABP-deficient (KO), FABP-
deficient reconstituted with aP2 (KO 1 aP2), and FABP-deficient with
vector (KO 1 GFP) adipocyte cell lines.
b, Fatty acid uptake using
3
H-
stearate in adipocyte cell lines.
c, Blood glucose levels in ob/ob mice
treated with vehicle (n 5 6) or aP2 inhibitor (n 5 6) at the fed state after 2
weeks of treatment and at the fasting state after 6 weeks of treatment.
d, e, Plasma levels of insulin (d) and adiponectin (e)inob/ob mice treated
with vehicle (n 5 6) or aP2 inhibitor (n 5 6) for 6 weeks.
f, Glucose
tolerance tests performed after 4 weeks of treatment in ob/ob mice with
vehicle (open circle, n 5 6) or aP2 inhibitor (closed circle, n 5 6).
g, Insulin
tolerance tests performed after 5 weeks of treatment in ob/ob mice with
vehicle (open circle, n 5 6) or aP2 inhibitor (closed circle, n 5 6).
hj, Hyperinsulinaemic–euglycaemic clamp studies performed in ob/ob mice
treated with vehicle (n 5 7) or aP2 inhibitor (n 5 9) for 4 weeks. Basal and
clamp hepatic glucose production (HGP) (
h), glucose disposal rate (Rd) and
glucose infusion rate (GIR) (
i), and tissue glucose uptake in gastrocnemius
muscle and epididymal fat (
j). Data are shown as the mean 6 s.e.m.
*P , 0.05, **P , 0.01.
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reduced in the aP2-inhibitor-treated mice compared with vehicle-
treated controls (Fig. 5b, c). Obesity leads to increased production of
several chemoattractant and inflammatory cytokines, which have a
critical role in obesity-associated inflammation and metabolic
pathologies. Expression of Mcp-1, Il1b, Il6 and Tnf in adipose tissue
was significantly reduced in ob/ob mice treated with the aP2 inhibitor
compared with those treated with the vehicle (Fig. 5d–g).
Obesity-induced Jun N-terminal kinase (JNK) 1 activity is critical
in the generation of inflammatory responses and inhibition of
insulin action
16,17
. To examine whether aP2 inhibition modifies the
inflammatory profile and insulin action by this mechanism, we
determined JNK1 activity in the adipose tissue of vehicle- and aP2-
inhibitor-treated ob/ob mice. There was a significant attenuation
(40%) of obesity-induced adipose tissue JNK1 activity in mice
treated with aP2 inhibitor compared with vehicle-treated controls
(Fig. 5h).
We next examined whether inhibition of aP2 and the alterations
seen in inflammatory mediators and JNK activity in adipose tissue
resulted in enhanced insulin action at this site. Insulin receptor sig-
nalling capacity was examined biochemically in intact mice following
insulin administration. Insulin-stimulated tyrosine 1162/1163 phos-
phorylation of insulin receptor b subunit (IRb or INSRb subunit)
and serine 473 phosphorylation of AKT were significantly increased
in the adipose tissue of aP2-inhibitor-treated ob/ob mice compared
with that of vehicle-treated controls (Fig. 5i). These results dem-
onstrate that aP2 inhibition reduced inflammation and increased
insulin sensitivity in the adipose tissues of ob/ob mice.
Effects of aP2 inhibition on liver in ob/ob mice
In genetic aP2-deficiency, there is a striking molecular compensation
through increased expression of mal1 in adipose tissue
6
. Owing to
this compensation, the phenotype of aP2-deficiency is much milder
than aP2–mal1 combined deficiency
18
. We show here that the com-
pensatory increase in mal1 expression of adipose tissue in the genetic
absence of aP2 also occurs in the ob/ob background (Fig. 6a). Because
the aP2-inhibitor-treated animals exhibit a significant protection
against metabolic disease, we asked whether the compensatory
increase in mal1 expression was present or not under these circum-
stances. Interestingly, after 6 weeks of the aP2-inhibitor treatment,
there was no change in levels of aP2 or mal1 protein in the adipose
tissue (Fig. 6a). This is a critical observation contributing to the
efficacy of chemical inhibition of aP2 action in adult animals. For
example, in genetic aP2-deficiency, there is no protection against
fatty liver disease but a profound protection is seen in aP2–mal1
combined deficiency
18,19
. In the aP2-inhibitor-treated ob/ob mice,
fatty infiltration of the liver was attenuated (Fig. 6b) with a significant
reduction in total liver triglyceride content (Fig. 6c). This reduction
in fatty liver disease on aP2 inhibition was associated with dimi-
nished expression of key lipogenic enzymes in liver, including the
stearoyl-CoA desaturase 1 (Scd1), fatty acid synthase (Fasn), and
acetyl-CoA carboxylase 1 (Acaca) (Fig. 6d–f). This phenotype is
reminiscent of aP2–mal1 combined deficiency rather than isolated
aP2-deficiency
18,19
.
In a similar way to adipose tissue, total JNK1 activity in the liver
tissue of aP2-inhibitor-treated ob/ob mice was significantly reduced
(43%) compared with that of vehicle-treated control mice (Fig. 6g).
Suppression of fatty liver infiltration and inflammatory responses in
aP2-inhibitor-treated ob/ob mice also resulted in enhanced insulin
action in the liver. Insulin-stimulated tyrosine 1162/1163 phosphory-
lation of IRb and serine 473 phosphorylation of AKT were signifi-
cantly increased in the liver tissue of aP2-inhibitor-treated ob/ob
mice compared with vehicle-treated controls (Fig. 6h).
Discussion
A principal mechanistic core of obesity, type 2 diabetes and athero-
sclerosis resides at the interface of metabolic and inflammatory path-
ways
1
. However, this mechanistic platform has not yet been exploited
for the development of effective therapeutic strategies.
a
df
bc
e
h
g
i
p-IRβ
p-AKT
AKT
IRβ
aP2 inhibitor: +++
++++Insulin:
Vehicle
aP2 inhibitor
+++
p-c-jun
JNK1
aP2 inhibitor
**
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
F4/80 /18s (AU)
Vehicle aP2 inhibitor
*
0
0.2
0.4
0.6
0.8
1.0
1.2
Tnf/18s (AU)
0
0.5
1.0
1.5
2.0
2.5
3.0
Il6/18s (AU)
*
*
0
0.5
1.0
1.5
2.0
2.5
3.0
Il1β/18s (AU)
0
0
0.5
1.0
1.5
2.0
2.5
0.5
1.0
1.5
2.0
Vehicle
aP2 inhibitor
p-IRβ/IRβ (AU)
Insulin –+ –+
Vehicle
aP2 inhibitor
Insulin
–+ –+
*
p-AKT/AKT (AU)
*
*
0
0.2
0.4
0.6
0.8
1.0
1.2
Vehicle aP2 inhibitor
p-c-jun/JNK1(AU)
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Cd68/18s (AU)
Vehicle aP2 inhibitor
0
0.2
0.4
0.6
0.8
Mcp-1/18s (AU)
*
Vehicle
aP2 inhibitor
Vehicle
aP2 inhibitor
Vehicle
aP2 inhibitor
Vehicle
aP2 inhibitor
Figure 5
|
Effects of aP2 inhibitor in adipose tissue of ob/ob mice.
a
, Haematoxylin and eosin staining of the adipose tissue in ob/ob mice
treated with vehicle or aP2 inhibitor. Scale bar, 200 mm.
bg, Expression of
F4/80 (
b), Cd68 (c), Mcp-1 (d), Il1b (e), Il6 (f), and Tnf (g) in the adipose
tissue of ob/ob mice treated with vehicle (n 5 6) or aP2 inhibitor (n 5 6).
h, JNK1 activity in the adipose tissue of ob/ob mice. Quantification is shown
in the graph below.
i, Insulin-stimulated IRb tyrosine 1162/1163 and AKT
serine 473 phosphorylation (p) in the adipose tissues of ob/ob mice. The
graphs on the right of each blot show the quantification. Data are shown as
the mean 6 s.e.m. *P , 0.05, **P , 0.01.
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The locus for the adipocyte/macrophage FABP aP2 is critical in the
regulation and dysregulation of metabolic and inflammatory res-
ponses as they relate to metabolic diseases
6–9,13,15
. In addition to
cell-autonomous effects in macrophages and adipocytes, aP2 also
acts to coordinate the functional interactions between these two
critical cell types in adipose tissue. Genetic deletion of the aP2 gene
in mice has demonstrated a strong role for this molecule in several
chronic metabolic diseases, most notably, atherosclerosis and type 2
diabetes, and has raised the possibility of using aP2 as a potential drug
target. Here, we have provided a critical proof of principle in mice
that aP2 could be successfully targeted by an orally active, small-
molecule inhibitor to generate a profile reminiscent of genetic defi-
ciency in vitro and in vivo.
There are several indications that FABPs might be involved in meta-
bolic homeostasis in a similar fashion in humans. First, the expression
and regulation patterns of aP2 are very similar in both adipocytes and
macrophages between mice and humans
2,6,9
. Expression of aP2 is
highly regulated on macrophage activation and, interestingly, sup-
pressed by a statin in vitro
20
.Inbothmouseandhumanmacrophages,
aP2 expression modulates inflammatory responses, foam cell forma-
tion and cholesterol efflux
9,15
. Atherosclerotic lesions express high
levels of aP2 in both mice and humans
9,13,21
. Finally, aP2 expression
is increased in obesity
22
. Hence, it is possible that aP2 function may be
similar in humans as well. In fact, in a recent study, we produced
genetic support for this concept in humans
23
. A rare genetic variant
was identified at the promoter region of the human aP2 orthologue
FABP4, coinciding with the binding site for C/EBP. This particular
mutation alters C/EBP binding and significantly reduces the trans-
criptional activity of the human aP2 promoter and the expression
level of aP2 in the tissues of the carriers. In a large population
sampling, individuals with the aP2 variant had lower triglyceride levels,
exhibited reduced cardiovascular disease risk and were protected from
obesity-induced type 2 diabetes. This study offers a critical insight and
indicates that the metabolic function of aP2 in humans may be similar
to that observed in mouse models. It is therefore possible that chemical
inhibition of aP2 in humans might also show beneficial effects against
diabetes and cardiovascular disease.
METHODS SUMMARY
The synthetic agent BMS309403 is a selective, high-affinity inhibitor of aP2.
Information on synthesis and chemical properties has recently been reported
14
.
Human monocytic leukaemia THP-1 cells were obtained from ATCC.
Immortalized aP2
1/1
and aP2
2/2
mouse macrophage cell lines were generated
in our laboratory as described
9,15
. Wild-type (aP2
1/1
mal1
1/1
) and FABP-defi-
cient (aP2
2/2
mal1
2/2
) pre-adipocytes were developed using a previously
described protocol
24
. The FABP-deficient pre-adipocytes were reconstituted by
lentivirus with exogenous aP2 or with control empty vector including green
fluorescent protein. All Apoe
2/2
and ob/ob mice were from Jackson
Laboratory, and aP2
2/2
mal1
2/2
mice were generated as previously described
18
.
All mice have the C57BL/6J genetic background. The aP2 inhibitor was adminis-
tered by oral gavage. Quantification of atherosclerotic lesions was performed as
previously described
25
. Hyperinsulinaemic–euglycaemic clamps were performed
by modification of a described procedure
26
.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 21 November 2006; accepted 12 April 2007.
Published online 6 June 2007.
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a
c
b
ed
gh
p-IRβ
IRβ
p-AKT
AKT
aP2 inhibitor: +++
++++Insulin:
Vehicle
aP2 inhibitor
aP2
mal1
Actin
aP2 inhibitor
– – – – – – + + + + + +
A
+
A
f
– – – + + +
p-c-jun
JNK1
aP2 inhibitor
p-AKT/AKT (AU)
Vehicle aP2 inhibitor
Insulin
*
0
0
1.0
2.0
3.0
2
4
6
8
Vehicle
aP2 inhibitor
p-IRβ/IRβ (AU)
Insulin
+
+
+
+
*
*
0
5
10
15
20
25
30
Vehicle aP2 inhibitor
Liver TG content
(mg g
–1
of tissue)
*
0
0.2
0.4
0.6
0.8
1.0
1.2
Scd1/18s (AU)
Vehicle aP2 inhibitor
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Fasn/18s (AU)
Vehicle aP2 inhibitor
*
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0.2
0.4
0.6
0.8
1.0
Acaca/18s (AU)
Vehicle aP2 inhibitor
*
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Vehicle aP2 inhibitor
p-c-jun/JNK1(AU)
Figure 6
|
Effects of aP2 inhibitor in liver of ob/ob mice. a, aP2 and mal1
protein in the adipose tissue of ob/ob mice treated with vehicle or aP2
inhibitor. For control, the adipose tissue of ob/ob;aP2
1/1
(A
1
) and ob/
ob;aP2
2/2
(A
2
) mice was used. b, Haematoxylin and eosin staining of the
liver of ob/ob mice treated with vehicle or aP2 inhibitor. Scale bar, 200 mm.
cf, Triglyceride (TG) content (c) and mRNA expression of Scd1 (d), Fasn
(
e), and Acaca (f) in the liver of ob/ob mice treated with vehicle (n 5 6) or aP2
inhibitor (n 5 6).
g, JNK1 activity in the liver of ob/ob mice treated with
vehicle or aP2 inhibitor. The graph below the blot shows quantification.
h, Insulin-stimulated IRb tyrosine 1162/1163 and AKT serine 473
phosphorylation in the liver tissues of ob/ob mice treated with vehicle or aP2
inhibitor. The graphs demonstrate the quantification of phosphorylation of
each molecule. Data are shown as the mean 6 s.e.m. *P , 0.05.
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4. Melki, S. A. & Abumrad, N. A. Expression of the adipocyte fatty acid-binding
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6. Hotamisligil, G. S. et al. Uncoupling of obesity from insulin resistance through a
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7. Uysal, K. T., Scheja, L., Wiesbrock, S. M., Bonner-Weir, S. & Hotamisligil, G. S.
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12. Pelton, P. D., Zhou, L., Demarest, K. T. & Burris, T. P. PPARc activation induces the
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1691 (2002).
14. Sulsky, R. et al. Potent and selective biphenyl azole inhibitors of adipocyte fatty
acid binding protein (aFABP). Bioorg. Med. Chem. Lett. (in the press)
15. Makowski, L., Brittingham, K. C., Reynolds, J. M., Suttles, J. & Hotamisligil, G. S.
The fatty acid-binding protein, aP2, coordinates macrophage cholesterol
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements This work was supported in part by grants from the NIH and
the American Diabetes Association. M.F. is supported by a JSPS Postdoctoral
Fellowship for Research Abroad from the Japan Society for the Promotion of
Science. G.T. is supported by a fellowship from the Iacocca Foundation.
Author Contributions G.S.H. designed and supervised experiments and analysed
data. M.F. designed and performed experiments and analysed data. G.T., C.Z.G.,
E.V. and K.K. performed experiments. L.M. and G.A. developed cell lines from mice.
V.R.B., S.F. and M.F.L. analysed lipoprotein profiles and advised on experiments.
R.S., J.A.R. and R.A.P developed the aP2 inhibitor, BMS309403. M.F. and G.S.H
wrote the manuscript. All authors discussed the results and commented on the
manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare competing financial interests:
details accompany the paper at www.nature.com/nature. Correspondence and
requests for materials should be addressed to G.S.H.
(ghotamis@hsph.harvard.edu).
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METHODS
Biochemical reagents. All biochemical reagents were purchased from Sigma-
Aldrich (Saint Louis) unless indicated.
The compound of aP2 inhibitor BMS309403. A synthetic agent, BMS309403,
was developed and provided by Bristol–Myers Squibb Pharmaceutical Research
Institute. This agent is a selective, high affinity inhibitor of aP2 with the following
chemical properties: 2-(29-(5-ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphenyl-3-
yloxy) acetic acid (BMS309403): m.p: 174–176 uC;
1
H-NMR (500 MHz,
CDCl
3
): d 9.20 p.p.m. (br s, 1H), 7.65 (d, J 5 7.1 Hz, 1H), 7.6–7.5 (m, 3H), 7.4
(m, 2H), 7.3–7.1 (m, 5H), 7.04 (d, J 5 16.6 Hz, 2H), 6.90 (d, J 5 7.7 Hz, 1H), 6.87
(dd, J 5 2.2, 8.2 Hz, 1H), 6.66 (s, 1H), 4.36 (s, 2H), 2.05 (br s, 2H), 0.58 (t,
J 5 7.2 Hz);
13
C-NMR (125 MHz, CDCl
3
): d172.2, 157.36, 149.1, 145.1, 139.5,
139.0, 137.2, 133.6, 132.7, 130.3, 130.2, 129.7, 129.5, 128.6, 128.4, 128.1, 128.0,
127.5, 126.8, 122.0, 119.0, 114.9, 113.6,64.6, 17.6, 13.0; IR (KBr): 1710 cm
21
;
analysis (% calculated, % found for C
31
H
26
N
2
O
3
): C (78.46, 78.30), H (5.52,
5.51), N (5.90, 5.69). Additional information on synthesis and chemical prop-
erties has recently been reported
14
.
Cells. Human monocytic leukaemia THP-1 cells were obtained from ATCC and
cultured in Gibco RPMI 1640 medium (Invitrogen) supplemented with 10%
heat-inactivated fetal bovine serum (Hyclone), 50 U ml
21
penicillin and
50 mgml
21
streptomycin (Invitrogen) at 37 uCin5%CO
2
. THP-1 monocytes
were differentiated into macrophages with 100 nM phorbol 12-myristate 13-
acetate for 24 h. Immortalized aP2
1/1
and aP2
2/2
mouse macrophage cell lines
were generated in our laboratory by a modification of a described procedure (refs
9, 27). Reconstitution of aP2 expression into aP2
2/2
macrophages to produce
the aP2
2/2
R cells was performed as described
15
. The levels of aP2 protein, as
assessed by western blot, were similar in the aP2
2/2
R cell line as compared with
the aP2
1/1
macrophage line. Human THP-1 macrophages and mouse macro-
phage cell lines, aP2
1/1
, aP2
2/2
and aP2
2/2
R, were incubated in RPMI 1640
supplemented with heat-inactivated 10% FBS or 5% lipoprotein-deficient serum
(Biomedical Technologies) in the absence or presence of aP2 inhibitor dissolved
in dimethyl sulphoxide (DMSO) at the indicated concentrations. The incuba-
tion periods varied according to the experimental protocol. Each experiment was
done in at least triplicate.
We generated aP2
1/1
mal1
1/1
(WT) and aP2
2/2
mal1
2/2
(KO) pre-adipocytes
from mouse models using a previously described protocol
24
. The KO pre-
adipocytes were also reconstituted by lentivirus with exogenous aP2
(KO1aP2) or with control empty vector including green fluorescent protein
(KO1GFP). These cell lines were maintained and propagated in Dulbeco’s
Modified Eagle’s Media (Invitrogen) with 10% cosmic calf serum (Hyclone),
50 U ml
21
penicillin and 50 mgml
21
streptomycin at 37 uC in 10% CO
2
.
Differentiation was then initiated (day 0) by incubation in induction medium
(1 mM dexamethasone, 0.5 mM isobutylmethyxanthine, 1 mM rosiglitazone and
5 mgml
21
insulin). Following a 4-day induction period (two 48-h incubations),
the medium was changed to a post-induction medium (1 mM rosiglitazone and
5 mgml
21
insulin) for an additional